U.S. patent application number 13/179936 was filed with the patent office on 2012-01-12 for sub-diffraction image resolution and other imaging techniques.
This patent application is currently assigned to President and Fellows of Harvard College. Invention is credited to Wilfred M. Bates, Graham Thomas Dempsey, Bo Huang, Michael J. Rust, Xiaowei Zhuang.
Application Number | 20120009589 13/179936 |
Document ID | / |
Family ID | 39668443 |
Filed Date | 2012-01-12 |
United States Patent
Application |
20120009589 |
Kind Code |
A1 |
Zhuang; Xiaowei ; et
al. |
January 12, 2012 |
SUB-DIFFRACTION IMAGE RESOLUTION AND OTHER IMAGING TECHNIQUES
Abstract
The present invention generally relates to sub-diffraction limit
image resolution and other imaging techniques. In one aspect, the
invention is directed to determining and/or imaging light from two
or more entities separated by a distance less than the diffraction
limit of the incident light. For example, the entities may be
separated by a distance of less than about 1000 nm, or less than
about 300 nm for visible light. In one set of embodiments, the
entities may be selectively activatable, i.e., one entity can be
activated to produce light, without activating other entities. A
first entity may be activated and determined (e.g., by determining
light emitted by the entity), then a second entity may be activated
and determined. The entities may be immobilized relative to each
other and/or to a common entity. The emitted light may be used to
determine the positions of the first and second entities, for
example, using Gaussian fitting or other mathematical techniques,
and in some cases, with sub-diffraction limit resolution. The
methods may thus be used, for example, to determine the locations
of two or more entities immobilized relative to a common entity,
for example, a surface, or a biological entity such as DNA, a
protein, a cell, a tissue, etc. The entities may also be determined
with respect to time, for example, to determine a time-varying
reaction. Other aspects of the invention relate to systems for
sub-diffraction limit image resolution, computer programs and
techniques for sub-diffraction limit image resolution, methods for
promoting sub-diffraction limit image resolution, methods for
producing photoswitchable entities, and the like.
Inventors: |
Zhuang; Xiaowei; (Cambridge,
MA) ; Bates; Wilfred M.; (Goettingen, DE) ;
Rust; Michael J.; (Medford, MA) ; Huang; Bo;
(US) ; Dempsey; Graham Thomas; (US) |
Assignee: |
President and Fellows of Harvard
College
Cambridge
MA
|
Family ID: |
39668443 |
Appl. No.: |
13/179936 |
Filed: |
July 11, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12795423 |
Jun 7, 2010 |
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13179936 |
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12012524 |
Feb 1, 2008 |
7838302 |
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12795423 |
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PCT/US2007/017618 |
Aug 7, 2007 |
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12012524 |
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11605842 |
Nov 29, 2006 |
7776613 |
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PCT/US2007/017618 |
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11605842 |
Nov 29, 2006 |
7776613 |
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PCT/US2007/017618 |
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60836167 |
Aug 7, 2006 |
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60836170 |
Aug 8, 2006 |
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Current U.S.
Class: |
435/6.19 ;
435/7.1 |
Current CPC
Class: |
G01N 2201/12 20130101;
G02B 21/16 20130101; G01N 2021/6441 20130101; G01N 2201/06113
20130101; G01N 2021/6439 20130101; G01N 2021/6421 20130101; G01N
21/6458 20130101; C09K 11/06 20130101; G01N 33/582 20130101; C09K
2211/1475 20130101; G02B 27/58 20130101; C09K 2211/1044 20130101;
G02B 21/0076 20130101; G01N 15/1475 20130101; G01N 15/1429
20130101; G02B 21/367 20130101; G01N 21/6408 20130101; G01N 21/6428
20130101; C09K 2211/1018 20130101; G01N 2015/0065 20130101 |
Class at
Publication: |
435/6.19 ;
435/7.1 |
International
Class: |
G01N 21/64 20060101
G01N021/64; C12Q 1/68 20060101 C12Q001/68 |
Goverment Interests
GOVERNMENT FUNDING
[0002] This invention was made with U.S. Government support under
GM068518 awarded by the National Institutes of Health and under
N66001-04-1-8903 awarded by U.S. Navy SPAWAR/SD. The U.S.
Government has certain rights in the invention.
Claims
1. A method of determining spatial information about fluorescent
probes in a sample, the method comprising: (a) providing a sample
labeled with a plurality of fluorescent probes capable of emitting
light having a wavelength, at least some of the fluorescent probes
being separated by a distance of separation less than the
wavelength of the emitted light; (b) applying incident light to the
sample, wherein the incident light is able to to cause a
statistical subset of the plurality of fluorescent probes to emit
light, and to subsequently deactivate the statistical subset of the
plurality of fluorescent probes; (c) determining the light emitted
by the statistical subset of the plurality of fluorescent probes;
(d) repeating (b) and (c) one or more times, each time causing a
statistically different subset of the fluorescent probes to emit
light; and (f) determining the positions of at least some of the
fluorescent probes within the sample, to a precision smaller than
the wavelength of the emitted light, by using the light emitted by
the statistical subsets of the fluorescent probes.
2. The method of claim 1, further comprising constructing an image
using the positions of at least some of the fluorescent probes
determined in (f).
3. The method of claim 1, wherein the act of determining the light
emitted by the statistical subset of the plurality of fluorescent
probes comprises acquiring an image of the light emitted by the
statistical subset of the plurality of fluorescent probes.
4. The method of claim 1, wherein the act of determining the
positions of at least some of the fluorescent probes comprises
using Gaussian fitting of the light emitted by the statistical
subset of the plurality of fluorescent probes.
5. The method of claim 1, wherein the act of determining the
positions of at least some of the fluorescent probes comprises
using drift correction to determine the positions of at least some
of the fluorescent probes.
6. The method of claim 5, wherein the act of using drift correction
comprises using fiduciary markers to determine drift.
7. The method of claim 1, comprising determining the positions of
at least some of the fluorescent probes as a function of time.
8. The method of claim 1, wherein at least some of the fluorescent
probes comprise Cy5, Cy5.5, Cy7, Alexa Fluor 647, Alexa Fluor 680,
Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, DiD, DiR,
YOYO-3, YO-PRO-3, TOT-3, and/or TO-PRO-3.
9. The method of claim 1, wherein substantially all of the
fluorescent probes in the sample are essentially identical.
10. The method of claim 1, wherein at least some of the plurality
of fluorescent probes are separated by a distance of less than
about 1000 nm.
11. The method of claim 1, wherein at least some of the plurality
of fluorescent probes are photoswitchable.
12. A method of determining spatial information about fluorescent
probes in a sample, the method comprising: (a) providing a sample
labeled with a plurality of fluorescent probes capable of emitting
light having a wavelength, at least some of the fluorescent probes
being separated by a distance of separation less than the
wavelength of the emitted light; (b) exposing the plurality of
fluorescent probes to incident light to cause a statistical subset
of the plurality of fluorescent probes to emit light; (c)
determining the light emitted by the subset of the plurality of
fluorescent probes; (d) deactivating the subset of the plurality of
fluorescent probes by exposing the plurality of fluorescent probes
to incident light having substantially the same frequency as in
(b); (e) repeating (b) through (d) one or more times, each time
causing a statistically different subset of the fluorescent probes
to emit light; and (f) determining the positions of at least some
of the fluorescent probes within the sample, to a precision smaller
than the wavelength of the emitted light, by using the light
emitted by the subsets of the fluorescent probes.
13. The method of claim 12, further comprising constructing an
image using the positions of at least some of the fluorescent
probes determined in (f).
14. The method of claim 12, wherein at least some of the
fluorescent probes comprise Cy5, Cy5.5, Cy7, Alexa Fluor 647, Alexa
Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, DiD,
DiR, YOYO-3, YO-PRO-3, TOT-3, and/or TO-PRO-3.
15. The method of claim 12, wherein at least some of the plurality
of fluorescent probes are separated by a distance of less than
about 1000 nm.
16. A method of determining spatial information about fluorescent
probes in a sample, the method comprising: (a) providing a sample
labeled with a plurality of fluorescent probes capable of emitting
light having a wavelength, at least some of the fluorescent probes
being separated by a distance of separation less than the
wavelength of the emitted light; (b) causing a statistical subset
of the plurality of fluorescent probes to emit light; (c)
determining the light emitted by the statistical subset of the
plurality of fluorescent probes; (d) deactivating the statistical
subset of fluorescent probes by waiting for at least a time
sufficient to allow the subset to substantially spontaneously
deactivate; (e) repeating (b) through (d) one or more times, each
time causing a statistically different subset of the fluorescent
probes to emit light; and (f) determining the positions of at least
some of the fluorescent probes within the sample, to a precision
smaller than the wavelength of the emitted light, by using the
light emitted by the statistical subsets of the fluorescent
probes.
17. The method of claim 16, wherein the act of causing a
statistical subset of the plurality of fluorescent probes to emit
light comprises causing a statistical subset of the plurality of
fluorescent probes to emit light by applying incident light having
a sufficiently weak intensity that only the statistical subset of
the plurality of fluorescent probes is able to emit light.
18. The method of claim 16, further comprising constructing an
image using the positions of at least some of the fluorescent
probes determined in (f).
19. The method of claim 16, wherein at least some of the
fluorescent probes comprise Cy5, Cy5.5, Cy7, Alexa Fluor 647, Alexa
Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, DiD,
DiR, YOYO-3, YO-PRO-3, TOT-3, and/or TO-PRO-3.
20. The method of claim 16, wherein at least some of the plurality
of fluorescent probes are separated by a distance of less than
about 1000 nm.
Description
RELATED APPLICATIONS
[0001] This application claims priority to all of the following
according to the following to recitation of priority relationships.
This application is a continuation of U.S. patent application Ser.
No. 12/795,423, filed Jun. 7, 2010, which is a continuation of U.S.
patent application Ser. No. 12/012,524, filed Feb. 1, 2008, which
is a continuation-in-part of International Patent Application No.
PCT/US2007/017618, filed Aug. 7, 2007, entitled "Sub-Diffraction
Limit Image Resolution and other Imaging Techniques," which is a
continuation-in-part of U.S. patent application Ser. No.
11/605,842, filed Nov. 29, 2006, entitled "Sub-Diffraction Image
Resolution and other Imaging Techniques," which claims the benefit
of U.S. Provisional Patent Application Ser. No. 60/836,167, filed
Aug. 7, 2006, entitled "Sub-Diffraction Image Resolution," and the
benefit of U.S. Provisional Patent Application Ser. No. 60/836,170,
filed Aug. 8, 2006, entitled "Sub-Diffraction Image Resolution."
Said Ser. No. 12/012,524 is also a continuation-in-part of said
Ser. No. 11/605,842, which claims the benefit of said Ser. Nos.
60/836,167 and 60/836,170. Each of the above is incorporated herein
by reference.
FIELD OF INVENTION
[0003] The present invention generally relates to sub-diffraction
limit image resolution and other imaging techniques.
BACKGROUND
[0004] Fluorescence microscopy is widely used in molecular and cell
biology and other applications for non-invasive, time-resolved
imaging. Despite these advantages, standard fluorescence microscopy
is not useful for ultra-structural imaging, due to a resolution
limit set by the diffraction of light. Several approaches have been
employed to try to pass this diffraction limit, including
near-field scanning optical microscopy (NSOM), multi-photon
fluorescence, stimulated emission depletion (STED), reversible
saturable optical linear fluorescence transition (RESOLFT), and
saturated structured-illumination microscopy (SSIM), but each has
certain unsatisfactory limitations. Electron microscopy is often
used for high resolution imaging of biological samples, but
microscopy uses electrons, rather than light, and is difficult to
use with biological samples due to its preparation requirements.
Accordingly, new techniques are needed to harness the benefits of
fluorescence microscopy for ultra-resolution imaging of biological
and other samples.
SUMMARY OF THE INVENTION
[0005] The present invention generally relates to sub-diffraction
limit image resolution to and other imaging techniques. The subject
matter of the present invention involves, in some cases,
interrelated products, alternative solutions to a particular
problem, and/or a plurality of different uses of one or more
systems and/or articles.
[0006] The invention is a method, in one aspect. In one set of
embodiments, the method includes acts of providing a first entity
and a second entity separated by a distance of less than about 1000
nm, determining light emitted by the first entity, determining
light emitted by the second entity, and determining the positions
of the first entity and the second entity by using the light
emitted by the first entity and the light emitted by the second
entity. In another set of embodiments, the method includes acts of
providing a first entity and a second entity separated by a
distance of less than about 1000 nm, activating the first entity
but not the second entity, determining light emitted by the first
entity, activating the second entity, determining light emitted by
the second entity, and determining the positions of the first
entity and the second entity by using the light emitted by the
first entity and the light emitted by the second entity.
[0007] The method, according to yet another set of embodiments,
includes acts of providing a plurality of entities able to emit
light, at least some of which are separated by a distance of less
than about 1000 nm, activating a fraction of the plurality of
entities to emit light, determining the emitted light, deactivating
the activated fraction of the plurality of entities, and repeating
the acts of activating and deactivating the plurality of entities
to determine the positions of the plurality of entities.
[0008] In another set of embodiments, the method includes acts of
providing a first entity and a second entity separated by a
distance of separation, determining light emitted by the first
entity, the light emitted by the first entity having a wavelength
greater than the distance of separation, determining light emitted
by the second entity, and determining the positions of the first
entity and the second entity by using the light emitted by the
first entity and the light emitted by the second entity. The
method, in yet another set of embodiments, includes acts of
providing a first entity and a second entity separated by a
distance of separation, activating the first entity but not the
second entity, determining light emitted by the first entity, the
light emitted by the first entity having a wavelength greater than
the distance of separation, activating the second entity,
determining light emitted by the second entity, and determining the
positions of the first entity and the second entity by using the
light emitted by the first entity and the light to emitted by the
second entity.
[0009] In one set of embodiments, the method includes acts of
providing a plurality of entities able to emit light, at least some
of which are separated by a distance of separation less than the
wavelength of the emitted light, activating a fraction of the
plurality of entities to emit light, determining the emitted light,
deactivating the activated fraction of the plurality of entities,
and repeating the acts of activating and deactivating the plurality
of entities to determine the positions of the plurality of
entities.
[0010] The method, in another set of embodiments, includes acts of
providing a first entity and a second entity separated by a
distance of less than about 1000 nm where the first entity and the
second entity each are immobilized relative to a common entity,
determining the positions of the first entity and the second entity
at a first point of time, determining the positions of the first
entity and the second entity at a second point of time, and
determining movement and/or structural changes of the common entity
using the positions of the first and second entities at the first
and second points of time. In some cases, the entities may be
resolved or imaged in time. One or both of these entities may be
photoactivatable or photoswitchable in some cases. The two entities
may be chemically identical or distinct, for example, to allow
multi-color imaging. In yet another set of embodiments, the method
includes acts of providing a first entity and a second entity
separated by a distance of separation where the first entity and
the second entity are each immobilized relative to a common entity,
determining the positions of the first entity and the second entity
at a first point of time using light emitted by the first entity
and light emitted by the second entity where the light emitted by
the first entity has a wavelength greater than the distance of
separation, determining the positions of the first entity and the
second entity at a second point of time, and determining movement
and/or structural changes of the common entity using the positions
of the first and second entities at the first and second points of
time. In some cases, the entities may be resolved or imaged in
time. One or both of these entities may be photoactivatable or
photoswitchable in some cases. The two entities may be chemically
identical or distinct, for example, to allow multi-color
imaging.
[0011] In still another set of embodiments, the method includes
acts of identifying, within a series of images in time, one or more
light-emission regions, each generated by a single entity; for each
light-emission region, identifying the center of the light-emission
to region; and for each light-emission region, reconstructing the
position of the single entity generating the light-emission region
at a resolution greater than the wavelength of the light emitted by
the single entity. Some or all of these entities may be
photoactivatable or photoswitchable. The entities may be chemically
identical or distinct for example, to allow multi-color
imaging.
[0012] In another aspect, the invention is directed to an article
including a translation stage for a microscope having a drift of
less than about 100 nm/min, and/or an article including
time-modulated light sources that can be switched on and off
periodically and/or in a programmed fashion, and/or an article
including detectors for detecting fluorescence emission.
[0013] Still another aspect of the invention is directed to an
imaging composition. The composition, according to one set of
embodiments, includes a light-emitting entity, capable of being
reversibly or irreversibly switched between a first state able to
emit light at a first, emission wavelength and a second state that
does not substantially emit light at the first wavelength. In one
embodiment, the light-emitting entity comprises a first portion
that is capable of emitting light at the first wavelength, and a
second portion that activates the first portion upon exposure to an
external stimulus, thereby causing the first portion to emit light
at the first wavelength.
[0014] In another aspect, the present invention is directed to a
system for performing one or more of the embodiments described
herein. In another aspect, the present invention is directed to
computer programs and techniques for performing one or more of the
embodiments described herein. For example, one embodiment of the
invention is directed to a machine-readable medium comprising a
program, embodied in the medium, for causing a machine to perform a
method comprising acts of identifying, within a series of images in
time, one or more light-emission regions, each generated by a
single entity; for each light-emission region, identifying the
center of the light-emission region; and for each light-emission
region, reconstructing the position of the single entity generating
the light-emission region at a resolution greater than the
wavelength of the light emitted by the single entity.
