U.S. patent application number 14/595579 was filed with the patent office on 2015-07-16 for light sheet generator.
This patent application is currently assigned to APPLIED SCIENTIFIC INSTRUMENTATION, INC.. The applicant listed for this patent is Applied Scientific Instrumentation, Inc.. Invention is credited to Gary D. Rondeau.
Application Number | 20150198794 14/595579 |
Document ID | / |
Family ID | 53521237 |
Filed Date | 2015-07-16 |
United States Patent
Application |
20150198794 |
Kind Code |
A1 |
Rondeau; Gary D. |
July 16, 2015 |
LIGHT SHEET GENERATOR
Abstract
Systems and methods for generating light sheets suitable for use
in single plane illumination microscopy may include a series of
chambers in sequential communication with each other, the series of
chambers including an optics system configured to convert a beam of
light, such as a laser beam, into a planar light sheet. The series
of chambers may include chambers having respective long axes
oriented at an acute angle to each other to form a compact zig-zag
pattern.
Inventors: |
Rondeau; Gary D.; (Eugene,
OR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Applied Scientific Instrumentation, Inc. |
Eugene |
OR |
US |
|
|
Assignee: |
APPLIED SCIENTIFIC INSTRUMENTATION,
INC.
Eugene
OR
|
Family ID: |
53521237 |
Appl. No.: |
14/595579 |
Filed: |
January 13, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61927179 |
Jan 14, 2014 |
|
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Current U.S.
Class: |
359/390 ;
359/385 |
Current CPC
Class: |
G02B 21/10 20130101;
G02B 21/0076 20130101; G02B 21/06 20130101; G02B 26/0833 20130101;
G02B 27/30 20130101; G02B 21/16 20130101; G02B 21/0032 20130101;
G02B 21/367 20130101; G02B 21/0048 20130101 |
International
Class: |
G02B 21/00 20060101
G02B021/00; G02B 26/08 20060101 G02B026/08; G02B 21/26 20060101
G02B021/26 |
Claims
1. A system for generating a light sheet for use in a microscope,
the system comprising: a housing including a plurality of elongate
internal chambers in sequential communication with each other and
containing an optics system, such that a first chamber contains a
first lens, a second chamber extends from the first chamber and
contains a second lens, a third chamber extends from the second
chamber, and a fourth chamber extends from the third chamber and
contains a third lens, each chamber having a respective long axis;
and a light source mount at a first end of the housing, the mount
configured to receive a light source and direct a light beam down
the long axis of the first chamber; wherein the first lens is
configured to focus the laser beam onto a first mirror, the second
lens is configured to collimate the laser beam after reflecting
from the first mirror, the second mirror is configured to steer the
collimated laser beam into a fan shape, and the third lens
configured to collimate the fan shaped laser into a light sheet;
and wherein the long axes of each pair of consecutive cavities form
an acute angle.
2. The system of paragraph 1, wherein the first mirror is an
anti-striping mirror.
3. The system of paragraph 1, wherein the second mirror is a
scanning mirror.
4. The system of paragraph 1, further including a third mirror
between the second mirror and the third lens.
5. The system of paragraph 1, further including a collimation
device disposed between the laser source and the first lens.
6. The system of paragraph 1, wherein the first lens and the second
lens form a 4f lens pair.
7. The system of paragraph 1, wherein the first lens has a focal
length, and the first lens is spaced from the first mirror by a
distance equivalent to the focal length.
8. A system for conducting light sheet microscopy comprising: a
microscope having an objective including an objective axis; a
sample apparatus configured to hold a sample spaced from the
objective along the objective axis; a light sheet generating
assembly configured to generate a planar sheet of collimated light
intersecting the sample at an angle transverse to the objective
axis; wherein the light sheet generating assembly includes a laser
source operatively connected to a first elongate chamber containing
a first lens, a first reflector disposed at a terminal end of the
first chamber, a second elongate chamber containing a second lens
and arranged at an acute angle with respect to the first chamber, a
second reflector disposed at a terminal end of the second chamber,
a third elongate chamber arranged at an acute angle with respect to
the second chamber, a third reflector disposed at a terminal end of
the third chamber, and a fourth elongate chamber containing a third
lens and arranged at an acute angle with respect to the third
chamber; wherein the second reflector is a scanning mirror.
9. The system of paragraph 8, wherein the first reflector is an
anti-striping scanning mirror.
10. The system of paragraph 8, wherein the first lens has a focal
length and the first lens is positioned a distance from the first
reflector corresponding to the focal length.
11. The system of paragraph 8, wherein the third reflector is
mounted at an adjustable angle with respect to the third
chamber.
12. The system of paragraph 8, wherein the scanning mirror includes
a micromirror device.
13. The system of paragraph 8, wherein the light sheet generating
assembly is modular.
14. The system of paragraph 8, wherein the light sheet generating
assembly includes a housing and the elongate chambers are contained
in sequential communication within the housing.
15. The system of paragraph 14, wherein the housing includes a
laser source interface at a first end and a microscope interface at
a second end.
16. The system of paragraph 15, wherein the microscope interface
includes a C-mount.
17. A method for generating a light sheet suitable for use in a
microscope, the method comprising: generating a light beam using a
light beam source; directing the beam into an optics system
disposed in a housing by directing the beam into a first chamber of
a series of elongate chambers contained by the housing, a long axis
of each successive chamber being oriented at an acute angle with
respect to the long axis of the immediately preceding chamber;
focusing the beam, using a first lens, onto a first mirror;
redirecting the beam, using the first mirror, toward a second lens;
collimating the beam using the second lens; redirecting and fanning
the beam using a scanning second mirror; collimating the fanned
beam using a third lens.
18. The method of paragraph 17, further including reflecting the
fanned beam toward the third lens using a third mirror.
19. The method of paragraph 17, further including reducing a
striping effect in the microscope by rapidly pivoting the first
mirror.
20. The method of paragraph 19, wherein rapid pivoting of the first
mirror is performed using a micromirror device.
21. The method of paragraph 17, wherein the scanning second mirror
includes a micromirror device.
22. The method of paragraph 17, further including collimating the
light beam after the beam is generated by the light beam source.
Description
CROSS-REFERENCES
[0001] This application is based upon and claims the benefit under
35 U.S.C. .sctn.119(e) of U.S. Provisional Patent Application Ser.
No. 61/927,179, filed Jan. 14, 2014.
[0002] The complete disclosure of the above-identified patent
application is hereby incorporated by reference for all
purposes.
FIELD
[0003] This disclosure relates to sample illumination for
microscopes. More specifically, the disclosed embodiments relate to
systems and methods for generation of light sheets in fields such
as single plane illumination microscopy (SPIM).
INTRODUCTION
[0004] Single plane illumination microscopy (SPIM), or light sheet
microscopy, utilizes a microscope objective having a first axis of
observation. A light sheet is generated to illuminate a sample, and
the light sheet consists of a collimated plane of light oriented
transverse to the first axis. In some examples, the first axis is
orthogonal to the plane of the light sheet. The objective is
typically focused at the plane of the light sheet. In some
examples, the sample is also repositioned relative to the light
sheet in order to observe various levels/planes of the sample.
Multiple images may be acquired in this fashion in order to create
a 3-D image of the sample. If the image is changing over time,
multiple such 3-D image compilations may also be taken over time,
resulting in so-called 4-D imaging. A light sheet generating device
and method are described below and in the attached materials.
[0005] In the last decade, light sheet fluorescence microscopy
(LSFM) has emerged as a powerful imaging tool for cell and
developmental biology. LSFM systems excite the sample with a thin
light sheet and collect the resulting fluorescence along a
perpendicular detection axis. Imaging volumes are collected by
sweeping the light sheet and detection plane through the sample. As
only the focal plane is illuminated at any instant, these
microscopes provide highly efficient `optical sectioning`; unlike
confocal microscopes that use a pinhole to reject background,
little fluorescence is wasted on its way to the detector and
photodamage/bleaching are confined to the vicinity of the focal
plane. Since a wide field detector (camera) is used to collect
information from the entire imaging plane simultaneously, high
signal-to-noise ratio (SNR) images may be obtained with low
excitation intensities, minimizing undesirable effects like dye
saturation. Collectively, these advantages result in instruments
that are much faster, much gentler, and provide images with much
better SNR than laser scanning confocal microscopy. LSFM has been
particularly beneficial in long-term 4D imaging studies, as in the
embryogenesis of model organisms such as C. elegans, zebrafish, and
Drosophila. Recent efforts have improved the spatiotemporal
resolution of LSFM, and have enabled the study of fast,
intracellular dynamics that would have been otherwise impossible to
capture with other 4D imaging systems.
[0006] Modern LSFM systems use one objective lens to deliver the
excitation sheet and another to collect the fluorescence. The
required perpendicularity between excitation and detection forces
the use of relatively long working distance objectives and
constrains the sample geometry relative to single objective epi- or
confocal fluorescence microscopy. Many LSFM implementations embed
the sample in agarose, translating the resultant gel appropriately
to the common focal point of the objectives. By rotating the
sample, specimen views acquired at different angles may be fused
together into a composite volume, increasing the overall image
quality by masking the effects of scattering and light sheet
degradation that plague individual views. Moreover, multiview
deconvolution can be applied to compensate for the poor axial
resolution of any single view with the much better lateral
resolution inherent to a corresponding perpendicular view,
improving resolution isotropy.
[0007] While appropriate for large embryos like zebrafish or
Drosophila, embedding the sample in agarose is cumbersome for a
large variety of samples that are more easily grown or deposited on
conventional glass coverslips. Inverted selective plane
illumination microscopy (iSPIM) is a version of LSFM that is
compatible with glass cover slips. In iSPIM, two perpendicular,
water dipping objectives are placed above a sample mounted in an
inverted microscope stand. A light sheet is introduced with one
objective and scanned through the sample, and the second objective
is translated with a piezoelectric stage in order to keep the
imaging plane in focus during scanning. The sample can be easily
found with a low magnification objective mounted in the
epi-fluorescence port of the inverted microscope, and translated to
the focus of each objective. A modified version of iSPIM may be
utilized to capture a second specimen view, by alternating
excitation and detection between the two objectives. The resulting
dual-view inverted selective plane illumination microscope (diSPIM)
may provide isotropic spatial resolution (down to 330 nm) at high
speed (200 images/second, 0.5 seconds for a 50 plane volume).