[0015] Other advantages and novel features of the present invention
will become apparent from the following detailed description of
various non-limiting embodiments of the invention when considered
in conjunction with the accompanying figures. In cases to where the
present specification and a document incorporated by reference
include conflicting and/or inconsistent disclosure, the present
specification shall control. If two or more documents incorporated
by reference include conflicting and/or inconsistent disclosure
with respect to each other, then the document having the later
effective date shall control.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] Non-limiting embodiments of the present invention will be
described by way of example with reference to the accompanying
figures, which are schematic and are not intended to be drawn to
scale. In the figures, each identical or nearly identical component
illustrated is typically represented by a single numeral. For
purposes of clarity, not every component is labeled in every
figure, nor is every component of each embodiment of the invention
shown where illustration is not necessary to allow those of
ordinary skill in the art to understand the invention. In the
figures:
[0017] FIGS. 1A-1B illustrate the principle of sub-diffraction
limit resolution imaging, according to one embodiment of the
invention, and a photoswitchable Cy3-Cy5 moiety;
[0018] FIGS. 2A-2D illustrate the localization of molecular
entities according to another embodiment of the invention;
[0019] FIGS. 3A-3D illustrate the sub-diffraction limit
localization of photoswitchable molecular entities according to yet
another embodiment of the invention;
[0020] FIG. 4 illustrates the repeated switching of Cy3-Cy5-labeled
antibody, in accordance with one embodiment of the invention;
[0021] FIGS. 5A-5B illustrate drift correction in another
embodiment of the invention;
[0022] FIG. 6 illustrates the repeated switching of two molecular
switches, in yet another embodiment of the invention;
[0023] FIGS. 7A-7E respectively illustrate the structures of Cy3,
Cy5, Cy5.5, Cy7, and an example of a linked Cy3-Cy5 moiety;
[0024] FIGS. 8A-8B illustrate various photoswitchable entities,
according to certain embodiments of the invention;
[0025] FIGS. 9A-9C illustrate absorption and emission spectra of
various moieties, according to certain embodiments of the
invention;
[0026] FIGS. 10A-10F illustrate sub-diffraction limit imaging of
cells with to photoswitchable entities, according to certain
embodiments of the invention;
[0027] FIGS. 11A-11C illustrate multi-color, sub-diffraction limit
imaging of cells with photoswitchable entities, according to
certain embodiments of the invention;
[0028] FIGS. 12A-12F illustrate multi-color, sub-diffraction limit
localization of multiple types of photoswitchable molecular,
entities according to yet another embodiment of the invention;
[0029] FIGS. 13A-13H illustrate various chemical structures and
properties of certain entities of the invention;
[0030] FIG. 14 illustrates photoswitching behavior of Alexa
405-Cy7, in one embodiment of the invention;
[0031] FIG. 15 is a conventional fluorescence image of certain DNA
constructs labeled with Cy3-Cy5, Cy2-Cy5, or Alexa 405-Cy5, in an
embodiment of the invention;
[0032] FIG. 16 shows crosstalk analysis for a three-color image of
a DNA sample, in another embodiment of the invention;
[0033] FIG. 17 shows localization accuracy for a single-color image
of a cell, in one embodiment of the invention;
[0034] FIG. 18 shows localization accuracy for a two-color image of
a cell, in another embodiment of the invention;
[0035] FIGS. 19A-B shows images of various clathrin-coated pits, in
yet another embodiment of the invention; and
[0036] FIGS. 20A-20F show various emissive entities containing
succinimide moieties.
DETAILED DESCRIPTION
[0037] The present invention generally relates to sub-diffraction
limit image resolution and other imaging techniques. In one aspect,
the invention is directed to determining and/or imaging light from
two or more entities separated by a distance less than the
diffraction limit of the incident light. For example, the entities
may be separated by a distance of less than about 1000 nm, or less
than about 300 nm for visible light. In one set of embodiments, the
entities may be selectively activatable, i.e., one entity can be
activated to produce light, without activating other entities. A
first entity may be activated and determined (e.g., by determining
light emitted by the entity), then a second entity may be activated
and determined. The entities may be immobilized relative to each
other and/or to a common entity. The emitted light may be used to
determine the positions of the first and second entities, for
example, using Gaussian fitting or other mathematical techniques,
and in some cases, with sub-diffraction limit resolution. The
methods may thus be used, for example, to determine the locations
of two or more entities immobilized relative to (directly or
indirectly, e.g., via a linker) a common entity, for example, a
surface, or a biological entity such as DNA, a protein, a cell, a
tissue, a biomolecular complex, etc. The entities may also be
determined with respect to time, for example, to determine a
time-varying reaction. Other aspects of the invention relate to
systems for sub-diffraction limit image resolution, computer
programs and techniques for sub-diffraction limit image resolution,
methods for promoting sub-diffraction limit image resolution,
methods for producing photoswitchable entities, and the like.
[0038] In various aspects of the invention, any entity able to emit
light may be used. The entity may be a single molecule in some
cases. Non-limiting examples of emissive entities include
fluorescent entities (fluorophores) or phosphorescent entities, for
example, cyanine dyes (e.g., Cy2, Cy3, Cy5, Cy5.5, Cy7, etc.) metal
nanoparticles, semiconductor nanoparticles or "quantum dots," or
fluorescent proteins such as GFP (Green Fluorescent Protein). Other
light-emissive entities are readily known to those of ordinary
skill in the art. As used herein, the term "light" generally refers
to electromagnetic radiation, having any suitable wavelength (or
equivalently, frequency). For instance, in some embodiments, the
light may include wavelengths in the optical or visual range (for
example, having a wavelength of between about 400 nm and about 700
nm, i.e., "visible light"), infrared wavelengths (for example,
having a wavelength of between about 300 micrometers and 700 nm),
ultraviolet wavelengths (for example, having a wavelength of
between about 400 nm and about 10 nm), or the like. In certain
cases, as discussed in detail below, more than one entity may be
used, i.e., entities that are chemically different or distinct, for
example, structurally. However, in other cases, the entities may be
chemically identical or at least substantially chemically
identical.
[0039] In one set of embodiments, the entity is "switchable," i.e.,
the entity can be switched between two or more states, at least one
of which emits light having a desired wavelength. In the other
state(s), the entity may emit no light, or emit light at a
different wavelength. For instance, an entity may be "activated" to
a first state able to produce to light having a desired wavelength,
and "deactivated" to a second state. An entity is
"photoactivatable" if it can be activated by incident light of a
suitable wavelength. As a non-limiting example, Cy5, can be
switched between a fluorescent and a dark state in a controlled and
reversible manner by light of different wavelengths, i.e., 633 nm
red light can switch or deactivate Cy5 to a stable dark state,
while 532 nm green light can switch or activate the Cy5 back to the
fluorescent state. In some cases, the entity can be reversibly
switched between the two or more states, e.g., upon exposure to the
proper stimuli. For example, a first stimuli (e.g., a first
wavelength of light) may be used to activate the switchable entity,
while a second stimuli (e.g., a second wavelength of light) may be
used to deactivate the switchable entity, for instance, to a
non-emitting state. Any suitable method may be used to activate the
entity. For example, in one embodiment, incident light of a
suitable wavelength may be used to activate the entity to emit
light, i.e., the entity is "photoswitchable." Thus, the
photoswitchable entity can be switched between different
light-emitting or non-emitting states by incident light, e.g., of
different wavelengths. The light may be monochromatic (e.g.,
produced using a laser) or polychromatic. In another embodiment,
the entity may be activated upon stimulation by electric field
and/or magnetic field. In other embodiments, the entity may be
activated upon exposure to a suitable chemical environment, e.g.,
by adjusting the pH, or inducing a reversible chemical reaction
involving the entity, etc. Similarly, any suitable method may be
used to deactivate the entity, and the methods of activating and
deactivating the entity need not be the same. For instance, the
entity may be deactivated upon exposure to incident light of a
suitable wavelength, or the entity may be deactivated by waiting a
sufficient time.
[0040] Typically, a "switchable" entity can be identified by one of
ordinary skill in the art by determining conditions under which an
entity in a first state can emit light when exposed to an
excitation wavelength, switching the entity from the first state to
the second state, e.g., upon exposure to light of a switching
wavelength, then showing that the entity, while in the second state
can no longer emit light (or emits light at a much reduced
intensity) when exposed to the excitation wavelength.
[0041] In one set of embodiments, as discussed, a switchable entity
may be switched upon exposure to light. In some cases, the light
used to activate the switchable entity may come from an external
source (e.g., a light source such as a fluorescent light source,
another light-emitting entity proximate the switchable entity,
etc.). The second, light emitting entity, in some cases, may be a
fluorescent entity, and in certain embodiments, the second,
light-emitting entity may itself also be a switchable entity.
In some embodiments, the switchable entity includes a first,
light-emitting portion (e.g., a fluorophore), and a second portion
that activates or "switches" the first portion. For example, upon
exposure to light, the second portion of the switchable entity may
activate the first portion, causing the first portion to emit
light. Examples of activator portions include, but are not limited
to, Alexa Fluor 405 (Invitrogen), Alexa Fluor 488 (Invitrogen), Cy2
(GE Healthcare), Cy3 (GE Healthcare), Cy3B (GE Healthcare), Cy3.5
(GE Healthcare), or other suitable dyes. Examples of light-emitting
portions include, but are not limited to, Cy5, Cy5.5 (GE
Healthcare), Cy7 (GE Healthcare), Alexa Fluor 647 (Invitrogen),
Alexa Fluor 680 (Invitrogen), Alexa Fluor 700 (Invitrogen), Alexa
Fluor 750 (Invitrogen), Alexa Fluor 790 (Invitrogen), DiD, DiR,
YOYO-3 (Invitrogen), YO-PRO-3 (Invitrogen), TOT-3 (Invitrogen),
TO-PRO-3 (Invitrogen) or other suitable dyes. These may linked
together, e.g., covalently, for example, directly, or through a
linker, e.g., forming compounds such as, but not limited to,
Cy5-Alexa Fluor 405, Cy5-Alexa Fluor 488, Cy5-Cy2, Cy5-Cy3,
Cy5-Cy3.5, Cy5.5-Alexa Fluor 405, Cy5.5-Alexa Fluor 488, Cy5.5-Cy2,
Cy5.5-Cy3, Cy5.5-Cy3.5, Cy7-Alexa Fluor 405, Cy7-Alexa Fluor 488,
Cy7-Cy2, Cy7-Cy3, Cy7-Cy3.5, Alexa Fluor 647-Alexa Fluor 405, Alexa
Fluor 647-Alexa Fluor 488, Alexa Fluor 647-Cy2, Alexa Fluor
647-Cy3, or Alexa Fluor 647-Cy3.5. The structures of Cy3, Cy5,
Cy5.5, and Cy7 are shown in FIG. 7, with a non-limiting example of
a linked version of Cy3-Cy5 shown in FIG. 7E; those of ordinary
skill in the art will be aware of the structures of these and other
compounds, many of which are available commercially. The portions
may be linked via a covalent bond, or by a linker, such as those
described in detail below. Other light-emitting or activator
portions may include portions having two quaternized nitrogen atoms
joined by a polymethine chain, where each nitrogen is independently
part of a heteroaromatic moiety, such as pyrrole, imidazole,
thiazole, pyridine, quinoine, indole, benzothiazole, etc., or part
of a nonaromatic amine. In some cases, there may be 5, 6, 7, 8, 9,
or more carbon atoms between the two nitrogen atoms.
[0042] In certain cases, the light-emitting portion and the
activator portions, when isolated from each other, may each be
fluorophores, i.e., entities that can emit light of a certain,
emission wavelength when exposed to a stimulus, for example, an
excitation wavelength. However, when a switchable entity is formed
that comprises the first fluorophore and the second fluorophore,
the first fluorophore forms a first, light-emitting portion and the
second fluorophore forms an activator portion that switches that
activates or "switches" the first portion in response to a
stimulus. For example, the switichable entity may comprise a first
fluorophore directly bonded to the second fluorophore, or the first
and second entity may be connected via a linker or a common entity.
Whether a pair of light-emitting portion and activator portion
produces a suitable switchable entity can be tested by methods
known to those of ordinary skills in the art. For example, light of
various wavelength can be used to stimulate the pair and emission
light from the light-emitting portion can be measured to determined
wither the pair makes a suitable switch.
[0043] As a non-limiting example, Cy3 and Cy5 may be linked
together to form such an entity. Such a procedure is described in
more detail in the Examples, below. In this example, Cy3 is an
activator portion that is able to activate Cy5, the light-emission
portion. Thus, light at or near the absorption maximum (e.g., near
532 nm light for Cy3) of the activation or second portion of the
entity may cause that portion to activate the first, light-emitting
portion, thereby causing the first portion to emit light (e.g.,
near 633 nm for Cy5). As previously described, the first,
light-emitting portion can subsequently be deactivated by any
suitable technique (e.g., by directing 633 nm red light to the Cy5
portion of the molecule).