[0008] The devices described herein may be compatible with
fiber-coupled laser excitation (making it much easier to align the
excitation path, making the device compatible with a broad array of
commercial laser excitation sources, and assuring collinearity
between different excitation wavelengths). Instead of controlling
multiple galvanometric mirrors, compact scan-heads (one for each
specimen view) may be used to generate and sweep the excitation
sheets through the sample, also aiding system alignment. Freely
available LabVIEW data acquisition software may be utilized for
generating and sweeping the light sheets, controlling and
synchronizing objective piezos, and recording images from
scientific complementary metal-oxide-semiconductor (CMOS)
cameras.
SUMMARY
[0009] The present disclosure provides systems and methods for
light sheet generation. In some embodiments, a light sheet
generating system may include a housing including a plurality of
elongate internal chambers in sequential communication with each
other and containing an optics system. A first chamber may contain
a first lens, a second chamber may extend from the first chamber
and contain a second lens, a third chamber may extend from the
second chamber, and a fourth chamber may extend from the third
chamber and contain a third lens, each chamber having a respective
long axis. A light source mount may be included at a first end of
the housing, the mount configured to receive a light source and
direct a light beam down the long axis of the first chamber. The
first lens may be configured to focus the laser beam onto a first
mirror. The second lens may be configured to collimate the laser
beam after reflecting from the first mirror. The second mirror may
be configured to steer the collimated laser beam into a fan shape.
The third lens may be configured to collimate the fan shaped laser
into a light sheet. The long axes of each pair of consecutive
cavities may form an acute angle.
[0010] In some embodiments, a system for conducting light sheet
microscopy may include a microscope having an objective including
an objective axis, a sample apparatus configured to hold a sample
spaced from the objective along the objective axis, and a light
sheet generating assembly configured to generate a planar sheet of
collimated light intersecting the sample at an angle transverse to
the objective axis. The light sheet generating assembly may include
a laser source operatively connected to a first elongate chamber
containing a first lens, a first reflector disposed at a terminal
end of the first chamber, a second elongate chamber containing a
second lens and arranged at an acute angle with respect to the
first chamber, a second reflector disposed at a terminal end of the
second chamber, a third elongate chamber arranged at an acute angle
with respect to the second chamber, a third reflector disposed at a
terminal end of the third chamber, and a fourth elongate chamber
containing a third lens and arranged at an acute angle with respect
to the third chamber. The second reflector may include a scanning
mirror.
[0011] In some embodiments, a method for generating a light sheet
suitable for use in a microscope may include generating a light
beam using a light beam source. The beam may be directed into an
optics system disposed in a housing by directing the beam into a
first chamber of a series of elongate chambers contained by the
housing, a long axis of each successive chamber being oriented at
an acute angle with respect to the long axis of the immediately
preceding chamber. The beam may be focused, using a first lens,
onto a first mirror. The beam may be redirected, using the first
mirror, toward a second lens. The beam may be collimated using the
second lens. The beam may be redirected and fanned using a scanning
second mirror. The fanned beam may be collimated using a third
lens.
[0012] Features, functions, and advantages may be achieved
independently in various embodiments of the present disclosure, or
may be combined in yet other embodiments, further details of which
can be seen with reference to the following description and
drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 is a schematic diagram of an illustrative light sheet
generation device in accordance with aspects of the present
disclosure.
[0014] FIG. 2 is an illustrative embodiment of a light sheet
generator in accordance with aspects of the present disclosure.
[0015] FIG. 3 is another illustrative embodiment of light sheet
generator in accordance with aspects of the present disclosure.
[0016] FIG. 4 is an illustration of steps performed in an exemplary
method for generating a light sheet for use in light sheet
microscopy or the like.
DESCRIPTION
Overview
[0017] Various embodiments of light sheet generation systems and
methods are described below and illustrated in the associated
drawings. Unless otherwise specified, a light sheet generation
system and/or its various components may, but are not required to,
contain at least one of the structure, components, functionality,
and/or variations described, illustrated, and/or incorporated
herein. Furthermore, the structures, components, functionalities,
and/or variations described, illustrated, and/or incorporated
herein in connection with the present teachings may, but are not
required to, be included in other light sheet generation systems
and methods. The following description of various embodiments is
merely exemplary in nature and is in no way intended to limit the
disclosure, its application, or uses. Additionally, the advantages
provided by the embodiments, as described below, are illustrative
in nature and not all embodiments provide the same advantages or
the same degree of advantages.
[0018] Light sheet generation devices and systems may be
interchangeably termed light sheet scanners, light sheet
generators, and/or light sheet scan units or modules. A light sheet
scanner generates a planar sheet of light by passing a collimated
laser beam through a folded optics system. The folded optics system
includes a plurality of lenses, one or more folding mirrors, and/or
one or more scanning mirrors arranged to generate a light sheet
suitable for use by a SPIM (and/or iSPIM and/or diSPIM)
microscope.
[0019] A light sheet generator is shown schematically in FIG. 1,
and generally indicated at 10. Light sheet generator 10 may include
a light source interface 12 configured to receive a light source
14, such as a laser. Interface 12 may be configured to direct a
light (e.g., laser) beam from light source 14 into and through a
first chamber 16 within light sheet generator 10. For example,
light source interface 12 may be coaxial with first chamber 16.
[0020] A series of elongate cavities or internal tunnels similar to
chamber 16 may be arranged within generator 10 (e.g., within a
housing 17 or other enclosure), and each successive pair of
chambers may be in communication with each other. For example, as
shown in FIG. 1, first chamber 16 may be followed by a second
chamber 18, and a third chamber 20. Optionally, a fourth chamber 22
(shown in dashed lines) may proceed from third chamber 20. In some
examples, additional chambers may be included beyond three or four.
The respective long axis of each of the chambers may be arranged
such that the chambers collectively form a zig-zag pattern. More
specifically, the intersection of each pair of long axes associated
with successive chambers may form a respective acute angle. This
relationship is indicated, for example, at angles 24 and 26. Such a
zig-zag pattern may result in a more compact device.
[0021] An optics system may be housed within light sheet generator
10. A portion of the optics system may be disposed within or
adjacent to chambers 16, 18, 20, and 22 (if present). For example,
a plurality of lenses may be included. For example, first chamber
16 may include a first lens 28, second chamber 18 may include a
second lens 30, and third chamber 20 may include a third lens 32.
Optionally, third lens 32 may be located within fourth chamber 22,
if present.
[0022] Light sheet generator 10 may further include a plurality of
mirrors to direct and shape the beam as it passes through the
chambers. For example, a first mirror 34 may be disposed at a
terminal end of first chamber 16, and a second mirror 36 may be
disposed at a terminal end of second chamber 36. If fourth chamber
22 is included, a third mirror 38 (e.g., a fold mirror) may be
included to direct the beam into the fourth chamber. Each of these
mirrors may include any suitable structure or device configured to
reflect a beam of light at a selected angle. In some examples, the
selected angle may be adjustable. In some examples, the selected
angle may be altered with respect to time, such as in a scanning
mirror.
[0023] An aperture 40, such as an iris or diaphragm, may be
included in first cavity 16 between light source interface 12 and
first lens 28. Aperture 40 may be configured to stop down the beam
from light source 14, and may be adjustable.
[0024] The optics system, including lenses 28, 30, and 32 as well
as mirrors 34, 36, and 38 (if present), are configured to receive a
beam (e.g., laser beam) entering the system at light source
interface 12 and convert the beam into a planar light sheet at a
system exit 42. The planar light sheet would be usable by a device
44 such as a microscope. Accordingly, light sheet generator 10 may
include a microscope interface 46. Microscope interface 46 may
include any suitable structure configured to facilitate secure
attachment of light sheet generator 10 to a light receiving portion
of microscope 44. Microscope 44 may include an objective axis, and
a sample apparatus configured to hold a sample spaced from the
objective along the objective axis. Light sheet generator 10 may be
configured to generate a planar sheet of collimated light
intersecting the sample at an angle transverse to the objective
axis.
[0025] Lenses 28 and 30 may be arranged as a 4f pair. In other
words, a distance A from first lens 28 to first mirror 34 may be
equal to the focal length of first lens 28. Similarly, a distance B
from first mirror 34 to second lens 30 may be equal to the focal
length of second lens 30. Accordingly, the effective distance
between first lens 28 and second lens 30 (i.e., A+B) is the sum of
the respective focal lengths. Excluding other optical effects, this
arrangement of the lenses results in a beam exiting second lens 30
that has similar or identical characteristics to the beam that
entered first lens 28. A similar arrangement may exist between
lenses 30 and 32.
[0026] First mirror 34 and/or second mirror 36 may be configured to
produce selected optical effects on the beam. For example, first
mirror 34 may be configured as an anti-striping mirror (explained
further below), such that the first mirror is caused to tilt
rapidly on one or more axes by an actuator 48. In some embodiments,
first mirror 34 may be stationary, functioning simply as a folding
mirror to redirect the beam. In some embodiments, the angle of
first mirror 34 may be adjustable.
[0027] Second mirror 36 may be configured as a scanning mirror,
such that the mirror tilts rapidly back and forth in two dimensions
and creates a fanning effect in the beam. In other words, a point
created by the beam impinging on a surface would be reflected back
and forth by mirror 36 such that the beam would instead create a
line segment on the surface. The tilting of mirror 36 may be driven
by a scanning actuator 50. This fanning of the beam may be
collimated by third lens 32. Accordingly, the beam remains at a
selected width following the third lens, and the fanning effect
does not continue beyond the third lens.
[0028] Third mirror 38, if present, may include a stationary (e.g.,
adjustable) reflector between scanning mirror 36 and third lens 32.
Mirror 38 may function to fold the beam, reflecting the beam down
the length of chamber 22.
[0029] Light sheet generator 10 may be described as compact due to
the zig-zag pattern created by chambers arranged at acute angles.
Generator 10 may be described as modular because it may be separate
from, and attachable to, any suitable light source and/or
microscope.
Examples, Components, and Alternatives
[0030] The following examples describe selected aspects of
exemplary light sheet generators, as well as related systems and/or
methods. These examples are intended for illustration and should
not be interpreted as limiting the entire scope of the present
disclosure. Each example may include one or more distinct
inventions, and/or contextual or related information, function,
and/or structure.
Example 1
[0031] FIGS. 2 and 3 show illustrative light sheet scanner devices
100 and 100'. Light sheet scanners 100 and 100' are embodiments of
light sheet generator 10, and may therefore include corresponding
components. Such corresponding components will have corresponding
names, and may be identified below and/or indicated by appropriate
reference numbers in FIGS. 2 and 3.