[0044] Other non-limiting examples of potentially suitable
activator portions include 1,5 IAEDANS, 1,8-ANS,
4-Methylumbelliferone, 5-carboxy-2,7-dichlorofluorescein,
5-Carboxyfluorescein (5-FAM), 5-Carboxynapthofluorescein,
5-Carboxytetramethylrhodamine (5-TAMRA), 5-FAM
(5-Carboxyfluorescein), 5-HAT (Hydroxy Tryptamine), 5-Hydroxy
Tryptamine (HAT), 5-ROX (carboxy-X-rhodamine), 5-TAMRA
(5-Carboxytetramethylrhodamine), 6-Carboxyrhodamine 6G, 6-CR 6G,
6-JOE, 7-Amino-4-methylcoumarin, 7-Aminoactinomycin D (7-AAD),
7-Hydroxy-4-methylcoumarin, 9-Amino-6-chloro-2-methoxyacridine,
ABQ, Acid Fuchsin, ACMA (9-Amino-6-chloro-2-methoxyacridine),
Acridine Orange, Acridine Red, Acridine Yellow, Acriflavin,
Acriflavin Feulgen SITSA, Alexa Fluor 350, Alexa Fluor 405, Alexa
Fluor 430, Alexa Fluor 488, Alexa Fluor 500, Alexa Fluor 514, Alexa
Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa
Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 635,
Alizarin Complexon, Alizarin Red, AMC, AMCA-S, AMCA
(Aminomethylcoumarin), AMCA-X, Aminoactinomycin D, Aminocoumarin,
Aminomethylcoumarin (AMCA), Anilin Blue, Anthrocyl stearate,
APTRA-BTC, APTS, Astrazon Brilliant Red 4G, Astrazon Orange R,
Astrazon Red 6B, Astrazon Yellow 7GLL, Atabrine, ATTO 390, ATTO
425, ATTO 465, ATTO 488, ATTO 495, ATTO 520, ATTO 532, ATTO 550,
ATTO 565, ATTO 590, ATTO 594, ATTO 610, ATTO 611X, ATTO 620, ATTO
633, ATTO 635, ATTO 647, ATTO 647N, ATTO 655, ATTO 680, ATTO 700,
ATTO 725, ATTO 740, ATTO-TAG CBQCA, ATTO-TAG FQ, Auramine,
Aurophosphine G, Aurophosphine, BAO 9 (Bisaminophenyloxadiazole),
BCECF (high pH), BCECF (low pH), Berberine Sulphate, Bimane,
Bisbenzamide, Bisbenzimide (Hoechst), bis-BTC, Blancophor FFG,
Blancophor SV, BOBO-1, BOBO-3, Bodipy 492/515, Bodipy 493/503,
Bodipy 500/510, Bodipy 505/515, Bodipy 530/550, Bodipy 542/563,
Bodipy 558/568, Bodipy 564/570, Bodipy 576/589, Bodipy 581/591,
Bodipy 630/650-X, Bodipy 650/665-X, Bodipy 665/676, Bodipy Fl,
Bodipy FL ATP, Bodipy Fl-Ceramide, Bodipy R6G, Bodipy TMR, Bodipy
TMR-X conjugate, Bodipy TMR-X, SE, Bodipy TR, Bodipy TR ATP, Bodipy
TR-X SE, BO-PRO-1, BO-PRO-3, Brilliant Sulphoflavin FF, BTC,
BTC-5N, Calcein, Calcein Blue, Calcium Crimson, Calcium Green,
Calcium Green-1 Ca.sup.2+ Dye, Calcium Green-2 Ca.sup.2+, Calcium
Green-5N Ca.sup.2+, Calcium Green-C18 Ca.sup.2+, Calcium Orange,
Calcofluor White, Carboxy-X-rhodamine (5-ROX), Cascade Blue,
Cascade Yellow, Catecholamine, CCF2 (GeneBlazer), CFDA, Chromomycin
A, Chromomycin A, CL-NERF, CMFDA, Coumarin Phalloidin, CPM
Methylcoumarin, CTC, CTC Formazan, Cy2, Cy3.1 8, Cy3.5, Cy3, Cy5.1
8, cyclic AMP Fluorosensor (FiCRhR), Dabcyl, Dansyl, Dansyl Amine,
Dansyl Cadaverine, Dansyl Chloride, Dansyl DHPE, Dansyl fluoride,
DAPI, Dapoxyl, Dapoxyl 2, Dapoxyl 3' DCFDA, DCFH
(Dichlorodihydrofluorescein Diacetate), DDAO, DHR (Dihydrorhodamine
123), Di-4-ANEPPS, Di-8-ANEPPS (non-ratio), DiA (4-Di-16-ASP),
Dichlorodihydrofluorescein Diacetate (DCFH), DiD-Lipophilic Tracer,
DiD (DiIC18(5)), DIDS, Dihydrorhodamine 123 (DHR), DiI (DiIC18(3)),
Dinitrophenol, DiO (DiOC18(3)), DiR, DiR (DiIC18(7)), DM-NERF (high
pH), DNP, Dopamine, DTAF, DY-630-NHS, DY-635-NHS, DyLight 405,
DyLight 488, DyLight 549, DyLight 633, DyLight 649, DyLight 680,
DyLight 800, ELF 97, Eosin, Erythrosin, Erythrosin ITC, Ethidium
Bromide, Ethidium homodimer-1 (EthD-1), Euchrysin, EukoLight,
Europium to (III) chloride, Fast Blue, FDA, Feulgen
(Pararosaniline), FIF (Formaldehyd Induced Fluorescence), FITC,
Flazo Orange, Fluo-3, Fluo-4, Fluorescein (FITC), Fluorescein
Diacetate, Fluoro-Emerald, Fluoro-Gold (Hydroxystilbamidine),
Fluor-Ruby, Fluor X, FM 1-43, FM 4-46, Fura Red (high pH), Fura
Red/Fluo-3, Fura-2, Fura-2/BCECF, Genacryl Brilliant Red B,
Genacryl Brilliant Yellow 10GF, Genacryl Pink 3G, Genacryl Yellow
5GF, GeneBlazer (CCF2), Gloxalic Acid, Granular blue,
Haematoporphyrin, Hoechst 33258, Hoechst 33342, Hoechst 34580,
HPTS, Hydroxycoumarin, Hydroxystilbamidine (FluoroGold),
Hydroxytryptamine, Indo-1, high calcium, Indo-1, low calcium,
Indodicarbocyanine (DiD), Indotricarbocyanine (DiR), Intrawhite Cf,
JC-1, JO-JO-1, JO-PRO-1, LaserPro, Laurodan, LDS 751 (DNA), LDS 751
(RNA), Leucophor PAF, Leucophor SF, Leucophor WS, Lissamine
Rhodamine, Lissamine Rhodamine B, Calcein/Ethidium homodimer,
LOLO-1, LO-PRO-1, Lucifer Yellow, Lyso Tracker Blue, Lyso Tracker
Blue-White, Lyso Tracker Green, Lyso Tracker Red, Lyso Tracker
Yellow, LysoSensor Blue, LysoSensor Green, LysoSensor Yellow/Blue,
Mag Green, Magdala Red (Phloxin B), Mag-Fura Red, Mag-Fura-2,
Mag-Fura-5, Mag-Indo-1, Magnesium Green, Magnesium Orange,
Malachite Green, Marina Blue, Maxilon Brilliant Flavin 10 GFF,
Maxilon Brilliant Flavin 8 GFF, Merocyanin, Methoxycoumarin,
Mitotracker Green FM, Mitotracker Orange, Mitotracker Red,
Mitramycin, Monobromobimane, Monobromobimane (mBBr-GSH),
Monochlorobimane, MPS (Methyl Green Pyronine Stilbene), NBD, NBD
Amine, Nile Red, Nitrobenzoxadidole, Noradrenaline, Nuclear Fast
Red, Nuclear Yellow, Nylosan Brilliant Iavin EBG, Oregon Green,
Oregon Green 488-X, Oregon Green, Oregon Green 488, Oregon Green
500, Oregon Green 514, Pacific Blue, Pararosaniline (Feulgen),
PBFI, Phtoxin B (Magdala Red), Phorwite AR, Phorwite BKL, Phorwite
Rev, Phorwite RPA, Phosphine 3R, PKH26 (Sigma), PKH67, PMIA,
Pontochrome Blue Black, POPO-1, POPO-3, PO--PRO-1, PO-PRO-3,
Primuline, Procion Yellow, Propidium Iodid (PI), PyMPO, Pyrene,
Pyronine, Pyronine B, Pyrozal Brilliant Flavin 7GF, QSY 7,
Quinacrine Mustard, Resorufin, RH 414, Rhod-2, Rhodamine, Rhodamine
110, Rhodamine 123, Rhodamine 5 GLD, Rhodamine 6G, Rhodamine B,
Rhodamine B 200, Rhodamine B extra, Rhodamine BB, Rhodamine BG,
Rhodamine Green, Rhodamine Phallicidine, Rhodamine Phalloidine,
Rhodamine Red, Rhodamine WT, Rose Bengal, S65A, S65C, S65L, S65T,
SBFI, Serotonin, Sevron Brilliant Red 2B, Sevron Brilliant Red 4G,
Sevron to Brilliant Red B, Sevron Orange, Sevron Yellow L, SITS,
SITS (Primuline), SITS (Stilbene Isothiosulphonic Acid), SNAFL
calcein, SNAFL-1, SNAFL-2, SNARF calcein, SNARF1, Sodium Green,
SpectrumAqua, SpectrumGreen, SpectrumOrange, Spectrum Red, SPQ
(6-methoxy-N-(3-sulfopropyl)quinolinium), Stilbene, Sulphorhodamine
B can C, Sulphorhodamine Extra, SYTO 11, SYTO 12, SYTO 13, SYTO 14,
SYTO 15, SYTO 16, SYTO 17, SYTO 18, SYTO 20, SYTO 21, SYTO 22, SYTO
23, SYTO 24, SYTO 25, SYTO 40, SYTO 41, SYTO 42, SYTO 43, SYTO 44,
SYTO 45, SYTO 59, SYTO 60, SYTO 61, SYTO 62, SYTO 63, SYTO 64, SYTO
80, SYTO 81, SYTO 82, SYTO 83, SYTO 84, SYTO 85, SYTOX Blue, SYTOX
Green, SYTOX Orange, Tetracycline, Tetramethylrhodamine (TAMRA),
Texas Red, Texas Red-X conjugate, Thiadicarbocyanine (DiSC3),
Thiazine Red R, Thiazole Orange, Thioflavin 5, Thioflavin S,
Thioflavin TCN, Thiolyte, Thiozole Orange, Tinopol CBS (Calcofluor
White), TMR, TO-PRO-1, TO-PRO-3, TO-PRO-5, TOTO-1, TOTO-3, TRITC
(tetramethylrodamine isothiocyanate), True Blue, TruRed, Ultralite,
Uranine B, Uvitex SFC, WW 781, X-Rhodamine, XRITC, Xylene Orange,
Y66F, Y66H, Y66W, YO-PRO-1, YO-PRO-3, YOYO-1, YOYO-3, SYBR Green,
Thiazole orange (interchelating dyes), or combinations thereof.
[0045] Accordingly, in one embodiment of the invention, a
light-emitting switchable entity is provided, comprising a first,
light emitting portion and a second, activation portion. The entity
has a maximum emission wavelength determined by the first, light
emitting portion and a maximum activation wavelength determined by
the second, activation portion. Notably, the two wavelengths are
not controlled by the same molecular entity, and are effectively
decoupled. In some cases, the same wavelength light can be used
both for activating the emitting portion to a fluorescent state and
for exciting emission from and deactivating the emitting portion.
Further, multiple types of switchable entities within a sample may
be independently determined. For example, two switchable entities
having the same activator portions but different light-emission
portions can be activated by the same wavelength light applied to
the sample, but emit at different wavelengths due to different
light-emission portions and can be easily distinguished, even at
separation distances of less than sub-diffraction limit
resolutions. This can effectively yield two colors in the image.
Similarly, two switchable entities having the same light-emission
portions but different activator portions can be activated to by
different wavelength light applied to the sample, due to the
different activator portions, and the light-emission portions may
emit at same wavelengths and can thus be distinguished, even at
separation distances of less than sub-diffraction limit
resolutions. This also can effectively yield two colors in the
image. When these methods are combined, four (or more) color images
can be readily produced. Using this principle, multi-color imaging
can be scaled up to 6 colors, 9 colors, etc., depending on the
switchable and/or activator entities. This multi-color imaging
principle may also be used with the imaging methods described
herein to yield sub-diffraction limit resolutions, and/or used to
obtained multi-color images with other imaging methods not limited
to sub-diffraction limit resolutions.
[0046] In contrast, fluorescent dyes commonly used by those of
ordinary skill in the art (e.g., isolated Cy5) inherently have a
maximum excitation wavelength and a maximum emission wavelength
that are each determined by the nature and structure of the
fluorescent dye, and cannot be independently controlled. Thus, the
invention, in another set of embodiments, provides a range of
switchable entities where the first, light emitting portion and the
second, activation portion are each independently selected.
Accordingly, in some embodiments, a larger set of colors is
provided, e.g., with respect to conventional imaging methods.
[0047] Any suitable method may be used to link the first,
light-emitting portion and the second, activation portion. For
instance, the light-emitting and the activation portions may be
covalently bonded to each other, for example, using the techniques
described below. In some cases, a linker is used, and is chosen
such that the distance between the first and second portions is
sufficiently close to allow the activator portion to activate the
light-emitting portion as desired, e.g., whenever the
light-emitting portion has been deactivated in some fashion.
Typically, the portions will be separated by distances on the order
of 500 nm or less, for example, less than about 300 nm, less than
about 100 nm, less than about 50 nm, less than about 20 nm, less
than about 10 nm, less than about 5 nm, less than about 2 nm, less
than about 1 nm, etc. Examples of linkers include, but are not
limited to, carbon chains (e.g., alkanes or alkenes), optionally
including one or more heteroatoms, polymer units, a biological
molecule such as a nucleic acid (DNA, RNA, PNA, LNA, or the like),
a lipid molecule, a protein or a polypeptide, a carbohydrate or
polysaccaride molecule, or the like.
[0048] In some cases, a linker comprising a rigid portion may be
used. As used herein, a "rigid" portion means a portion of a
molecule, the ends of which are separated by a distance which
cannot change (outside of normal molecule-scale changes in
temperature, etc.) without breaking at least one bond. Examples of
rigid portions include aryl or alkyne groups. For example, the
linker may include phenyl, pyridinyl, biphenyl, xylyl, acetylene,
or the like. The light-emitting portion and/or the activator
portion may be attached to the linker using any suitable technique.
In some embodiments, the technique may include the use of
attachment systems including an electrophile-nucleophile
combination. For example, the light-emitting portion and/or the
activator portion may comprise an electrophilic atom, which refers
to an atom which may be attacked by, and forms a new bond to, a
nucleophilic atom (e.g., an atom having a reactive pair of
electrons). In some cases, the electrophilic atom may comprise a
suitable leaving group. The electrophilic atom can be attached to
(e.g., reacted with) a linker comprising a nucleophilic atom. In
some embodiments, the technique includes reacting a light-emitting
portion and/or the activator portion comprising a nucleophilic atom
with a linker comprising an electrophilic atom. Non-limiting
examples of functional groups comprising electrophilic atoms
include a carbonyl group such as an aldehyde, an ester, a
carboxylic acid, a ketone, an amide (e.g., iodoacetamide), an
anhydride, an acid chloride, a hydrazone, a succinimide, a
maleimide group, or an alpha,beta-unsaturated ketone. Examples of
functional groups comprising nucleophilic atoms include, but are
not limited to, a thiol, a hydroxyl group, an amine, a hydrazide,
or the like.
[0049] Non-limiting examples of potentially useful attachment
systems include succinimide-amine (e.g., producing an amide),
maleimide-thiol or iodoacetamide-thiol (e.g., producing a thiol
ester or a thiol ether), amine-carboxylic acid, hydrazide-aldehyde
or hydrazide-ketone (e.g., producing an amine or an imine),
disulfide bonds, or the like. As an example, a light-emitting
portion may contain a succinimide moiety (positioned anywhere in
the light-emitting portion), and be reacted with a nucleic acid
linker (e.g., DNA) containing one or more amine groups, a protein
linker (e.g., an antibody or an enzyme) containing one or more
amine groups, etc. Similarly, an activator portion may be attached
to the linker using the same, or different techniques. For
instance, the activator portion may contain a maleimide moiety,
which can react with a thiol on the protein linker. In some cases,
such moieties can be commercially obtained. As a to specific,
non-limiting example, certain dyes such as Cy3 and Cy5 are
commercially sold conjugated to succinimide moieties, and certain
nucleic acids are sold modified to include various amine groups at
certain locations, such that the dyes can be conjugated to the
nucleic acids via succinimide-amine reactions. As another example,
an amine-modified Alexa 647, which may be obtained commercially,
can be directly bonded to a bis-functional Cy3 NHS
(N-hydroxysuccinimide) ester (also obtainable commercially) via a
succidimide-amine attachment method.
[0050] Thus, in some cases, a succinimide moiety and/or a maleimide
moiety may be attached to the light-emitting portion and/or the
activator portion, and the succinimide moiety and/or maleimide
moiety may be covalently bonded to amines such as primary amines,
e.g., on a linker. Non-limiting examples of light-emitting or
activators containing such moieties are shown in FIGS. 20A-20F. The
light-emitting portion and/or the activator portion may be bonded
to a linker, or to each other, using such moieties. Examples of
compounds having such moieties include those discussed above;
structural formulae of some of these compounds can be seen in the
figures. As used herein, a "succinimide moiety" is a moiety having
a general succinimide structure, e.g.:
##STR00001##
Similarly, a "maleimide moiety" is a moiety having a general
maleimide structure, e.g.:
##STR00002##
where each of R.sup.1, R.sup.2, and R.sup.3 in the above structures
independently is a hydrogen atom (i.e., succinimide or maleimide,
respectively) or represents other, non-hydrogen atoms or group of
atoms, for example, halogens, alkyls, alkoxyls, etc. In some cases,
at least one of R.sup.1, R.sup.2, and R.sup.3 may indicate
attachment of the succinimide or maleimide moiety to a linker.
[0051] Those of ordinary skill in the art would be able to select
other attachment systems suitable for use in the context of the
invention. For example, the light-emitting portion and/or the
activator portion may be attached to the linker via pericyclic
reactions (e.g., Diels-Alder reactions, cycloadditions, etc.),
Wittig reactions, metal-catalyzed reactions (e.g., cross-coupling
reactions), and the like.
[0052] In some cases, the light-emitting portion and/or the
activator portion may contain more than one functional group, one
or more of which may be used to attach the portion to the linker.
For example, the light-emitting portion and/or the activator
portion may comprise two functional groups (e.g., a bi-functional
portion), which may be the same or different. Those of ordinary
skill in the art would be able to synthesize compositions of the
invention utilizing such bi-functional, tri-functional, or other
multi-functional portions. For example, the synthesis may comprise
the use of one or more protecting groups to alter the reactivity of
one functional group relative to another functional group, such
that the functional groups may be reacted in a particular manner at
a selected point in the synthesis. In an illustrative embodiment, a
light-emitting portion may comprise two amine groups, where one
amine may be reacted with di-tert-butyl dicarbonate to form a
"protected" amine (e.g., N-tert-butoxycarbonyl- or t-BOC-protected)
having reduced reactivity relative to the un-protected amine, under
a particular set of conditions. The phrase "protecting group," as
used herein, refers to temporary substituents which protect a
potentially reactive functional group from undesired chemical
transformations. Examples of such protecting groups include esters
of carboxylic acids, silyl ethers of alcohols, and acetals and
ketals of aldehydes and ketones, respectively. Other protecting
groups are described in, for example, Greene, T. W., Wuts, P. G. M.
Protective Groups in Organic Synthesis, 2.sup.nd ed., Wiley: New
York, 1991.
[0053] In one set of embodiments of the invention, the
light-emitting switchable entity can also include other switchable
fluorescent probes that do not necessarily include the two
portions, such as, but not limited to, photoactivatable or
photoswitchable dye molecules, natural or engineered fluorescent
proteins that are photoactivatable or photoswitchable,
photoactivatable or photoswitchable inorganic particles, or the
like. In some cases, the switchable entity may include a first,
light-emitting portion and a second, activation portion, as
discussed herein.
[0054] In one set of embodiments, the switchable entity can be
immobilized, e.g., covalently, with respect to a binding partner,
i.e., a molecule that can undergo binding with a particular
analyte. Binding partners include specific, semi-specific, and
non-specific binding partners as known to those of ordinary skill
in the art. The term "specifically binds," when referring to a
binding partner (e.g., protein, nucleic acid, antibody, etc.),
refers to a reaction that is determinative of the presence and/or
identity of one or other member of the binding pair in a mixture of
heterogeneous molecules (e.g., proteins and other biologics). Thus,
for example, in the case of a receptor/ligand binding pair, the
ligand would specifically and/or preferentially select its receptor
from a complex mixture of molecules, or vice versa. Other examples
include, but are not limited to, an enzyme would specifically bind
to its substrate, a nucleic acid would specifically bind to its
complement, an antibody would specifically bind to its antigen. The
binding may be by one or more of a variety of mechanisms including,
but not limited to ionic interactions, and/or covalent
interactions, and/or hydrophobic interactions, and/or van der Waals
interactions, etc. By immobilizing a switchable entity with respect
to the binding partner of a target molecule or structure (e.g., DNA
or a protein within a cell), the switchable entity can be used for
various determination or imaging purposes. For example, a
switchable entity having an amine-reactive group may be reacted
with a binding partner comprising amines, for example, antibodies,
proteins or enzymes. A non-limiting example of the immobilization
of a switchable entity to an antibody is discussed in the Examples,
below.