[0032] Light sheet scanners 100 and 100' include similar components
having similar descriptions. Accordingly, both scanners will be
described simultaneously using unprimed and primed respective
reference numbers, and with selected differences being identified
as needed. Light sheet scanner 100, 100' may include a body 102,
102' having a laser mount 104, 104' at one end, a microscope mount
106, 106' at another end, and a housing 108, 108' containing an
optics system 110, 110'. Body 102, 102' may include housing 108,
108' as well as mounting hardware, adjustment devices, openings,
threaded connections, and/or mounting surfaces suitable for
containing the remaining components of scanner 100, 100' and for
attaching scanner 100, 100' to another device such as a microscope,
camera, and/or support base.
[0033] Body 102, 102' may include a plurality of connected internal
cavities or pathways (also referred to as chambers), through which
a beam 103, 103' travels. Housing 108, 108' may be constructed of
rigid material, or a combination of rigid components with
semi-rigid and/or resilient material. Housing 108, 108' may be
configured to protect internal components from the environment. For
example, housing 108, 108' may be dust-proof, water resistant,
and/or resistant to mechanical shock. In some examples, housing
108, 108' may be configured to prevent or resist build up of a
static electrical charge. In some examples, housing 108, 108' may
be configured to exclude unwanted external light from the internal
pathways.
[0034] Laser mount 104, 104' may include any suitable structure
configured to enable attachment of a laser-generating device in a
predetermined orientation. For example, a laser mount 104 may be
configured to allow a coaxially mounted laser source 112, as shown
in FIG. 2. In other examples, such as the one shown in FIG. 3, a
laser mount 104' may be configured to connect with a
transverse-mounted laser source 112'.
[0035] Laser mount 104, 104' may include a collimation device 114,
114'. In other examples, collimation device 114, 114' may be part
of or integral with laser source 112, 112'. Collimation device 114,
114' may include any suitable device configured to collimate light
emitted by the laser source. In some examples, such as the one
shown in FIG. 3, multiple laser sources may be included.
Collimation device 114' may be configured to collimate the light
from all of the sources into a single collimated beam. Together,
laser mount 104, 104', laser source 112, 112', and collimation
device 114, 114' are configured to create a collimated laser beam
103, 103' directed down a path parallel to the long axis of a first
internal pathway 116, 116' in body 102, 102'.
[0036] Microscope mount 106, 106' may include any suitable
structure configured to enable attachment of scanner 100, 100' to a
microscope or other device, wherein the generated light sheet is
provided to the device through the mount. For example, microscope
mount 106, 106' may include a standard C-mount. For example, a
typical C-mount may have a 1-inch diameter, threaded aperture
having 32 threads per inch. Other mounting devices and arrangements
may be included.
[0037] Optics system 110, 110' may include any suitable devices and
systems configured to convert a collimated beam of light from a
laser source into a light sheet suitable for use in a SPIM
microscope. In the example shown in FIGS. 2 and 3, optics system
110, 110' includes an iris 118, 118', a first lens 120, 120', a
first mirror 122, 122', a second lens 124, 124', a scan mirror 126,
126', a fold mirror 128, 128', and a third lens 130, 130'. In other
examples, iris 118, 118' may be absent and/or more or fewer lenses
and/or mirrors may be present.
[0038] Iris 118, 118' (also referred to as an aperture, e.g.,
aperture 40) may include any suitable structure or device
configured to stop down or resize the collimated laser beam from
laser source 112, 112' by a desired amount. For example, iris 118,
118' may include an aperture or opening in an otherwise opaque
structure. In some examples, iris 118, 118' includes a diaphragm
that may be adjusted to alter the size of the opening in iris 118,
118' as desired.
[0039] First lens 120, 120' is disposed in the path of the
collimated laser beam within first internal pathway 116, 116'.
First lens 120, 120' may include any suitable lens-like optical
structure configured to focus the collimated laser beam onto first
mirror 122, 122'. For example, first lens 120, 120' may include a
compound lens. For example, first lens 120, 120' may include a
doublet, such as a concave and a convex lens in contact with each
other. In the example shown in FIGS. 2 and 3, first lens 120, 120'
includes a convex and concave lens doublet with the convex lens
oriented toward the laser source. In some examples, first lens 120,
120' may comprise a 4f lens pair with second lens 124, 124', as
described above, such that the effective lens separation distance
is approximately equal to the sum of the focal lengths of the first
and second lenses.
[0040] First mirror 122, 122' may be disposed at the end (or an end
portion) of first internal pathway 116, 116', and may be oriented
to redirect beam 103, 103' down a second internal pathway 132,
132', which may be in communication with first internal pathway
116, 116'. First mirror 122, 122' may include any suitable device,
structure, and/or surface configured to reflect the beam focused
thereon by first lens 120, 120' and in so reflecting, pass the beam
down the long axis of second internal pathway 132, 132' toward
second lens 124, 124'.
[0041] In some examples, first mirror 122, 122' includes an
adjustable, static, angled mirror. In some examples, first mirror
122, 122' includes a scanning mirror configured to prevent or
reduce striping, alternatively referred to as an anti-striping scan
mirror and/or anti-striping mirror. Striping refers to the
undesired shadowing effect of a light sheet that has been blocked
at certain points by nontransparent structures within the sample.
This effect may be overcome or reduced by rapidly alternating the
angle of the light sheet (within the plane of the light sheet) to
essentially steer light around the obstacles, first on one side
then on the other.
[0042] Accordingly, first mirror 122, 122' may include a scanning
feature that rapidly and symmetrically alternates the angle of the
mirror. This scanning may be accomplished by any suitable means. In
some examples, mirror scanning of mirror 122, 122' may be effected
using a suitable galvanometer scanner to steer the moveable mirror.