[0055] In some embodiments, more than one switchable entity may be
used, and the entities may be the same or different. In some cases,
the light emitted by a first entity and the light emitted by a
second entity have the same wavelength. The entities may be
activated at different times and the light from each entity may be
determined separately. This allows the location of the two entities
to be determined separately and, in some cases, the two entities
may be spatially resolved, as discussed in detail below, even at
distances of separation that are less than the light emitted by the
entities or below the diffraction limit of the emitted light (i.e.,
"sub-diffraction limit" resolutions). In certain instances, the
light emitted by a first entity and the light emitted by a second
entity have different wavelengths (for example, if the first entity
and the second entity are chemically different, and/or are located
in different environments). The entities may be spatially resolved
even at distances of separation that are less than the light
emitted by the entities or below the diffraction limit of the
emitted light. In certain instances, the light emitted by a first
entity and the light emitted by a second entity have substantially
the same wavelengths, but the two entities may be activated by
light of different wavelengths and the light from each entity may
be determined separately. The entities may be spatially resolved
even at distances of separation that are less than the light
emitted by the entities, or below the diffraction limit of the
emitted light.
[0056] In some embodiments, the first, light-emitting portion and
the second, activation portion as described above may not be
directly covalently bonded or linked via a linker, but are each
immobilized relative to a common entity. In other embodiments, two
or more of the switchable entities (some of which can include, in
certain cases, a first, light-emitting portion and a second,
activation portion linked together directly or through a linker)
may be immobilized relative to a common entity in some aspects of
the invention. The common entity in any of these embodiments may be
any nonbiological entity or biological entity, for example, a cell,
a tissue, a substrate, a surface, a polymer, a biological molecule
such as a nucleic acid (DNA, RNA, PNA, LNA, or the like), a lipid
molecule, a protein or a polypeptide, or the like, a biomolecular
complex, or a biological structure, for example, an organelle, a
microtubule, a clathrin-coated pit, etc. The common entity may
accordingly be determined in some fashion, e.g., imaged. As another
example, two or more entities may be immobilized relative to a DNA
strand or other nucleic acid strand (e.g., using antibodies,
substantially complementary oligonucleotides labeled with one or
more entities, chemical reactions or other techniques known to
those of ordinary skill in the art), and their locations along the
strand detected. In some cases, the number of base pairs (bp)
separating the entities along the nucleic acid strand may be
determined.
[0057] In some cases, the entities may be independently switchable,
i.e., the first entity may be activated to emit light without
activating a second entity. For example, if the entities are
different, the methods of activating each of the first and second
entities may be different (e.g., the entities may each be activated
using incident light of different wavelengths). As another
non-limiting example, incident light having a sufficiently weak
intensity may be applied to the first and second entities such that
only a subset or fraction of the entities within the incident light
are activated, i.e., on a stochastic or random basis. Specific
intensities for activation can be determined by those of ordinary
skill in the art using no more than routine skill. By appropriately
choosing the intensity of the incident light, the first entity may
be activated without activating the second entity.
[0058] The second entity may be activated to emit light, and
optionally, the first entity may be deactivated prior to activating
the second entity. The second entity may be activated by any
suitable technique, as previously described, for instance, by
application of suitable incident light.
[0059] In some cases, incident light having a sufficiently weak
intensity may be applied to a plurality of entities such that only
a subset or fraction of the entities within the incident light are
activated, e.g., on a stochastic or random basis. The amount of
activation may be any suitable fraction, e.g., about 5%, about 10%,
about 15%, about 20%, about 25%, about 30%, about 35%, about 40%,
about 45%, about 50%, about 55%, about 60%, about 65%, about 70%,
about 75%, about 80%, about 85%, about 90%, or about 95% of the
entities may be activated, depending on the application. For
example, by appropriately choosing the intensity of the incident
light, a sparse subset of the entities may be activated such that
at least some of them are optically resolvable from each other and
their positions can be determined. Iterative activation cycles may
allow the positions of all of the entities, or a substantial
fraction of the entities, to be determined. In some cases, an image
with sub-diffraction limit resolution can be constructed using this
information.
[0060] The two or more entities may be resolved, even at distances
of separation that are less than the wavelength of the light
emitted by the entities or below the diffraction limit of the
emitted light, according to another aspect of the invention. The
resolution of the entities may be, for instance, on the order of 1
micrometer (1000 nm) or less, as described herein. For example, if
the emitted light is visible light, the resolution may be less than
about 700 nm. In some cases, two (or more) entities may be resolved
even if separated by a distance of less than about 500 nm, less
than about 300 nm, less than about 200 nm, less than about 100 nm,
less than about 80 nm, less than about 60 nm, less than about 50
nm, or less than about 40 nm. In some cases, two or more entities
separated by a distance of at least about 20 nm or less than 10 nm
can be resolved using embodiments of the present invention.
[0061] Light emitted by each of the switchable entities may be
determined, e.g., as an image or matrix. For example, the first
entity may be activated and the light emitted by the first entity
determined, and the second entity may be activated (with or without
deactivating the first entity) and light emitted by the second
entity may be determined. The light emitted by each of the
plurality of entities may be at the same or different wavelengths.
Any suitable method may be used to determine the emitted light. For
instance, a camera such as a CCD camera, a photodiode, a
photomultiplier, or a spectrometer may be used; those of ordinary
skill in the art will know of other suitable techniques. Additional
non-limiting example of a suitable technique for determining light
produced by the entities is discussed in the Examples, below. In
some cases, multiple images (or other determinations) may be used,
for example, to improve resolution and/or to reduce noise. For
example, at least 2, at least 5, at least 10, at least 20, at least
25, at least 50, at least 75, at least 100, etc. images may be
determined, depending on the application.
[0062] In some aspects, the light may be processed to determine the
spatial positions of the two or more entities. In some cases, the
positions of one or more entities, distributed within an image, may
each be individually determined. In one set of embodiments, the
emitted light may be processed, using Gaussian fitting or other
suitable techniques, to localize the position of each of the
emissive entities. Details of one suitable Gaussian fit technique
are described in the Examples, below; those of ordinary skill in
the art will be able to identify other suitable image-processing
techniques with the benefit of the present disclosure.
[0063] Another example of an image-processing technique follows, in
accordance with another embodiment of the invention. Starting with
a series of images of a sample (e.g., a movie), each light-emission
peak (e.g., through fluorescence, phosphorescence, etc.) is
identified, and the times which the peak is present are determined.
For example, a peak may be present with approximately the same
intensity in a series of images with respect to time. Peaks may be
fit, in some cases, to Gaussian and/or elliptical Gaussian
functions to determine their centroid positions, intensities,
widths, and/or ellipticities. Based on these parameters, peaks
which are too dim, too wide, too skewed, etc. to yield satisfactory
localization accuracy may be rejected in certain cases from further
analysis. Peaks which are sporadic, moving, discontinuously
present, etc. may also be discarded. By determining the center
position of the peak, for example, using least-squares fitting to
to a 2-dimensional Gaussian function of the peak intensities, the
location of the source of the peak (e.g., any entity or entities
able to emit light, as discussed herein) can be determined. This
process may be repeated as necessary for any or all of the peaks
within the sample.
[0064] In another non-limiting example of a suitable imaging
processing technique of the present invention, a series of images
of a sample (e.g. a movie) may include a repetitive sequence of
activation frames (e.g., in which the activation light is on) and
imaging frames (e.g., in which the imaging light is on). For one or
more of the imaging frames, fluorescent peaks can be identified and
fit to Gaussian and/or elliptical Gaussian functions to determine
their centroid positions, intensities, widths, and/or
ellipticities. Based on these parameters, peaks that are too dim,
too wide, too skewed, etc. to yield satisfactory localization
accuracy may be rejected from further analysis. Peaks which are
sporadic, moving, discontinuously present, etc. may also be
discarded in some cases. By determining the center position of the
peak, for example, using least-squares fitting to a 2-dimensional
Gaussian function of the peak intensities, the location of the
source of the peak (e.g., any entity or entities able to emit
light, as discussed herein) can be determined. Peaks appearing in
an imaging frame immediately after an activation frame can be
recognized as a controlled activation event and may be color-coded
according to the activation laser color, in some embodiments. This
process may also be repeated as necessary for any or all of the
peaks within the sample.
[0065] Other image-processing techniques may also be used to
facilitate determination of the entities, for example, drift
correction or noise filters may be used. Generally, in drift
correction, for example, a fixed point is identified (for instance,
as a fiduciary marker, e.g., a fluorescent particle may be
immobilized to a substrate), and movements of the fixed point
(i.e., due to mechanical drift) are used to correct the determined
positions of the switchable entities. In another example method for
drift correction, the correlation function between images acquired
in different imaging frames or activation frames can be calculated
and used for drift correction. In some embodiments, the drift may
be less than about 1000 nm/min, less than about 500 nm/min, less
than about 300 nm/min, less than about 100 nm/min, less than about
50 nm/min, less than about 30 nm/min, less than about 20 nm/min,
less than about 10 nm/min, or less than 5 nm/min. Such drift may be
achieved, for example, in a microscope having a translation stage
to mounted for x-y positioning of the sample slide with respect to
the microscope objective. The slide may be immobilized with respect
to the translation stage using a suitable restraining mechanism,
for example, spring loaded clips. In addition, a buffer layer may
be mounted between the stage and the microscope slide. The buffer
layer may further restrain drift of the slide with respect to the
translation stage, for example, by preventing slippage of the slide
in some fashion. The buffer layer, in one embodiment, is a rubber
or polymeric film, for instance, a silicone rubber film.
Accordingly, one embodiment of the invention is directed to a
device, comprising a translation stage, a restraining mechanism
(e.g., a spring loaded clip) attached to the translation stage able
to immobilize a slide, and optionally, a buffer layer (e.g., a
silicone rubber film) positioned such that a slide restrained by
the restraining mechanism contacts the buffer layer.
[0066] Multiple locations on a sample may each be analyzed to
determine the entities within those locations. For example, a
sample may contain a plurality of various entities, some of which
are at distances of separation that are less than the wavelength of
the light emitted by the entities or below the diffraction limit of
the emitted light. Different locations within the sample may be
determined (e.g., as different pixels within an image), and each of
those locations independently analyzed to determine the entity or
entities present within those locations. In some cases, the
entities within each location may be determined to resolutions that
are less than the wavelength of the light emitted by the entities
or below the diffraction limit of the emitted light, as previously
discussed.
[0067] As noted, in some embodiments, more than one type of
switchable entity may be used in a sample, and the positions of
each of the entities may be independently determined, in some
cases, at sub-diffraction limit resolutions, e.g., to generate a
multi-color image with sub-diffraction limit resolution. For
instance, by repeatedly activating and deactivating particular
switchable entities in a sample, the positions of a plurality of
switchable entities may be determined, in some cases, to
resolutions that are less than the wavelength of the light emitted
by the entities or below the diffraction limit of the emitted
light.
[0068] As a non-limiting example, a sample may contain two types of
light emitting portions (e.g., Cy5 and Cy5.5) and two types of
activation portions (e.g., Cy3 and Cy2), for a total of four
(2.times.2) types of switchable entities: Cy5-Cy3, Cy5-Cy2,
Cy5.5-Cy3, and Cy5.5-Cy2. The Cy3-containing entities can be
activated by applying light at a suitable wavelength without
activating the Cy2-containing entities (since different activation
wavelengths are required) while light emitted by the Cy5-containing
molecules can be distinguished from light emitted by the
Cy5.5-containing molecules (which emits light at a different
wavelength). Thus, only the Cy5-Cy3 entities will be determined,
while the other entities are either not activated, or are activated
but the light emitted by those entities is not used. By using the
above-described techniques, the positions of the Cy5-Cy3 entities
may be determined within a sample, even to resolutions that are
less than the wavelength of the light emitted by Cy5. Additionally,
by repeating this procedure using suitable activation and emission
wavelengths, the positions of the other entities may also be
determined, e.g., to sub-diffraction limit resolutions. In another
example, the sample may include three (or more) types of light
emitting portions (e.g., Cy5, Cy5.5 and Cy7) and/or three (or more)
types of activation portions (e.g., Alexa Fluor 405, Cy2 and Cy3).
Accordingly, multi-color imaging (e.g., 4 colors, 6 colors, 8
colors, 9 colors, 10 colors, 12 colors, etc.) with sub-diffraction
limit resolutions may be realized.
[0069] In some embodiments of the invention, the entities may also
be resolved as a function of time. For example, two or more
entities may be observed at various time points to determine a
time-varying process, for example, a chemical reaction, cell
behavior, binding of a protein or enzyme, etc. Thus, in one
embodiment, the positions of two or more entities may be determined
at a first point of time (e.g., as described herein), and at any
number of subsequent points of time. As a specific example, if two
or more entities are immobilized relative to a common entity, the
common entity may then be determined as a function of time, for
example, time-varying processes such as movement of the common
entity, structural and/or configurational changes of the common
entity, reactions involving the common entity, or the like. The
time-resolved imaging may be facilitated in some cases since a
switchable entity can be switched for multiple cycles, each cycle
give one data point of the position of the entity.
[0070] Another aspect of the invention is directed to a
computer-implemented method. For instance, a computer and/or an
automated system may be provided that is able to automatically
and/or repetitively perform any of the methods described herein. As
used herein, "automated" devices refer to devices that are able to
operate without human direction, i.e., an automated device can
perform a function during a period of time after any human has
finished taking any action to promote the function, e.g. by
entering instructions into a computer. Typically, automated
equipment can perform repetitive functions after this point in
time. The processing steps may also be recorded onto a
machine-readable medium in some cases.
[0071] Still another aspect of the invention is generally directed
to a system able to perform one or more of the embodiments
described herein. For example, the system may include a microscope,
a device for activating and/or switching the entities to produce
light having a desired wavelength (e.g., a laser or other light
source), a device for determining the light emitted by the entities
(e.g., a camera, which may include color-filtering devices, such as
optical filters), and a computer for determining the spatial
positions of the two or more entities. In some cases, mirrors (such
as dichroic mirror or a polychroic mirror), prisms, lens,
diffraction gratings, or the like may be positioned to direct light
from the light source. In some cases, the light sources may be
time-modulated (e.g., by shutters, acoustic optical modulators, or
the like). Thus, the light source may be one that is activatable
and deactivatable in a programmed or a periodic fashion. In one
embodiment, more than one light source may be used, e.g., which may
be used to illuminate a sample with different wavelengths or
colors. For instance, the light sources may emanate light at
different frequencies, and/or color-filtering devices, such as
optical filters or the like may be used to modify light coming from
the light sources such that different wavelengths or colors
illuminate a sample.
[0072] In some embodiments, a microscope may be configured so to
collect light emitted by the switchable entities while minimizing
light from other sources of fluorescence (e.g., "background
noise"). In certain cases, imaging geometry such as, but not
limited to, a total-internal-reflection geometry a spinning-disc
confocal geometry, a scanning confocal geometry, an
epi-fluorescence geometry, etc., may be used for sample excitation.
In some embodiments, a thin layer or plane of the sample is exposed
to excitation light, which may reduce excitation of fluorescence
outside of the sample plane. A high numerical aperture lens may be
used to gather the light emitted by the sample. The light may be
processed, for example, using filters to remove excitation light,
resulting in the collection of emission light from the sample. In
some cases, the magnification factor at which the image is
collected can be optimized, for example, when the edge length of
each pixel of the image corresponds to the length of a standard to
deviation of a diffraction limited spot in the image.
[0073] In some cases, a computer may be used to control excitation
of the switchable entities and the acquisition of images of the
switchable entities. In one set of embodiments, a sample may be
excited using light having various wavelengths and/or intensities,
and the sequence of the wavelengths of light used to excite the
sample may be correlated, using a computer, to the images acquired
of the sample containing the switchable entities. For instance, the
computer may apply light having various wavelengths and/or
intensities to a sample to yield different average numbers of
activated switchable elements in each region of interest (e.g., one
activated entity per location, two activated entities per location,
etc). In some cases, this information may be used to construct an
image of the switchable entities, in some cases at sub-diffraction
limit resolutions, as noted above.
[0074] In other aspects of the invention, the systems and methods
described herein may also be combined with other imaging techniques
known to those of ordinary skill in the art, such as
high-resolution fluorescence in situ hybridization (FISH) or
immunofluorescence imaging, live cell imaging, confocal imaging,
epi-fluorescence imaging, total internal reflection fluorescence
imaging, etc.
[0075] The following are incorporated herein by reference: U.S.
Provisional Patent Application Ser. No. 60/836,167, filed Aug. 7,
2006, entitled "Sub-Diffraction Image Resolution"; and U.S.
Provisional Patent Application Ser. No. 60/836,170, filed Aug. 8,
2006, entitled "Sub-Diffraction Image Resolution."
[0076] The following examples are intended to illustrate certain
embodiments of the present invention, but do not exemplify the full
scope of the invention.
EXAMPLES
[0077] This example shows a high-resolution optical microscopy,
stochastic optical reconstruction microscopy ("STORM"), in which a
fluorescence image is constructed from high-accuracy localization
of individual fluorescent entities ("fluorophores") that are
switched on and off using light of different colors, in accordance
with one embodiment of the invention. The STORM imaging process in
this example includes a series of imaging cycles (FIG. 1A). In each
cycle, a fraction of the fluorophores in the field of view are
switched on or activated, such that each of the active fluorophores
is optically resolvable from the rest, i.e. their images are not
overlapping. This allows the to position of these fluorophores to
be determined with high accuracy. Repeating this process for
multiple cycles, each causing a stochastically different subset of
fluorophores to be turned on or activated, enables the positions of
many fluorophores to be determined and thus an overall image to be
reconstructed. In these examples, an imaging resolution of
approximately 20 nm is demonstrated, an improvement of more than 10
times over the resolution of conventional fluorescence microscopy,
using a simple total-internal-reflection fluorescence microscope,
low-power continuous-wave lasers, and a photoswitchable cyanine
dye.
[0078] FIG. 1A shows that a STORM imaging sequence using a
hypothetical hexameric object labeled with fluorophores can be
switched between a fluorescent and a dark state by a green and a
red laser, respectively. In this non-limiting illustration, all
fluorophores were first switched "off" to the dark state
("deactivated") by a strong red laser pulse. In each imaging cycle,
a green laser pulse was used to switch on ("activate") only a
fraction of the fluorophores to give an optically resolvable set of
active fluorophores. Next, under red illumination ("image"), these
molecules emitted fluorescence until they were switched off,
allowing their positions (indicated by white crosses) to be
determined with relatively high accuracy. The overall image was
then reconstructed from the fluorophore positions obtained from
multiple imaging cycles.
[0079] Cyanine dye, Cy5, can be switched between a fluorescent and
a dark state in a controlled and reversible manner by light of
different wavelengths. Red laser light that produces fluorescent
emission from Cy5 can also switch the dye to a stable dark state.
Exposure to green laser light converts Cy5 back to the fluorescent
state, but the recovery rate may depend in some cases on the close
proximity of a secondary dye, Cy3. The Cy3-Cy5 dye pair can also be
referred to as a switch. Under illumination conditions allowing
single-molecule detection, it was found that such a switch, when
attached or immobilized to nucleic acids or proteins, can be cycled
on and off hundreds of times before permanent photobleaching occurs
(FIG. 1B and FIG. 4). In FIG. 1B, a red laser (633 nm, 30
W/cm.sup.2) was used to excite fluorescence (black line) from Cy5
and to switch Cy5 to the dark state. A green laser (532 nm, 1
W/cm.sup.2) was used to return Cy5 to the fluorescent state. The
alternating red and green line indicates the laser excitation
pattern. In some cases, the recovery rate of Cy5 appeared to depend
on the close proximity of Cy3.
[0080] FIG. 4 shows goat anti-mouse secondary antibody labeled with
the cyanine switch exhibits photoswitching behavior similar to
switch-labeled DNA. The antibody was labeled with Cy3 and Cy5 (see
below) and bound to a quartz slide coated with unlabeled mouse
anti-transferrin primary antibody. The trace shows the Cy5
fluorescence intensity detected from a single labeled antibody as
it switched on and off until permanent photobleaching occurs after
about 230 seconds. The sample was excited with a sequence of
alternating green and red laser pulses (0.5 s green followed by 2 s
red).
[0081] Other photoswitchable dye pairs can be constructed using
similar principles. For instance, in addition to Cy5, Cy5.5, Cy7,
and Alexa Fluor 647 can also be switched between a fluorescent and
a dark state in a controlled and reversible manner by light of
different wavelengths. When paired with Cy3, upon illumination with
a red laser (633 nm or 657 nm), each of these four dyes (Cy5,
Cy5.5, Cy7, and Alexa Fluor 647) was initially fluorescent and then
quickly switched into a non-fluorescent, dark state. A brief
exposure to a green laser pulse (532 nm) led to reactivation of
these dyes back to the fluorescent state (see FIGS. 8A and 9).
Furthermore, different activating portion can be used to activate
the same emitting portion in some cases. For instance, using Cy5 as
an example for the emitting portion, Cy3, Cy2, and Alexa Fluor 405
could be paired with Cy5. Upon illumination with a red laser (633
nm or 657 nm), Cy5 was observed to be quickly switched into the
dark state. In this example, the reactivation of Cy5 required
different colored lasers corresponding to the absorption wavelength
of the activator (FIGS. 8B and 9). The Alexa 405-Cy5 pair was
efficiently activated by a violet laser (405 nm), but appeared to
be less sensitive to blue (457 nm) and green (532 nm) lasers.
Similarly, the Cy2-Cy5 pair was more sensitive to the blue light
than to violet or green light, whereas the Cy3-Cy5 pair was
appeared more sensitive to the green laser.
[0082] Examples of activator portions include, but are not limited
to, Alexa Fluor 405, Alexa Fluor 488, Cy2, Cy3, Cy3.5, or Cy5.
Examples of light-emitting portions include, but are not limited
to, Cy5, Cy5.5, Cy7, Alexa Fluor 647, Alexa Fluor 680, or Alexa
Fluor 700. These may be linked together to form photoswitchable
entities such as, but not limited to, Cy5-Alexa Fluor 405,
Cy5-Alexa Fluor 488, Cy5-Cy2, Cy5-Cy3, Cy5-Cy3.5, Cy5.5-Alexa Fluor
405, Cy5.5-Alexa Fluor 488, Cy5.5-Cy2, Cy5.5-Cy3, Cy5.5-Cy3.5,
Cy7-Alexa Fluor 405, Cy7-Alexa Fluor 488, Cy7-Cy2, Cy7-Cy3,
Cy7-Cy3.5, or to Cy7-Cy5.
[0083] In this example, the concept of STORM is shown using the
Cy5-C53 switch, but any suitably optically switched fluorophores
could also be used. The resolution of STORM may be limited in some
cases by the accuracy with which individual switches can be
localized during a switching cycle. As an example of determining
the localization accuracy, a switch was attached or immobilized to
a short double-stranded DNA, which was surface-immobilized at low
density so that single switches were resolvable. These switches
were periodically cycled on and off using green and red laser
light, and the red laser also served to excite fluorescence from
Cy5. No fluorescence from Cy3 was recorded in this process. The
high localization accuracy of individual switches during each
switching cycle defining the intrinsic resolution of STORM is shown
in FIG. 2. The fluorescence image from a single switch gave a
point-spread function shown in FIG. 2A. The point spread function
(PSF) of the emission from a single switch on DNA during a single
switching cycle. A Gaussian fit to this image (not shown) was used
to localize the centroid position of the PSF, indicating the
position of the switch. The positions determined from multiple
switching cycles showed a substantial spread (FIG. 2B), which was
significantly reduced by correcting for sample drift over the
course of the experiment (FIG. 2C, see below for additional
details). FIGS. 2B and 2C show the centroid positions of an
individual switch determined in 20 successive imaging cycles before
(FIG. 2B) and after (FIG. 2C) correction for sample drift. The
scale bars are 20 nm. The standard deviation of the drift-corrected
positions obtained from 20 imaging cycles was on average 8 nm for
individual switches (FIG. 2D). FIG. 2D shows a histogram of the
standard deviation of centroid positions. The standard deviation is
determined as (.sigma..sub.x+.sigma..sub.y)/2 ((sigma-x+sigma-y)/2)
for each switch using 20 imaging cycles, where .sigma..sub.x
(sigma-x) and .sigma..sub.y (sigma-y) are the standard deviations
of the centroid positions in the x and y dimensions. This histogram
was constructed from 29 switches. This value gave a measure of the
uncertainty in the localization of a single switch per imaging
cycle. Correspondingly, the experimentally measured spread of
switch positions follows a Gaussian distribution with a FWHM of 18
nm, as expected from the 8 nm standard deviation (FIG. 5).
Therefore, under these imaging conditions, two switches separated
by 20 nm could be resolved.
[0084] FIG. 5 shows the superimposed position distributions of 29
individual switches to before (FIG. 5A) and after (FIG. 5B)
correction for stage drift. The scale bar is 20 nm To obtain these
distributions, the image of each switch during a given imaging
cycle was fit to a two-dimensional Gaussian function, yielding the
centroid position of each switch during that cycle. Twenty imaging
cycles were carried out, giving twenty measured positions per
switch. The position data for each switch were realigned so that
the average position of each switch was at the origin, and the
measurements from different switches were then superimposed to give
the overall position distribution shown in the insets. The main
plots are histograms of these centroid positions. The uncorrected
distribution is fairly broad and skewed asymmetrically. After
correction for stage drift using fiducial markers (see below) the
distribution become significantly narrower. The fit of the
drift-corrected histogram to a Gaussian gave a full width at
half-maximum of 18.+-.2 nm.
[0085] To demonstrate the capability of STORM to resolve
fluorescent molecules in close proximity, standard samples of
linear, double-stranded DNA labeled were constructed with multiple
switches separated by a well-defined number of base pairs (see
methods, below, for details). The DNA strands were also labeled
with multiple biotins and attached to a high-density streptavidin
layer (see below), increasing the likelihood of multiple
attachments between the DNA and the surface so that the DNA was
immobilized in the plane. DNA strands were first examined
containing two switches separated by 135 base-pairs (FIG. 3A and
FIG. 6), corresponding to a length of 46 nm along the DNA contour.
A STORM image obtained using the imaging procedure described above
(see below for details) showed two clusters of measured switch
positions, indicating that the two switches were well-resolved
(FIG. 3A). This image shows two separated clusters of measured
switch positions (crosses), each corresponding to a single switch.
The center-of-mass position of each cluster is marked by a dot. The
inter-switch distances are 46 nm, 44 nm and 34 nm for these three
examples. The scale bars are 20 nm. The distance between the
centers of the two clouds had a mean value of 41 nm (FIG. 3B), in
quantitative agreement with the theoretical mean (40 nm) determined
using the known contour and persistence lengths of the DNA sample
(see below). FIG. 3B shows a comparison between the inter-switch
distances measured using STORM (grey column) and the predicted
distance distribution considering the flexibility of DNA (dashed
line). FIG. 3C shows STORM images of four switches attached to a
double-stranded DNA, pair-wise separated by a contour length of 46
nm. The measured switch positions were clustered by an automated
algorithm (see below for details) and different clusters are
indicated by different symbols. The scale bars are 20 nm.
[0086] FIG. 6 is an excerpt of a fluorescence trace (black line)
showing the cycling of Cy3-Cy5 switches separated by a contour
length of 46 nm on dsDNA (see also FIG. 3A). Each switching cycle
lasted for 5 seconds and included a brief green pulse (underlying
tick marks) to activate one or two switches followed by a long red
exposure (underlying bar) to excite Cy5 fluorescence and return the
switches to the off state. In cycles when one switch is activated
(e.g. 225-230 seconds), a single intensity level was apparent
before the return to the dark state. In cycles where two switches
are activated (e.g. 245-250 seconds), a "staircase" with two
decreasing levels corresponding to the sequential switching off of
each dye is observed. Regions where only a single switch is on and
all of the peak quality criteria are satisfied (see below) were
used for single-switch localization.
[0087] Longer DNA samples labeled with four switches evenly
separated by 46 nm along the contour were also imaged. The STORM
images revealed four clusters of switch positions following a bent
contour consistent with the persistence length of DNA and the
engineered separation between the switches. These results indicated
that STORM can image biological samples with sub-diffraction limit
resolution and that features separated by 40 nm are well within the
resolving power.
[0088] One advantage of STORM is its ability to localize a large
number of switches within a diffraction-limited spot by cycling the
switches on and off in a controlled manner, allowing this to be
used as a general biological imaging technique. To demonstrate this
capability, circular DNA plasmids were prepared and coated with
RecA protein and imaged using indirect immunofluorescence with
switch-labeled secondary antibody (FIG. 3D, see methods, below, for
details). Here the photoswitch comprise of Cy3 and Cy5. Cy3 serve
as the activating portion and Cy5 serve as the light-emitting
portion. STORM images of the RecA filaments revealed their circular
structure with greatly increased resolution when compared with
conventional wide-field images. Parts of the filament appear to be
unlabeled or kinked, which may correspond to regions of the plasmid
that remain uncoated by RecA. In FIG. 3D, the top panels show
indirect immunofluorescence images with switch-labeled secondary
antibody taken by a total internal reflection microscope. The
bottom panels are the reconstructed STORM images of the same
filaments. The scale bars are 300 nm.
[0089] The presence of multiple types of photoswitchable pairs of
light emitting portion and activating portion may also allow
multi-color STORM imaging, e.g., as described herein. This example
uses a selective activation scheme as an initial demonstration of
multicolor STORM imaging. Three different DNA constructs labeled
with Alexa 405-Cy5, Cy2-Cy5, or Cy3-Cy5 were mixed and immobilized
on a microscope slide at a high surface density such that
individual DNA molecules could not be resolved in a conventional
fluorescence image. To generate a STORM image, the sample was first
exposed to a red laser (633 nm) to switch off nearly all Cy5 dyes
in the field of view. The sample was then periodically excited with
a sequence of violet (405 nm), blue (457 nm), and green (532 nm)
laser pulses, each of which activated a sparse, optically
resolvable subset of fluorophores. In between activation pulses,
the sample was imaged with the red laser. The image of each
activated fluorescent spot was analyzed to determine its centroid
position (referred to as a localization), and a color was assigned
according to the preceding activation pulse. As the same imaging
laser and detection channel were used for all three dye pairs,
there was no need for correction of chromatic aberration. After
thousands of activation cycles, a STORM image was constructed by
plotting all of the colored localizations (FIGS. 12A-12C). The
STORM image showed separated clusters of localizations. Each
cluster corresponded to an individual DNA molecule and resulted
from the repetitive localization of a single Cy5 molecule over
multiple switching cycles. The majority of the localizations within
each cluster displayed the same color, identifying the type of
activator dye present on the DNA. There were two origins of
crosstalk between colors that were identified: false activation by
laser pulses of incorrect colors and non-specific activation by the
red imaging laser, but both effects were quantitatively small. The
localizations within each cluster approximately follow a Gaussian
distribution with a full-width-half-maximum (FWHM) of 26.+-.1 nm,
25.+-.1 nm, and 24.+-.1 nm for the three color channels (FIGS.
12D-12F), suggesting an imaging resolution of .about.25 nm. This
resolution is lower than the theoretical limit predicted from the
number of photons detected.
[0090] STORM imaging can also be performed on cell samples. To
demonstrate this capability, in this example, single-color
immunofluorescence imaging of microtubules, which are filamentous
cytoskeleton structures important for many cellular functions, were
performed. BS-C-1 cells were fixed and immunostained with primary
antibodies against microtubules and then with a switch-labeled
secondary antibody. Here, the photoswitch used comprised Cy3 and
Alexa Fluor 647, with Cy3 serving as the activating portion and
Alexa Fluor 647 serving as the light-emitting portion. The STORM
image showed a drastic improvement in the resolution of the
microtubule network as compared to the conventional fluorescence
image (FIG. 10). FIGS. 10A, 10C, and 10E are conventional images of
microtubules in regions of the cell, shown at different scales, and
FIGS. 10B, 10D, and 10F are STORM images of the same regions. In
the regions where microtubules were densely packed and undefined in
the conventional image, individual microtubule filaments were
resolved by STORM (FIGS. 10C-F). Whole-cell STORM images, including
.about.10.sup.6 single-molecule localizations, were acquired in 2
to 30 minutes. Super-resolution microtubule structures began to
emerge after only about 10 to 20 seconds of STORM imaging, although
this was not optimized. The effective imaging resolution was
affected both by the intrinsic dye localization accuracy and the
size of the antibody labels. Improvement in the effective
resolution may be achieved by using direct immunofluorescence
staining with dye-labeled primary antibodies or Fab fragments.
[0091] Using different photoswitchable pairs, multi-color STORM
imaging can also be performed on cell samples. To demonstrate this
capability, microtubules and clathrin-coated pits (CCPs), cellular
structures used for receptor-mediated endocytosis, were
simultaneously imaged. The microtubules and clathrin were
immunostained with primary antibodies and then with switch-labeled
secondary antibodies. Here, the photoswitches comprised Cy2 and
Alexa Fluor 647 for microtubule imaging, and Cy3 and Alexa Fluor
647 for clathrin imaging, with Cy2 or Cy3 serving as the activating
portion and Alexa Fluor 647 serving as the light-emitting portion.
The 457 nm and 532 nm lasers were used to selectively activate the
two pairs. Crosstalk between the two color channels due to false
and non-specific activations were subtracted from the image after
statistical analysis. FIG. 11 shows two-color super-resolution
STORM images of microtubules and clathrin-coated pits. FIG. 11
shows the two-color STORM image presented at different scales. The
green channel (457 nm activation) revealed filamentous structures
for microtubules (long thin structures in these images). The red to
channel reveals predominantly spherical structures in these images,
which were the clathrin-coated pits and vesicles.
[0092] In summary, this example demonstrates that STORM is capable
of imaging biological structures with sub-diffraction limit
resolution. The resolution of the technique is not limited to the
wavelength of light. For the Cy3-Cy5 switch, approximately 3,000
photons were detected per switching cycle, independent of red laser
intensity (data not shown), predicting a theoretical localization
accuracy of 4 nm. The difference between this theoretical
prediction and the measured accuracy of 8 nm is relatively small,
and the discrepancy may be due to imperfect correction of stage
drift and aberration due to focus drift in the measurements. This
measured localization accuracy corresponds to an imaging resolution
of approximately 20 nm (full width half maximum, FWHM). Indeed,
fluorescent switches separated by .about.40 nm were readily
resolved (FIG. 3).