In some examples, mirror 122, 122' may include a micromirror,
interchangeably referred to herein as a micromirror device. In some
examples including a micromirror device, an array of miniature or
microscopic mirrors may be responsive to an electromagnetic field,
and may be controlled by applying a voltage to one or more
electrodes adjacent to the array. In some preferred examples, the
micromirror device may include a single mirror rather than an
array. The micromirror device may be fabricated from a material
such as silicon using photolithographic methods similar to those
used to fabricate integrated electronic circuits. In the example
shown, each micromirror device has a single mirror that may be
tilted about one or more axes, such as two perpendicular axes, or
directions by applying appropriate electrical signals. The
micromirror device may be described as a micro-electro-mechanical
system (MEMS) that may be used to reflect light in a controlled
manner.
[0043] Second lens 124, 124' may be disposed in second internal
pathway 132, 132' in the path of the beam reflected by first mirror
122, 122'. Second lens 124, 124' may be positioned at a distance
from first mirror 122, 122' corresponding to the focal length of
second lens 124, 124'. Second lens 124, 124' may include any
suitable lens-like optical structure configured to collimate the
beam. For example, second lens 124, 124' may include a compound
lens. For example, second lens 124, 124' may include a doublet,
such as a concave and a convex lens in contact with each other.
[0044] In the example shown in FIG. 1, second lens 124, 124'
includes a convex and concave lens doublet with the convex lens
oriented away from the laser source. As explained above, first lens
120, 120' may comprise a 4f lens pair with second lens 124, 124'.
In some examples, second lens 124, 124' may comprise a 4f lens pair
with third lens 130, 130'. In some examples, second lens 124, 124'
may comprise a first 4f lens pair with first lens 120, 120' and a
second 4f lens pair with third lens 130, 130'.
[0045] Scan mirror 126, 126' may be disposed at the end (or an end
portion) of second internal pathway 132, 132', and may be oriented
to redirect the beam down a third internal pathway 134, 134', which
may be in communication with second internal pathway 132, 132'.
Scan mirror 126, 126' may include any suitable device, structure,
and/or surface configured to reflect the beam directed thereon by
second lens 124, 124' and in so reflecting, pass the beam down the
long axis of third internal pathway 134, 134' toward fold mirror
128, 128'.
[0046] Moreover, scan mirror 126, 126' may be configured as a
scanning mirror. Unlike first mirror 122, 122', which may steer the
beam to overcome striping, scan mirror 126, 126' may be configured
to scan such that the collimated beam is steered back and forth
within a plane, in a relatively wide, fan-like manner. Accordingly,
scan mirror 126, 126' may include a scanning feature that rapidly
and symmetrically alternates the angle of the mirror. This scanning
may be referred to as vector scanning, and may be accomplished by
any suitable means. In some examples, mirror scanning of mirror
126, 126' may be effected using a suitable galvanometer scanner to
steer the moveable mirror. In some examples, mirror 126, 126' may
include a micromirror device, as described above regarding mirror
122, 122'.
[0047] At the other end (or end portion) of third internal pathway
134, 134' lies fold mirror 128, 128'. Fold mirror 128, 128' may be
oriented to redirect the fanned beam down a fourth internal pathway
136, 136', which may be in communication with third internal
pathway 134, 134'. Fold mirror 128, 128' may include any suitable
device, structure, and/or surface configured to reflect the beam
directed thereon by scan mirror 126, 126' and in so reflecting,
pass the beam down the long axis of fourth internal pathway 136,
136' toward third lens 130, 130'. Fold mirror 128, 128' may
function to reduce the overall size or footprint of light sheet
scanner 100, 100' while maintaining the effective overall length of
the optical pathway therein. Fold mirror 128, 128' may be fixed at
a predetermined angle. In some examples, the angle and/or position
of fold mirror 128, 128' may be adjustable. In some examples,
mirror 128, 128' may be omitted.
[0048] Third lens 130, 130' may be disposed in fourth internal
pathway 136, 136' in the path of the fanned beam reflected by fold
mirror 128, 128'. Third lens 130, 130' may be positioned at a
distance from fold mirror 128, 128' corresponding to the focal
length of third lens 130, 130'. Third lens 130, 130' may include
any suitable lens-like optical structure configured to collimate
the beam. In other words, third lens 130, 130' may refract the
fanned beam such that a collimated plane of light, or light sheet,
is created. For example, third lens 130, 130' may include a
compound lens. For example, third lens 130, 130' may include a lens
doublet.
[0049] Once the beam has passed through third lens 130, 130', it is
in a condition suitable for use as a light sheet in a SPIM
microscope. The light sheet may pass through the aperture in
microscope mount 106, 106' and intersect the sample as desired.
Example 2
[0050] In some embodiments, a diSPIM microscope (e.g., device 44)
may utilize two light sheet scanners, such as light sheet
generators 10, 100, and/or 100', one for each objective. Each light
sheet scanner may be used in an arm portion of the microscope. The
scanners may use integrated 2D MEMS mirrors to provide light
weight, low vibration optical scanning of light from a single mode
fiber-coupled laser. The basic design of the unit may include a
series of 4f lens pair arrangements with the scan mirrors,
apertures, and focal planes located at the foci of the lenses, as
described above.