[0093] The cyanine switches could be turned on and off reliably for
hundreds of cycles before photobleaching, allowing STORM to be used
for resolving structures with many fluorophores in a potentially
time-resolved manner. The circular structure of RecA filaments
containing 10-20 switches and microtubule and clathrin-coated pits
structures in cells could be resolved in a few minutes or less. The
imaging speed may be improved, for instance, by increasing the
switching rate through stronger excitation or fluorophores with
faster switching kinetics, by using a camera with a faster frame
rate, by using a fast camera reading scheme, such as binning pixels
or only reading out a fraction of the pixels, or by other
technique. STORM is thus a valuable tool for high-resolution
imaging of biological or nonbiological samples. The STORM concept
is also applicable to other photoswitchable fluorophores and
fluorescent proteins, which will potentially allow high-resolution
live-cell imaging with endogenous labels, and any other imaging
applications in which sub-diffraction limit image resolution is
desired.
[0094] For additional details and examples, see W. M. Bates, et
al., "Multicolor Super-resolution Imaging with Photo-switchable
Fluorescent Probes" Science (in press), or M. J. Rust, et al.,
"Sub-diffraction-limit imaging by stochastic reconstruction optical
microscopy (STORM)," Nature Methods 3, 793-795 (2006).
[0095] Following are methods useful in the above description.
[0096] Preparation of Switch-Labeled DNA Constructs. Biotinylated
and/or Amine-modified DNA oligonucleotides ("oligos") were
purchased, PAGE purified, from Operon Amine-modified oligos were
labeled with amine reactive Cyanine dyes (Amersham Bioscience) or
Alexa dyes (Invitrogen) post-synthetically following the protocol
provided by the manufacturer. The dye-labeled oligos were purified
using reverse phase HPLC. Complementary strands of DNA were
annealed to form biotinylated double-stranded DNA (dsDNA) by mixing
equimolar amounts of the two complementary strands in 10 mM Tris-Cl
(pH 7.5), 50 mM NaCl, heating to 90.degree. C. for two minutes, and
then allowing the mixture to cool to room temperature in a heat
block over a period of one hour.
[0097] Biotinylated dsDNA of varying lengths having an intra-switch
distance of 135 bp were constructed by annealing complimentary
oligos as described above to form three different 45 bp dsDNA
segments denoted A, B, and C, followed by a ligation reaction. The
oligos were designed with specific sticky ends which only permit A
to ligate to B, B to ligate to C and C to ligate to A, reading in
the 5' to 3' direction. One oligo (A) contained amine-reactive
sites three base pairs apart on opposite strands which were
specifically labeled with Cy3 and Cy5 prior to annealing to form
the optical switch. Oligos B and C contain two internal biotin
modifications per strand to facilitate multivalent linkage to the
streptavidin surface. After annealing, the oligos were mixed at
equal concentrations and ligated overnight using T4 ligase (New
England Biolabs). The resulting ligation product was purified on a
1.5% agarose gel to select bands containing the desired
concatamerized dsDNA length.
[0098] Preparation of Switch-Labeled Antibodies. Goat Antimouse Igg
Secondary antibodies (Abcam or Invitrogen) and goat anti-rabbit
antibody (Abcam) were labeled non-specifically with amine-reactive
Cy2 or Cy3 (to serve as an activating portion) and Cy5 or Alexa 647
(to serve as a light-emitting portion). The average dye-to-antibody
ratio was .about.2:1 for Cy3 and .about.0.1:1 for Cy5 in the case
of Rec A imaging. In the case of microtubule and clathrin imaging,
dye-to-antibody ratio was .about.2:1 for Cy3 or Cy2 and
.about.0.4:1 for Alexa Fluor 647. Various Cy5-to-antibody or Alexa
647-to-antibody ratios may be used for STORM imaging, for example,
ratios up to or greater than 0.8. Various Cy3-to-antibody ratios
and Cy2-to-antibody ratios can also be used for efficiently STORM
imaging. The imaging quality appears not to be very sensitive the
Cy3 (or Cy2)-to-antibody ratio. These labeling ratios were chosen
to minimize the fraction of to antibodies labeled with more than
one Cy5 molecule, due to the inefficient switching observed for
antibodies labeled with multiple Cy5. The presence of more than one
Cy3 molecule on a single antibody did not interfere with switching.
With this labeling ratio, a significant fraction of the secondary
antibody did not carry Cy5, and were not observed. This lower
density of labels is typically not a problem for indirect
immunofluorescence imaging and, in particular, did not prevent
resolution of the circular structure of the RecA-plasmid filaments
or microtubules.
[0099] Preparation of RecA Filaments. Biotinylated RecA was
Prepared by Reacting purified recombinant RecA (New England
Biolabs) with amine-reactive biotin-XX (Invitrogen) in 0.1M
carbonate buffer at pH 8.3. The resulting biotinylated protein was
purified on a NAP-5 size exclusion column (Amersham). RecA
filaments were formed on .PHI.XRF-II (Phi-XRF) plasmid DNA (New
England Biolabs) by incubating RecA (25% biotinylated:75%
unbiotinylated, concentration of 80 micrograms/mL) with plasmid DNA
(2 micrograms/mL) in 10 mM Tris buffer at pH 7.0, 100 mM NaCl, 7 mM
MgCl.sub.2, and 0.8 mg/mL ATP-.gamma.-S (ATP-gamma-S) for 1 hour at
37.degree. C. The resulting RecA-DNA filaments were stored at
4.degree. C. and used for imaging the same day.
[0100] Microscope slide preparation. Quartz microscope slides (G.
Finkenbeiner) were cleaned using Alconox detergent, followed by
sonication for 15 minutes in acetone, 1 M aqueous KOH, ethanol, 1 M
aqueous KOH, sequentially. Slides being prepared for lipid bilayers
were submerged into a 5% HF solution for 2 hours after the second
KOH step. Finally, the slides were rinsed with deionized water, and
flame dried. The flow channels were prepared using two pieces of
double-sided adhesive tape (3M) and covered with a No. 1.5 glass
coverslip (VWR).
[0101] Oxygen scavenging system. All imaging buffers were
supplemented with the oxygen scavenging system, which included 10%
(w/v) glucose (Sigma), 0.1% (v/v) beta-mercaptoethanol (Sigma), 500
micrograms/mL glucose oxidase (Sigma), and 10 micrograms/mL
catalase (Roche). The oxygen scavenging system was important for
reliable photoswitching of the fluorophores. As low as 0.01% of
beta-mercaptoethanol can be used in some instances for efficiently
photoswitching of the cyanine dyes. The beta-mercaptoethanol can
also be replaced by other reducing reagents such as glutathione and
cysteins, in other embodiments.
[0102] Surface-immobilization of DNA and antibodies. To immobilize
the labeled dsDNA on a surface, quartz microscope slides (G.
Finkenbeiner) were cleaned using Alconox detergent, sonicated in 1
M KOH, ethanol, and 1 M KOH sequentially before being rinsed with
MilliQ water and flame dried. A biotinylated bovine serum albumin
(BSA, Sigma) solution (1.0 mg/mL) was first added to the slides,
followed by 0.25 mg/mL streptavidin (Invitrogen), and finally the
DNA sample at a low concentration (.about.30 pM) in order to obtain
a low surface density of DNA molecules such that individual
molecules were well separated and optically resolvable. The slides
were rinsed prior to the addition of each reagent.
[0103] To immobilize DNA constructs for the 3-color STORM imaging,
three different DNA constructs, each labeled with an Alexa 405-Cy5
pair, a Cy2-Cy5 pair, or a Cy3-Cy5 pair were mixed in solution and
co-immobilized onto a quartz slide as described above. A
concentration of 500 pM of DNA was used to reach a high surface
density of immobilized molecules.
[0104] To immobilize the DNA sample labeled with multiple switches
on a quartz slide, a lipid bilayer was first formed on the slide by
flowing in liposomes of egg PC and 5% biotin-PE (Avanti). The
liposomes were formed according to the manufacturer's instructions,
extruded through a 0.05 micrometer filter membrane at a
concentration of 5 mg lipids/mL in DI water, and then mixed with a
1:1 ratio with a buffer containing 10 mM Tris at pH 8.0, 100 mM
NaCl immediately before use. After a 2 hour incubation the bilayer
was rinsed extensively with 50 mM Tris at pH 8.0, 10 mM NaCl, and
the bilayer was incubated with streptavidin (0.25 mg/mL,
Invitrogen) for 30 minutes. After extensive washing, the
streptavidin surface was crosslinked in 4% v/v formaldehyde in PBS
for 1 hour and rinsed with Tris buffer before allowing the
biotinylated, switch-labeled DNA to bind. DNA was imaged in Tris
buffer (50 mM Tris-Cl at pH 7.5, 10 mM NaCl) with the oxygen
scavenging system described above.
[0105] To immobilized switch-labeled antibodies on a surface, the
quartz slides were cleaned as described and incubated for 5 minutes
with mouse anti-transferrin IgG (Abcam) allowing it to bind
non-specifically. The slide was then incubated with the labeled
secondary antibodies in PBS buffer containing 3% bovine serum
albumin (BSA) for 10 min. The buffer was replaced with 50 mM Tris,
10 mM NaCl, pH 7.5 containing the oxygen scavenging system for
imaging.
[0106] Immunofluorescence imaging of RecA-dsDNA filaments.
Biotinylated RecA-dsDNA filaments were attached via streptavidin
linkages to a quartz slide non-specifically coated with
biotinylated BSA. The surface was then washed with 50 mM Tris at pH
7.0, 100 mM NaCl, 7 mM MgCl.sub.2, 3% BSA w/v (block buffer) and
incubated for 30 minutes to block the surface against non-specific
antibody binding. The slide was then incubated in this block buffer
containing monoclonal mouse antibody against RecA (Stressgen) at 2
microgram/mL for 1 hour. After extensive washing with block buffer,
the slide was incubated in the block buffer containing
switch-labeled secondary antibody at 0.3 microgram/mL for 1 hour.
Finally, the sample was washed and imaged in 50 mM Tris at pH 7.5,
100 mM NaCl, 7 mM MgCl.sub.2, supplemented with the oxygen
scavenging system as described above.
[0107] Immunofluorescence imaging of microtubules and
clathrin-coated pits in cells. Green monkey kidney BS-C-1 cells
were plated in LabTek II 8 well chambered coverglass (Nunc) at a
density of 30k per well. After 16 to 24 hr, they were rinsed with
phosphate buffered saline (PBS) buffer, fixed with 3% formaldehyde,
and 0.1% glutaraldehyde at room temperature in PBS for 10 minutes,
and quenched with 0.1% sodium borohydride in PBS for 7 minutes to
reduce the unreacted aldehyde groups and fluorescent products
formed during fixation. The sodium borohydride solution was
prepared immediately before use to avoid hydrolysis. The fixed
sample was permeabalized in blocking buffer (3% BSA, 0.5% Triton
X-100 in PBS) for 10 min, stained with one or both of the primary
antibodies against tubulin and clathrin (2.5 micrograms/mL mouse
anti-beta (.beta.) tubulin, ATN01 from Cytoskeleton and/or 2
micrograms/mL rabbit anti-clathrin heavy chain, ab21679 from Abcam)
for 30 min in blocking buffer. The sample was then rinsed with
washing buffer (0.2% BSA, 0.1% Triton X-100 in PBS) three times.
Corresponding secondary antibodies labeled with photoswitchable
probes (2.5 micrograms/mL) were added to the sample in blocking
buffer and then thoroughly rinsed after 30 minutes. Cell imaging
was performed in a standard imaging buffer that contained 50 mM
Tris, pH 7.5, 10 mM NaCl, 0.5 mg/mL glucose oxidase (Sigma, G2133),
40 micrograms/mL catalase (Roche Applied Science, 106810), 10%
(w/v) glucose and 1% (v/v) beta-mercaptoethanol.
Beta-mercaptoethanol was found to be important for the observed
photoswitching behavior of Cy5, Cy5.5, and Cy7, but even a low
concentration of beta-mercaptoethanol (as low as 0.02% v/v, or
potentially lower) supported photoswitching. Beta-mercaptoethanol
at low concentrations (0.1%) was compatible with live cell imaging.
Photoswitching was also observed when beta-mercaptoethanol was
replaced with cysteine (100 mM), which was also compatible with
live cell imaging. Glucose oxidase was used as an oxygen scavenger
system to increase the photostablity of the cyanine dyes, and cells
were viable at the reported glucose oxidase concentration for at
least 30 minutes.
[0108] Goat anti-mouse antibody (Invitrogen) and goat anti-rabbit
antibody (Abcam) were each labeled with a mixture of amine-reactive
activators and reporters. Alexa 405, Cy2, and Cy3 were used as the
activating portion of the photoswitch. Alexa 647 (Invitrogen),
which has very similar structural and optical properties as Cy5,
was used as the light-emitting portion of the photoswitch. The
concentrations of the reactive dyes were controlled such that each
antibody had, on average, two activating dyes and 0.3-0.4
light-emitting dyes.
[0109] Imaging procedures. Photoswitch characterization, DNA
concatamer imaging and RecA-dsDNA imaging were performed with an
Olympus IX71 microscope. Single-molecule imaging was conducted in
the prism-type total internal-reflection fluorescence (TIRF)
imaging geometry. The samples were excited with a 657 nm laser, and
activated with a 532 nm, a 457 nm or a 405 nm laser. The
fluorescence emission of the dyes was collected with a N.A. 1.25
60X water immersion objective, and imaged onto an electron
multiplying CCD camera (Andor Ixon DV897) after passing through a
665 nm long pass filter (Chroma). To track motion of the sample
stage, 200 nm red fluorescent polystyrene beads (Invitrogen,
F-8810) were added to the slide in Tris buffer containing 10 mM
MgCl.sub.2 and allowed to bind to the surface. Data was acquired
using custom data acquisition software written in Labview, which
enabled sequences of alternating red and green laser excitation
pulses to be applied to the sample, switching the dyes on and off.
Laser excitation was synchronized with the camera exposure to 1 ms
accuracy.
[0110] Imaging experiments on DNA concatamers typically included of
60 laser pulse cycles to activate and deactivate the switches,
where each cycle lasted for 5 s, so that the final image took 5
minutes to acquire. The multiple cluster configuration of the
centroid position distribution is typically apparent within 2
minutes. The experiments on antibody-labeled RecA-dsDNA filaments
included 70 switch cycles, each lasting 10 s. The ring shape of the
RecA-dsDNA filaments typically became evident within 2 minutes,
although the position of every switch present in the sample had not
yet been identified by this time. As the rate of switching depended
linearly on the excitation intensity, the cycling time, and hence
the overall imaging time, could be shortened without substantially
affecting the localization accuracy by increasing the red laser
intensity. The green laser intensity was chosen so that typically
1-3 switches were switched on during the green pulse and, in most
cases, all activated switched were turned off during the red phase
of the cycle.
[0111] Raw images of RecA-dsDNA for comparison with STORM
reconstructed images were formed by taking the maximum fluorescence
value recorded for each pixel throughout the imaging sequence so
that each switch would be equally represented regardless of the
amount of time it spent in the fluorescent or dark states.
[0112] Three-color STORM imaging of the DNA sample was performed on
an Olympus IX71 inverted microscope in the prism-type TIRF
configuration. A 633 nm HeNe laser was used as the imaging laser
and the violet (405 nm), blue (457 nm), and green (532 nm) lasers
were used as the activation light sources. The sample was first
exposed to the red imaging light to switch off nearly all Cy5 dyes
in the field of view. Then the sample was periodically activated
with a sequence of violet, blue, and green laser pulses each of
which switched on a sparse, optically resolvable subset of
fluorophores which were then imaged with the red laser.
Fluorescence from these probes was detected with a CCD camera after
passing through a long pass emission filter (Chroma, HQ645LP).
During the STORM data acquisition, the camera recorded the
fluorescence signal at a constant frame rate of 19 Hz. In each
switching cycle, one of the activation lasers was turned on for 1
frame, followed by 9 frames of illumination with the red imaging
laser.
[0113] STORM imaging of cells was performed on the Olympus IX71
microscope with an objective-type TIRF imaging configuration. A
custom polychroic beamsplitter (z458/514/647rpc, Chroma) reflected
the excitation laser light onto the sample through an objective
(100.times. oil, NA 1.4, UPlanSApo, Olympus), and fluorescence
emission from the sample was collected by the same objective.
Emitted light was filtered with two stacked dual-band emission
filters (51007 m, Chroma, and 595-700 DBEM, Omega Optical) before
being imaged on the EMCCD camera. The use of a dual-band emitter
enables fluorescence from Cy3 to be collected in addition to the
fluorescence of the reporter dyes. Cy3 fluorescence collected
during frames in which the green activation to laser was on was
used for drift correction purposes and to generate the conventional
fluorescence image. For single-color STORM imaging with Alexa 647
as the light emitting portion of the photoswitch and Cy3 as the
activating portion, the red laser (657 nm) was used for imaging and
green (532 nm) laser pulses were for activation. For two-color
STORM imaging with Cy2 and Cy3 as the activating portion,
alternating blue and green (457 and 532 nm) laser pulses were used
for activation. Images were acquired at a frame rate of 19 Hz. In
each switching cycle, one of the activation lasers was turned on
for 1 frame, followed by 9 frames of illumination with the red
imaging laser. Typical laser powers used for STORM imaging were 40
mW for the red laser and 2 microwatts for each of the activation
lasers.