[0051] The input to each light sheet scanner may include a fiber
collimator (e.g., collimator 114, 114') that accepts a single-mode
FC/PC (or optionally FC/APC) connected fiber-coupled laser source
(e.g., source 112, 112'). The fiber collimator may be an
exchangeable part that allows some flexibility in the focal length,
and hence intrinsic beam diameter, and fiber connector type. The
collimated laser beam may be stopped down with an iris diaphragm
(e.g., iris 118, 118') if desired, thereby creating light sheets
with different thicknesses that are suitable for samples of varying
size.
[0052] The collimated laser is focused onto the first fold mirror
(e.g., mirror 122, 122'), which can optionally be a micromirror
scanner that is located in the equivalent image optical image plane
as the microscope objective specimen focus plane. In examples using
such a scanner, the direction of the focused laser beam may tilt
about the focus point, thus providing an anti-striping capability.
After reflecting from the first fold mirror, the focused light is
collimated again and projected onto the main 2D MEMS scanner
mirror. The main scan mirror may be in the equivalent optical plane
as the microscope objective back focal plane. Tilting the scan
mirror thus steers the focused laser beam to different positions in
the sample focus plane.
[0053] As suggested by the C-mount connection (e.g., mount 106,
106'), simply connecting scanner 10, 100, 100' to any properly
positioned microscope C-mount camera port will allow the scanner to
be used to position a focused laser spot at the microscope's sample
plane. For diSPIM applications, the camera tube lens may be
positioned to image collimated light into the objective back focal
plane so that the focused laser beam remains parallel to the
optical axis when scanning off-axis.
[0054] To use the scanners to create and sweep light sheets through
a sample, analog voltages may be applied to one or more/each axis
of the MEMS mirrors.
Example 3
[0055] This example describes a method for generating a light sheet
suitable for use in a SPIM microscope; see FIG. 4. Aspects of the
light sheet generators described above may be utilized in the
method steps described below. Where appropriate, reference may be
made to previously described components and systems that may be
used in carrying out each step. These references are for
illustration, and are not intended to limit the possible ways of
carrying out any particular step of the method.
[0056] FIG. 4 is a flowchart illustrating steps performed in an
illustrative method, and may not recite the complete process or all
steps of the method. FIG. 4 depicts multiple steps of a method,
generally indicated at 200, which may be performed in conjunction
with a light sheet generator according to aspects of the present
disclosure. Although various steps of method 200 are described
below and depicted in FIG. 4, the steps need not necessarily all be
performed, and in some cases may be performed in a different order
than the order shown.
[0057] At step 202, a beam may be generated using a light beam
source, such as a laser. Step 202 may include collimating the beam
after the beam is generated.
[0058] Step 204 includes directing the beam into an optics system
disposed in a housing containing a series of elongate chambers.
Directing the beam into the optics system may include directing the
beam into a first chamber of the series of elongate chambers. A
long axis of each successive chamber may be oriented at an acute
angle with respect to the long axis of the immediately preceding
chamber.
[0059] Step 206 includes focusing the beam onto a first mirror
using a first lens. The first mirror may be disposed adjacent to a
distal end of a first chamber of the series of elongate
chambers.
[0060] Step 208 includes redirecting the beam with the first mirror
toward a second lens. The second lens may be disposed in a second
chamber of the series of elongate chambers. Accordingly,
redirecting the beam may include directing the beam down the second
chamber in a direction substantially parallel to the long axis of
the chamber. In some examples, redirecting the beam with the first
mirror includes tilting or rapidly pivoting the first mirror in an
alternating fashion to reduce a striping effect in the microscope,
as described above. Rapid pivoting of the first mirror may be
performed using a micromirror device.
[0061] Step 210 includes collimating the beam with the second lens.
In other words, the beam may exit the second lens in a condition
substantially identical to the condition in which it entered the
first chamber. In some examples, the second lens may form a 4f lens
pair with the first lens.
[0062] Step 212 includes redirecting and fanning the beam with a
scanning second mirror. Scanning may include rapid tilting or
pivoting of the second mirror to cause the beam to fan out in a
selected plane. The scanning second mirror may include a
micromirror device. In some examples, step 212 may further include
reflecting the fanned laser beam toward a third lens using a third
mirror.
[0063] Step 214 includes collimating the fanned beam using the
third lens to create a light sheet. Collimating may include
refracting the fanned beam such that the width of the beam in the
selected plane would be essentially constant following the third
lens. In some examples, the third lens may form a 4f lens pair with
the second lens.
[0064] The light sheet may then be directed into or received by a
microscope, such as for use in SPIM microscopy or the like.
Example 4
[0065] This section describes additional aspects and features of
compact light sheet generators, presented without limitation as a
series of paragraphs, some or all of which may be alphanumerically
designated for clarity and efficiency. Each of these paragraphs can
be combined with one or more other paragraphs, and/or with
disclosure from elsewhere in this application, including the
materials incorporated by reference in the Cross-References, in any
suitable manner. Some of the paragraphs below expressly refer to
and further limit other paragraphs, providing without limitation
examples of some of the suitable combinations.