[0114] Image analysis. In one example of image analysis,
fluorescent structures in an averaged image were first isolated in
13.times.13 pixel square fitting window for data analysis. In a
given window, the total fluorescence intensity was integrated in
each frame to produce a fluorescence time trace (see FIG. 6).
Several criteria were used to ensure high accuracy localization of
single switches:
[0115] (1) In each switching cycle, only regions where a single
switch is on for at least three frames (0.3 seconds) were used for
localization analysis (see FIG. 6).
[0116] (2) The fluorescence images within these regions were fit by
nonlinear least-squares regression to a continuous ellipsoidal
Gaussian:
I(x,y)=A+I.sub.0e.sup.[-(x'/a).sup.2.sup.-(y'/b).sup.2.sup.]/2
where:
x'=(x-x.sub.0)cos .theta.-(y-y.sub.0)sin .theta.
y'=(x-x.sub.0)sin .theta.+(y-y.sub.0)cos .theta.
[0117] Here, A is the background fluorescence level, I.sub.0 is the
amplitude of the peak, a and b reflects the widths of the Gaussian
distribution along the x and y directions, x.sub.0 and y.sub.0
describe the center coordinates of the peak, and .theta. (theta) is
the tilt angle of the ellipse relative to the pixel edges. Based on
this fit, the peak ellipticity defined as |2(a-b)/(a+b)| was
computed. If this ellipticity exceeded 15%, indicating poor image
quality or the possible presence of multiple active switches, the
region was rejected from the analysis.
[0118] (3) The total number of counts collected in the peak was
calculated as 2.pi.ab I.sub.0 (2 pi ab I.sub.0) and then converted
to photoelectrons, and thus the number of photons to detected,
using the camera manufacturer's calibrated curve for the electron
multiplication and ADC gain settings used during imaging. If the
total number of photoelectrons in the peak was less than 2,000, the
region was rejected due to insufficient statistics to achieve high
localization accuracy.
[0119] The regions of the fluorescence traces that passed the above
tests were used for the final localization analysis. The
fluorescence images corresponding to these regions were subjected
to a final fit using a pixelated Gaussian function to determine the
centroid position. Because the CCD chip included square pixels of
finite size, for optimum accuracy, the image was fit to:
I ( x , y ) = A + .intg. x - .delta. x + .delta. X .intg. y -
.delta. y + .delta. Y I 0 [ - ( X - x 0 a ) 2 - ( Y - y 0 b ) 2 ] /
2 ##EQU00001##
which, for ease of evaluation, can be re-expressed in terms of
error functions as:
I ( x , y ) = A + I 0 ab .pi. 4 [ erf ( x + .delta. - x 0 a ) - erf
( x - .delta. - x 0 a ) ] [ erf ( y + .delta. - y 0 b ) - erf ( y -
.delta. - y 0 b ) ] ##EQU00002##
where A, I.sub.o, a, b, x.sub.0 and y.sub.0 are as defined
previously and .delta. (delta) is the fixed half-width of a pixel
in the object plane. The final centroid coordinates (x.sub.0,
y.sub.0) obtained from this fit were used as one data point in the
final STORM image.
[0120] In another example of image analysis, an image movie was
prepared that included a repetitive sequence of activation frames
(in which the activation laser is on) and imaging frames (in which
the imaging laser is on). For each imaging frame, fluorescent spots
were identified and fit to Gaussian and/or elliptical Gaussian
functions to determine their centroid positions, intensities,
widths, and ellipticities. Based on these parameters, peaks too
dim, too wide or too skewed to yield satisfactory localization
accuracy were rejected from further analysis. Peaks appearing in
consecutive imaging frames with a displacement smaller than one
camera pixel were considered to originate from the same fluorescent
molecule and centroid positions of these peaks were connected
across frames and organized into a data structure, which is
referred to as a "string." Each string represents a single
switching cycle for one fluorescent reporter molecule: the starting
point of the string is the frame in which the molecule is switched
on and its endpoint is the frame in which the molecule switches
off. The final localization of the molecule was determined as the
weighted average of the centroid positions across the to entire
string, weighted by the peak intensity of each frame. The total
number of photons detected for each switching cycle was used as an
additional filter to further reject localizations with low
accuracy. Strings starting in an imaging frame immediately after an
activation frame were recognized as a controlled activation event
and color-coded according to the activation laser color. Other
strings were identified as non-specific activations, most likely
induced by the red imaging laser.
[0121] To correct for mechanical drift in the microscope during
imaging, the same fitting algorithm was used to automatically track
the motion of several fluorescent beads in the field of view. The
beads served as fiducial marks and their positions were sampled
during each imaging cycle. The averaged motion of the beads was
subtracted from the coordinates obtained for each single switch
position, yielding a drift-corrected reconstructed image.
[0122] As another drift correction method, the activator
fluorophores was imaged during the activation frame and the
correlation function was calculated between the first activation
frame and all subsequent activation frames. By tracking the
centroid of the correlation function, the drift of the image could
be determined and corrected for in the STORM image. The correlation
functions obtained from the fiducial marker images may also be used
for drift correction. In some cases, it was also found that some
further drift correction was possible by analyzing the correlation
function of the STORM image itself as a function of time.
[0123] For quantitative analysis of images using switches equally
spaced on dsDNA, the coordinates in the reconstructed image were
classified using a k-means clustering algorithm, and inter-switch
distances were calculated as the distance between the cluster
centroids. The number of clusters input to the algorithm was chosen
according to the number of photobleaching steps observed during the
initial exposure to red light.
[0124] Prediction of DNA configuration. Possible configurations of
a 135 bp piece of dsDNA bound to the surface were computed by Monte
Carlo simulation of the DNA as a worm-like chain in the plane.
According to the Watson-Crick structure of dsDNA and accounting the
length of the C6 linkers attaching the Cy5 molecules to the
nucleotides, the expected contour length between neighboring Cy5
molecules of 46.+-.1 nm was calculated. The DNA was treated as a
series of 1 .ANG. (Angstrom) joints lying in the plane, each of
which was deflected by a random angle selected from a Gaussian
distribution chosen to give a persistence length of 50 nm. The
measured inter-switch spacing was compared to the distribution of
end-to-end distances of the polymer in this simulation.
[0125] Single-molecule imaging of photoswitchable
activator-reporter pairs. To characterize the switching kinetics of
the photoswitchable probes from above, the two fluorescent dye
molecules (activator and reporter) were conjugated to the end of a
double stranded DNA (dsDNA) construct, and the construct was
immobilized on a quartz surface for single-molecule imaging. The
DNA constructs were labeled as follows. Briefly, PAGE purified DNA
oligonucleotides with biotin and/or amine modification at the ends
were obtained from Operon. The oligos (30 base pairs (bp) in
length) were labeled with amine reactive dyes (Cy2, Cy3, Cy5,
Cy5.5, and Cy7 were obtained from GE Healthcare, and Alexa Fluor
405 and Alexa Fluor 647 were obtained from Invitrogen)
post-synthetically following the protocol provided by the
manufacturers. The dye-labeled oligos were purified using reverse
phase HPLC. Complementary strands of DNA, each labeled with an
activator or a reporter dye, were annealed to form biotinylated
dsDNA by mixing equimolar amounts of the two complementary strands
in 10 mM Tris-Cl (pH 7.5), 50 mM NaCl. This allowed a pair of
activator and reporter dyes to be brought into close proximity, as
illustrated in FIG. 13H, facilitating the immobilization of dye
pair to a microscope slide via biotin-straptavidin linkage. FIG.
13H is a schematic of double-stranded DNA and antibody molecules
labeled with a Cy3-Cy5 pair.
[0126] To immobilize the labeled dsDNA on a surface, quartz
microscope slides (G. Finkenbeiner) were cleaned using Alconox
detergent, sonicated in 1M KOH, ethanol, and 1M KOH sequentially
before being rinsed with MilliQ water and flame dried. A
biotinylated bovine serum albumin (b-BSA, Sigma) solution (1.0
mg/mL) was first added to the slides, followed by 0.25 mg/mL
streptavidin (Invitrogen), and finally the DNA sample at a low
concentration (.about.30 pM) in order to obtain a low surface
density of DNA molecules such that individual molecules were well
separated and optically resolvable from each other. The slides were
rinsed prior to the addition of each reagent. Single-molecule
imaging was performed in a standard imaging buffer that contains 50
mM Tris, pH 7.5, 10 mM NaCl, 0.5 mg/mL glucose oxidase (Sigma,
G2133), 40 micrograms/mL catalase (Roche Applied Science, 106810),
10% (w/v) glucose and 1% (v/v) beta-mercaptoethanol.
[0127] Single-molecule imaging was performed on an Olympus IX-71
inverted microscope equipped with prism-type total internal
reflection fluorescence (TIRF) configuration. A red 657 nm diode
laser (RCL-200-656, Crystalaser) was used to excite fluorescence
from the reporter fluorophore and to switch them off to the dark
state. A 532 nm diode-pumped solid state laser (GCL-200-L,
Crystalaser), the 457 nm line of an Ar ion laser (35-LAL-030-208,
Melles Griot), and a 405 nm diode laser (CUBE 405, Coherent) were
used to reactivate the reporters by exciting the different
activators. The fluorescence signal from the reporter dyes was
collected by a 60.times., NA 1.2 water immersion objective
(Olympus) and then imaged on to an EMCCD camera (Andor Ixon
DV897DCS-BV) after passing through a band pass fluorescence
emission filter (Chroma, HQ710/80m for Cy5 and Cy5.5 and HQ740LP
for Cy7). A 1.6.times. tube lens was used to set the final imaging
magnification to .about.100.times..
[0128] Switching kinetics analysis of the photoswitchable dyes. To
measure switching kinetics, the DNA samples were first illuminated
with the red imaging laser (657 nm) to switch the reporter
molecules into the dark state, and the rate at which they switched
off (k.sub.off) was measured by recording the number of fluorescent
molecules as a function of time and fitting it to a single
exponential function. For measurements of k.sub.on, after switching
the reporter fluorophores off and while the red imaging laser
remained on, the sample was exposed to the activation laser (405
nm, 457 nm, or 532 nm), which caused the fluorophores to switch
back on, reaching equilibrium between activation and deactivation.
The number of fluorophores in the fluorescent state at equilibrium
was measured, and the activation rate constant (k.sub.on) was then
calculated from the independently determined value of k.sub.off and
the fraction of molecules (F) in the fluorescent state at
equilibrium, according to the relation
F=k.sub.on/(k.sub.on+k.sub.off).
[0129] Photon number analysis of the photoswitchable dyes. The
number of photons detected per switching cycle for Cy5, Cy5.5, and
Cy7 were measured when they were paired with Cy3 as the activator
on DNA and antibody molecules. The average number of photons
detected per switching cycle was a constant independent of the
excitation laser intensity. The photon number, however, depended on
the emission filters and imaging geometry used. Using the
prism-type TIRF imaging geometry, 657 nm imaging laser, and two
stacked HQ665LP emission filters for Cy5 and Cy5.5 and a HQ740LP
emission filter for Cy7, the photon numbers detected were
.about.3000 for Cy5 and Cy5.5 and .about.500 for Cy7. These numbers
correspond to a theoretical limit of localization accuracy (in
terms of standard deviation or s.d.) of 3 nm for Cy5 and Cy5.5 and
9 nm for Cy7, calculated using the formula s.d.= {square root over
((S.sup.2+a.sup.2/12)/N+4 {square root over (.pi.)}{square root
over ((S.sup.2+a.sup.2/12)/N+4 {square root over
(.pi.)}S.sup.3b.sup.2/aN.sup.2)}. In the formula, S is the standard
deviation of the point spread function of the imaging setup, a is
the edge size of the area imaged on each CCD pixel, b is the
background noise level, and N is the number of photons detected
(S=173 nm for Cy5/Cy5.5 and =200 nm for Cy7, a=165 nm, b=6 for
Cy5/Cy5.5 and =1 for Cy7).
[0130] In this work, full-width-half-maximum (FWHM) was typically
used to describe imaging resolution. The FWHM values corresponding
to the localization accuracies quoted above are 8 nm for Cy5 and
Cy5.5 and 22 nm for Cy7. Using the objective-type TIRF imaging
geometry, 657 nm imaging laser, and stacked HQ665LP and HQ710/70BP
emission filters for Cy5 and Cy5.5 and stacked HQ740LP and 800WB80
emission filters for Cy7, the photon numbers detected were
.about.6000 for Cy5 and Cy5.5 and .about.1000 for Cy7, which were
approximately twice as high as the numbers obtained in the
prism-type TIRF geometry. The number of photons detected for Alexa
647, a cyanine dye with a similar structure to that of Cy5 (see
FIG. 13A), was within 10% of the number detected from Cy5. FIGS.
13A-13D illustrate structures of the photoswitchable reporters Cy5,
Alexa 647, Cy5.5, and Cy7. "R" stands for the place where DNA or
antibody was attached. The photon numbers detected from the
activator-reporter-labeled DNA samples were slightly smaller than
the numbers detected from the corresponding antibody samples. The
HQ665LP, HQ740LP, and HQ710/70BP filters were obtained from Chroma
and the 800WB80 filter was from Omega.
[0131] FIGS. 13E-13F illustrate normalized absorption and emission
spectra of Cy5, Alexa 647, Cy5.5, and Cy7 in aqueous solution. The
absorption spectra were normalized by the maximum absorption value,
and the emission spectra were normalized by the integrated peak
area. FIG. 13G illustrates normalized absorption spectra of
activator dyes, Alexa 405, Cy2, and Cy3 in aqueous solution. FIG.
14 illustrates photoswitching behavior of the Alexa 405-Cy7 pair.
The lower panel shows a fluorescence time trace of Cy7. The upper
panel shows the 405 nm laser pulses used to activate the dye pair.
A red laser (657 nm) was continuously on, serving to excite
fluorescence from the Cy7 and to switch it off to the dark
state.
[0132] FIG. 15 shows a conventional fluorescence image of a mixture
of three different DNA constructs, each labeled with Cy3-Cy5,
Cy2-Cy5, or Alexa 405-Cy5 and mixed at a high surface density on a
microscope slide. The fluorescence image was taken from all of the
Cy5 molecules in this region before the sample was subjected to any
photoswitching. A thermal color scheme is used here to illustrate
the intensity, with black indicating low intensity, red higher, and
yellow highest. A three-color image of the same region is shown in
FIG. 12A. The overall intensity profile may appear to be slightly
different for the two images due to the different numbers of
switching cycles exhibited by individual molecules.
[0133] Three-color imaging of a model DNA sample. Three different
DNA constructs, each labeled with an Alexa 405-Cy5 pair, a Cy2-Cy5
pair, or a Cy3-Cy5 pair were mixed in solution and co-immobilized
onto a quartz slide as described above. A concentration of 500 pM
of each DNA was used to reach a high surface density of immobilized
molecules. Due to a moderate Cy5 quenching effect that occurred
when a Cy3 molecule was positioned in very close proximity, in this
experiment these two dyes were separated by 9 base pairs on a 43 bp
dsDNA, instead of being attached to the end of a dsDNA. This Cy5
quenching effect was less significant when Alexa 405 or Cy2 was
positioned in very close proximity. Fluorescent beads (Molecular
probes, F8801) were added to the sample slide as fiducial markers
for the purpose of drift correction.
[0134] Imaging was performed on an Olympus IX71 inverted microscope
in the prism-type TIRF configuration. A 633 nm HeNe laser
(25-LHP-928-249, Melles Griot) was used as the imaging laser and
the violet (405 nm), blue (457 nm), and green (532 nm) lasers
mentioned above were used as the activation light sources. The
sample was first exposed to the red imaging light to switch off
nearly all Cy5 dyes in the field of view. Then the sample was
periodically activated with a sequence of violet, blue, and green
laser pulses each of which switched on a sparse, optically
resolvable subset of fluorophores which were then imaged with the
red laser. Fluorescence from these probes was detected with the CCD
camera after passing through a long pass emission filter (Chroma,
HQ645LP). During data acquisition, the camera recorded the
fluorescence signal at a constant frame rate of 19 Hz. In each
switching cycle, one of the activation lasers was turned on for 1
frame, followed by 9 frames of illumination with the red imaging
laser. Under typical imaging conditions, an average fluorophore
remains in the to fluorescent state for three frames after
activation, and .about.3000 photons per molecule were detected
during each switching cycle.
[0135] Imaging of microtubules and clathrin-coated pits in cells.
Green monkey kidney BS-C-1 cells were plated in LabTek II 8 well
chambered coverglass (Nunc) at a density of 30K per well. After 16
to 24 hr, cells were rinsed with phosphate buffered saline (PBS)
buffer, fixed with 3% formaldehyde, and 0.1% glutaraldehyde at room
temperature in PBS for 10 min, and quenched with 0.1% sodium
borohydride in PBS for 7 min to reduce the unreacted aldehyde
groups and fluorescent products formed during fixation. The sodium
borohydride solution was prepared immediately before use to avoid
hydrolysis. The fixed sample was permeabalized in blocking buffer
(3% BSA, 0.5% Triton X-100 in PBS) for 10 min, stained with one or
both of the primary antibodies against tubulin and clathrin (2.5
micrograms/mL mouse anti-beta tubulin, ATN01 from Cytoskeleton and
2 micrograms/mL rabbit anti-clathrin heavy chain, ab21679 from
Abcam) for 30 min in blocking buffer. The sample was then rinsed
with washing buffer (0.2% BSA, 0.1% Triton X-100 in PBS) three
times. Corresponding secondary antibodies labeled with
photoswitchable probes (2.5 micrograms/mL) were added to the sample
in blocking buffer and then thoroughly rinsed after 30 min. Cell
imaging was performed in a standard imaging buffer that contains 50
mM Tris, pH 7.5, mM NaCl, 0.5 mg/mL glucose oxidase, 40
micrograms/mL catalase, 10% (w/v) glucose and 1% (v/v)
beta-mercaptoethanol. It was found that beta-mercaptoethanol was
important for the observed photoswitching behavior of Cy5, Cy5.5,
and Cy7, but even low concentrations of beta-mercaptoethanol (as
low as 0.02% v/v) supported photoswitching. Beta-mercaptoethanol at
low concentrations (0.1% and 0.02%) was compatible with live cell
imaging. Photoswitching was also observed when beta-mercaptoethanol
was replaced with cysteine (100 mM), which was also compatible with
live cell imaging. Glucose oxidase was used as an oxygen scavenger
system to increase the photostablity of the cyanine dyes, and cell
morphology was normal at the reported glucose oxidase concentration
for at least 30 min. In this work, all imaging experiments were
performed on fixed cells.
[0136] Goat anti-mouse antibody (Invitrogen) and goat anti-rabbit
antibody (Abcam) were each labeled with a mixture of amine-reactive
activators and reporters. Cy2 and Cy3 were used as the activators.
Alexa 647 (Invitrogen), which has similar structural to and optical
properties to Cy5 (FIG. 13), was used as the reporter. The
concentrations of the reactive dyes were controlled such that each
antibody had, on average, two activator molecules and 0.3 to 0.4
reporter molecules. The photoswitching behavior was relatively
insensitive to the number of activators per antibody. The labeling
ratio of two activators per antibody was chosen to ensure that the
majority of antibodies had activators and thus to optimize the
staining efficiency. However, when more than one reporter molecule
was attached to the same antibody, it was found that the close
proximity of the reporter molecules lowered the off rate. To assess
this effect more quantitatively, dsDNA molecules labeled with two
Cy5 dyes of known separations were prepared. The off rate of the
construct having two Cy5 dyes separated by 2 nm was .about.5 times
slower than that for a construct with a single Cy5. For constructs
where the two Cy5 dyes were separated by 7 nm or 14 nm, the off
rates were roughly comparable to that of the single-Cy5 construct.
This self-interaction effect was slightly less pronounced for Alexa
647 as compared with Cy5. Practically, when labeling antibody, a
relatively low dye/protein ratio was chosen here for the reporter
(0.3 to 0.4) such that the majority of reporter-labeled antibody
molecules have only one reporter.
[0137] Imaging was performed on the Olympus IX71 microscope with an
objective-type TIRF configuration. A custom polychroic beamsplitter
(z458/514/647rpc, Chroma) reflected the excitation laser light onto
the sample through an objective (100.times. oil, NA 1.4, UPlanSApo,
Olympus), and fluorescence emission from the sample was collected
by the same objective. Emitted light was filtered with two stacked
dual-band emission filters (51007m, Chroma, and 595-700 DBEM, Omega
optical) before being imaged on the EMCCD camera. The use of a
dual-band emitter enables fluorescence from Cy3 to be collected in
addition to the fluorescence of the reporter dyes. Cy3 fluorescence
collected during frames in which the green activation laser was on
was used for drift correction purposes and to generate the
conventional fluorescence image. For single-color imaging with
Alexa 647 as the reporter and Cy3 as the activator, the red laser
(657 nm) was used for imaging and green (532 nm) laser pulses were
for activation. For two-color imaging with Cy2 and Cy3 as the
activators, alternating blue and green (457 and 532 nm) laser
pulses were used for activation. Images were acquired at a frame
rate of 19 Hz. In each switching cycle, one of the activation
lasers was turned on for 1 frame, followed by 9 frames of
illumination with the red imaging laser. Because the two stacked
dual-band emission filters (51007m and 595-700 DBEM) significantly
cut fluorescence signal from Alexa 647, only .about.3000 photons,
instead of .about.6000, were detected on average from one antibody
during each switching cycle. Typical laser powers used for imaging
were 40 mW for the red laser and 2 microwatts for each of the
activation lasers.
[0138] Image analysis. A typical image was generated from a
sequence of 2000 to 100000 image frames recorded at 19 Hz. The
movie included a repetitive sequence of activation frames (in which
the activation laser is on) and imaging frames (in which the
imaging laser is on). For each imaging frame, fluorescent spots
were identified and fit to a Gaussian or elliptical Gaussian
function to determine their centroid positions, intensities, widths
and ellipticities. Based on these parameters, peaks too dim, too
wide or too elliptical to yield satisfactory localization accuracy
were rejected from further analysis. Peaks appearing in consecutive
imaging frames with a displacement smaller than one camera pixel
were considered to originate from the same fluorescent molecule,
and centroid positions of these peaks were connected across frames
and organized into a data structure which is referred to here as a
"string." Each string represents a single switching cycle for one
fluorescent reporter molecule: the starting point of the string is
the frame in which the molecule is switched on and its endpoint is
the frame in which the molecule switches off. The final
localization of the molecule was determined as the weighted average
of the centroid positions across the entire string, weighted by the
number of photons detected in each frame. The total number of
photons detected for each switching cycle was used as an additional
filter to further reject localizations with low accuracy. Strings
starting in an imaging frame immediately after an activation frame
were recognized as a controlled activation event and color-coded
according to the activation laser color. Other strings were
identified as non-specific activations, most likely induced by the
red imaging laser as the amount of non-specific activation was
observed to increase with the red laser intensity (data not shown).
Nonspecific activation by the red imaging laser would also occur in
the first imaging frame and be counted as a controlled activation
event, giving one source of error for color crosstalk.
[0139] Besides the number of photons detected in one imaging cycle,
another factor that limits the localization accuracy was sample
drift during the course of the experiment. The drift was corrected
by two methods. The first method involved adding fiducial markers
(fluorescent beads) to track the drift of the sample and
subtracting the movement of the markers during image analysis. In
the second method, the activator fluorophores were imaged during
the activation frame and calculated the correlation function
between the first activation frame and all subsequent activation
frames. By tracking the centroid position of the correlation
function, the drift of the image can be determined and corrected
for in the image. The correlation functions obtained from the
fiducial marker images may also be used for drift correction. In
some cases, it was found that further drift correction was possible
by analyzing the correlation function of the image itself as a
function of time.
[0140] For image presentation, each localization was assigned as
one point in the image. These points were either represented by a
small marker (e.g. a cross) or rendered as a normalized 2D Gaussian
peak, the width of which was determined by its theoretical
localization accuracy calculated from the number of photons
detected for that localization event. For multicolor images, each
localization was also false-colored according to the color of the
activation laser pulse. The following color coding scheme was
typically used (although other coding schemes are possible):
activations by the violet (405 nm) laser were shown in blue, those
by the blue (457 nm) laser were shown in green, and those by the
green (532 nm) laser were shown in red.
[0141] Crosstalk between different color channels resulted mainly
from two effects: nonspecific activation and false activation. As
described earlier, nonspecific activations, mostly likely induced
by the red imaging laser, can be most easily identified if the
string did not start immediately after an activation frame.
However, such a nonspecific activation may also occur during the
frame immediately after an activation laser pulse and thus be
incorrectly assigned a color, although this mis-assignment will
occur with a relatively low probability. Three methods can be used
to reduce nonspecific activation-induced crosstalk: (1) increasing
the activation laser intensity, providing that the density of
activated probes remains low enough for single-molecule
localization; (2) using a faster frame rate which effectively
improves identification of those molecules activated by the
activation laser pulse; and (3) decreasing the imaging laser
intensity to reduce the non-specific activation rate, but at the
cost of reducing imaging speed and/or accuracy. The second source
of color crosstalk, false activations, stems from probes which were
switched on by the wrong activation laser. Combining these two
sources, the overall crosstalk ratios under the typical cell
imaging conditions used here were measured to be 15% to 25% for the
leakage of Cy2 signal into the Cy3 channel and 25 to 35% for the
leakage of Cy3 signal into the Cy2 channel. For the three-color
imaging of the DNA sample, crosstalk effects were observed to be
somewhat smaller because nonspecific activation was observed to be
less pronounced (FIG. 16), in part due to stronger activation laser
powers used.
[0142] FIG. 16 shows crosstalk analysis for the three-color image
of the DNA sample. The image shows separated clusters of
localizations, each cluster corresponding to an individual DNA
molecule (FIGS. 12A-12C). Each of the localizations was colored
according the activation laser used: localizations activated by the
405 nm laser were assigned the blue color, those activated by the
457 nm laser were assigned the green color and those activated by
the 532 nm laser were assigned the red color. The majority of the
localizations within each cluster displayed the same color,
identifying the type of activator dye (Alexa 405, Cy2, or Cy3)
present on the DNA molecule. The numbers of localizations of each
color were counted for individual clusters and the fractions of
localizations assigned to each color channel are plotted here for
the Alexa 405, Cy2; and Cy3 clusters. The crosstalk ratios can be
calculated from the ratios of incorrectly to correctly colored
localizations.
[0143] The crosstalk ratios between different color channels under
each imaging condition can be quantitatively determined using
samples singly labeled with only one of the photoswitchable probes.
Due to the clear separation between clathrin-coated pits and
microtubules, the two color cell image itself can also be used to
estimate crosstalk quantitatively. Using the crosstalk ratios,
crosstalk can be effectively subtracted from a multicolor image.
For instance, in the case of a two-color image, at any given
location:
{ D 1 = d 1 + C 2 -> 1 d 2 D 2 = C 1 -> 2 d 1 + d 2
##EQU00003##
where D.sub.1 and D.sub.2 are the observed local densities of spots
in color channels 1 and 2, respectively, and d.sub.1 and d.sub.2
are the corresponding true local densities. C.sub.1.fwdarw.2 and
C.sub.2.fwdarw.1 are the crosstalk ratios between the two channels.
The values of d.sub.1 and d.sub.2 can be solved from observed local
densities D.sub.1 and D.sub.2 and crosstalk ratios C.sub.1.fwdarw.2
and C.sub.2.fwdarw.1. Thus the probability of a localization at a
given position in channel 1 being assigned the wrong color is
simply P.sub.1=1-d.sub.1/D.sub.1. This point can thus be removed
according to this to probability. Similar treatment can be applied
to every points in channels 1 and 2. To correct color crosstalk in
the two-color images, a radius of 35 nm was chosen to calculate the
local densities. Due to the finite area required to reliably
calculate local densities, a slight erosion effect will arise from
the crosstalk subtraction where two different colored structures
overlap in space. According to simulations, for this example, if
the imaging resolution is 20 to 30 nm, such an operation will
reduce the spatial resolution by .about.20% when the crosstalk
ratio is 20% for both channels. A similar statistical approach can
also be used to assign colors to nonspecific activations (e.g. the
probability of a non-specific activation belong to color channel 1
is d.sub.1/(d.sub.1+d.sub.2), where d.sub.1 and d.sub.2 were
obtained from controlled activations as described above),
effectively increasing the overall localization point densities in
the images, which may help improve resolution in cases where the
resolution is point-density limited. Crosstalk subtraction and
nonspecific activation color assignment were applied in FIG.
11.
[0144] FIG. 17 shows localization accuracy for a single-color image
of the cell. The localization accuracy was determined from
point-like objects in the cell, appeared as small clusters of
localizations away from any discernable microtubule filaments.
Shown here is the spatial distribution of localizations within
these point-like clusters. The 2D histogram of localizations was
generated by aligning 170 clusters by their center of mass, each
cluster containing more than 8 localizations. Fitting the 2D
histogram with a Gaussian function gives a FWHM of 24 nm.
[0145] FIG. 18 shows localization accuracy for a two-color image of
the cell. The localization accuracy was also determined from
point-like objects in the cell, appeared as small clusters of
localizations away from any discernable microtubule or CCP
structures. Shown here is the spatial distribution of localizations
within these point-like clusters. The 2D histograms of
localizations were generated by aligning 187 clusters by their
center of mass, each cluster containing more than 8 localizations.
Fitting the 2D histogram with a Gaussian function gives a FWHM of
30 nm.
[0146] FIG. 19 shows images of clathrin-coated pits (CCPs). FIG.
19A shows a comparison of conventional fluorescence images (upper
panels) and the images generated here (lower panels). Nearly all
CCPs appear to adopt a spherical structure. The rightmost panel
shows two close-by CCPs that were resolved here, but appeared as a
single nearly diffraction-limited spot in the conventional
fluorescence image. FIG. 19B shows the size distribution of 300
CCPs determined from the images as shown in FIG. 19A.
[0147] While several embodiments of the present invention have been
described and illustrated herein, those of ordinary skill in the
art will readily envision a variety of other means and/or
structures for performing the functions and/or obtaining the
results and/or one or more of the advantages described herein, and
each of such variations and/or modifications is deemed to be within
the scope of the present invention. More generally, those skilled
in the art will readily appreciate that all parameters, dimensions,
materials, and configurations described herein are meant to be
exemplary and that the actual parameters, dimensions, materials,
and/or configurations will depend upon the specific application or
applications for which the teachings of the present invention
is/are used. Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. It is, therefore, to be understood that the foregoing
embodiments are presented by way of example only and that, within
the scope of the appended claims and equivalents thereto, the
invention may be practiced otherwise than as specifically described
and claimed. The present invention is directed to each individual
feature, system, article, material, kit, and/or method described
herein. In addition, any combination of two or more such features,
systems, articles, materials, kits, and/or methods, if such
features, systems, articles, materials, kits, and/or methods are
not mutually inconsistent, is included within the scope of the
present invention.
[0148] All definitions, as defined and used herein, should be
understood to control over dictionary definitions, definitions in
documents incorporated by reference, and/or ordinary meanings of
the defined terms.
[0149] The indefinite articles "a" and "an," as used herein in the
specification and in the claims, unless clearly indicated to the
contrary, should be understood to mean "at least one."
[0150] The phrase "and/or," as used herein in the specification and
in the claims, should be understood to mean "either or both" of the
elements so conjoined, i.e., elements that are conjunctively
present in some cases and disjunctively present in other cases.
Multiple elements listed with "and/or" should be construed in the
same fashion, i.e., "one or more" of the elements so conjoined.
Other elements may optionally be present other than the elements
specifically identified by the "and/or" clause, whether related or
unrelated to those elements specifically identified. Thus, as a
non-limiting example, a reference to "A and/or B", when used in
conjunction with open-ended language such as "comprising" can
refer, in one embodiment, to A only (optionally including elements
other than B); in another embodiment, to B only (optionally
including elements other than A); in yet another embodiment, to
both A and B (optionally including other elements); etc.
[0151] As used herein in the specification and in the claims, "or"
should be understood to have the same meaning as "and/or" as
defined above. For example, when separating items in a list, "or"
or "and/or" shall be interpreted as being inclusive, i.e., the
inclusion of at least one, but also including more than one, of a
number or list of elements, and, optionally, additional unlisted
items. Only terms clearly indicated to the contrary, such as "only
one of" or "exactly one of," or, when used in the claims,
"consisting of," will refer to the inclusion of exactly one element
of a number or list of elements. In general, the term "or" as used
herein shall only be interpreted as indicating exclusive
alternatives (i.e. "one or the other but not both") when preceded
by terms of exclusivity, such as "either," "one of," "only one of,"
or "exactly one of." "Consisting essentially of," when used in the
claims, shall have its ordinary meaning as used in the field of
patent law.
[0152] As used herein in the specification and in the claims, the
phrase "at least one," in reference to a list of one or more
elements, should be understood to mean at least one element
selected from any one or more of the elements in the list of
elements, but not necessarily including at least one of each and
every element specifically listed within the list of elements and
not excluding any combinations of elements in the list of elements.
This definition also allows that elements may optionally be present
other than the elements specifically identified within the list of
elements to which the phrase "at least one" refers, whether related
or unrelated to those elements specifically identified. Thus, as a
non-limiting example, "at least one of A and B" (or, equivalently,
"at least one of A or B," or, equivalently "at least one of A
and/or B") can refer, in one embodiment, to at least one,
optionally including more than one, A, with no B present (and
optionally including elements other than B); in another embodiment,
to at least one, optionally including more than one, B, with no A
present (and optionally including elements other than A); in yet
another embodiment, to at least one, optionally including more than
one, A, and at least one, optionally including more than one, B
(and optionally including other elements); etc.
[0153] It should also be understood that, unless clearly indicated
to the contrary, in any methods claimed herein that include more
than one step or act, the order of the steps or acts of the method
is not necessarily limited to the order in which the steps or acts
of the method are recited.
[0154] In the claims, as well as in the specification above, all
transitional phrases such as "comprising," "including," "carrying,"
"having," "containing," "involving," "holding," "composed of," and
the like are to be understood to be open-ended, i.e., to mean
including but not limited to. Only the transitional phrases
"consisting of" and "consisting essentially of" shall be closed or
semi-closed transitional phrases, respectively, as set forth in the
United States Patent Office Manual of Patent Examining Procedures,
Section 2111.03.
* * * * *