[0066] A0. A system for generating a light sheet for use in a
microscope, such as a SPIM, iSPIM, and/or diSPIM microscope, the
system comprising:
[0067] a housing including a plurality of elongate internal
chambers in sequential communication with each other and containing
an optics system, such that a first chamber contains a first lens,
a second chamber extends from the first chamber and contains a
second lens, a third chamber extends from the second chamber, and a
fourth chamber extends from the third chamber and contains a third
lens, each chamber having a respective long axis; and
[0068] a light source mount at a first end of the housing, the
mount configured to receive a light source and direct a light beam
down the long axis of the first chamber;
[0069] wherein the first lens is configured to focus the laser beam
onto a first mirror, the second lens is configured to collimate the
laser beam after reflecting from the first mirror, the second
mirror is configured to steer the collimated laser beam into a fan
shape, and the third lens configured to collimate the fan shaped
laser into a light sheet; and
[0070] wherein the long axes of each pair of consecutive cavities
form an acute angle.
[0071] A1. The system of paragraph A0, wherein the first mirror is
an anti-striping mirror.
[0072] A2. The system of any of paragraphs A0-A1, wherein the
second mirror is a scanning mirror.
[0073] A3. The system of any of paragraphs A0-A2, further including
a third mirror between the second mirror and the third lens.
[0074] A4. The system of any of paragraphs A0-A3, further including
a collimation device disposed between the laser source and the
first lens.
[0075] A5. The system of any of paragraphs A0-A4, wherein the first
lens and the second lens form a 4f lens pair.
[0076] A6. The system of any of paragraphs A0-A5, wherein the first
lens has a focal length, and the first lens is spaced from the
first mirror by a distance equivalent to the focal length.
[0077] B0. A system for conducting light sheet microscopy
comprising:
[0078] a microscope having an objective including an objective
axis;
[0079] a sample apparatus configured to hold a sample spaced from
the objective along the objective axis;
[0080] a light sheet generating assembly configured to generate a
planar sheet of collimated light intersecting the sample at an
angle transverse to the objective axis;
[0081] wherein the light sheet generating assembly includes a laser
source operatively connected to a first elongate chamber containing
a first lens, a first reflector disposed at a terminal end of the
first chamber, a second elongate chamber containing a second lens
and arranged at an acute angle with respect to the first chamber, a
second reflector disposed at a terminal end of the second chamber,
a third elongate chamber arranged at an acute angle with respect to
the second chamber, a third reflector disposed at a terminal end of
the third chamber, and a fourth elongate chamber containing a third
lens and arranged at an acute angle with respect to the third
chamber;
[0082] wherein the second reflector is a scanning mirror.
[0083] B1. The system of paragraph B0, wherein the first reflector
is an anti-striping scanning mirror.
[0084] B2. The system of any of paragraphs B0-B1, wherein the first
lens has a focal length and the first lens is positioned a distance
from the first reflector corresponding to the focal length.
[0085] B3. The system of any of paragraphs B0-B2, wherein the third
reflector is mounted at an adjustable angle with respect to the
third chamber.
[0086] B4. The system of any of paragraphs B0-B3, wherein the
scanning mirror includes a micromirror device.
[0087] B5. The system of any of paragraphs B0-B4, wherein the light
sheet generating assembly is modular.
[0088] B6. The system of any of paragraphs B0-B5, wherein the light
sheet generating assembly includes a housing and the elongate
chambers are contained in sequential communication within the
housing.
[0089] B7. The system of paragraph B6, wherein the housing includes
a laser source interface at a first end and a microscope interface
at a second end.
[0090] B8. The system of paragraph B7, wherein the microscope
interface includes a C-mount.
[0091] C0. A method for generating a light sheet suitable for use
in a microscope, the method comprising:
[0092] generating a light beam using a light beam source;
[0093] directing the beam into an optics system disposed in a
housing by directing the beam into a first chamber of a series of
elongate chambers contained by the housing, a long axis of each
successive chamber being oriented at an acute angle with respect to
the long axis of the immediately preceding chamber;
[0094] focusing the beam with a first lens onto a first mirror;
[0095] redirecting the beam, using the first mirror, toward a
second lens;
[0096] collimating the beam using the second lens;
[0097] redirecting and fanning the beam using a scanning second
mirror;
[0098] collimating the fanned beam using a third lens.
[0099] C1. The method of paragraph C0, further including reflecting
the fanned beam toward the third lens using a third mirror.
[0100] C2. The method of any of paragraphs C0-C1, further including
reducing a striping effect in the microscope by rapidly pivoting
the first mirror.
[0101] C3. The method of paragraph C2, wherein rapid pivoting of
the first mirror is performed using a micromirror device.
[0102] C4. The method of any of paragraphs C0-C3, wherein the
scanning second mirror includes a micromirror device.
[0103] C5. The method of any of paragraphs C0-C4, further including
collimating the light beam after the beam is generated by the light
beam source.
CONCLUSION
[0104] The disclosure set forth above may encompass multiple
distinct inventions with independent utility. Although each of
these inventions has been disclosed in its preferred form(s), the
specific embodiments thereof as disclosed and illustrated herein
are not to be considered in a limiting sense, because numerous
variations are possible.
[0105] The subject matter of the invention(s) includes all novel
and nonobvious combinations and subcombinations of the various
elements, features, functions, and/or properties disclosed herein.
The following claims particularly point out certain combinations
and subcombinations regarded as novel and nonobvious. Invention(s)
embodied in other combinations and subcombinations of features,
functions, elements, and/or properties may be claimed in
applications claiming priority from this or a related application.
Such claims, whether directed to a different invention or to the
same invention, and whether broader, narrower, equal, or different
in scope to the original claims, also are regarded as included
within the subject matter of the invention(s) of the present
disclosure.
* * * * *