U.S. patent application number 17/285497 was filed with the patent office on 2021-12-02 for light sheet microscope with movable container.
This patent application is currently assigned to LaVision BioTec GmbH. The applicant listed for this patent is LaVision BioTec GmbH. Invention is credited to Heinrich Spiecker.
Application Number | 20210373309 17/285497 |
Document ID | / |
Family ID | 1000005829649 |
Filed Date | 2021-12-02 |
United States Patent
Application |
20210373309 |
Kind Code |
A1 |
Spiecker; Heinrich |
December 2, 2021 |
LIGHT SHEET MICROSCOPE WITH MOVABLE CONTAINER
Abstract
The present application discloses a light sheet microscope for
imaging biological materials. The microscope uses a plurality of
light beams, focused to an overlapping line to excite a fluorescent
material within the biological sample. The laser-induced
fluorescence image is then analyzed and displayed.
Inventors: |
Spiecker; Heinrich;
(Bielefeld, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
LaVision BioTec GmbH |
Bielefeld |
|
DE |
|
|
Assignee: |
LaVision BioTec GmbH
Bielefeld
DE
|
Family ID: |
1000005829649 |
Appl. No.: |
17/285497 |
Filed: |
December 10, 2019 |
PCT Filed: |
December 10, 2019 |
PCT NO: |
PCT/EP2019/084314 |
371 Date: |
April 15, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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62783231 |
Dec 21, 2018 |
|
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62868917 |
Jun 30, 2019 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G02B 21/26 20130101;
G02B 21/0048 20130101; G02B 21/0072 20130101; G02B 21/008 20130101;
G02B 21/0032 20130101; H04N 5/2254 20130101; G02B 21/0076 20130101;
G02B 21/34 20130101; G02B 21/33 20130101 |
International
Class: |
G02B 21/00 20060101
G02B021/00; G02B 21/26 20060101 G02B021/26; G02B 21/34 20060101
G02B021/34; G02B 21/33 20060101 G02B021/33; H04N 5/225 20060101
H04N005/225 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 10, 2019 |
EP |
PCT/EP2019/084314 |
Claims
1. A light sheet microscope for imaging a biological sample,
located on a sample holder, comprising: a detector which forms an
image of the biological sample through imaging optics, wherein the
biological sample disposed in a focal plane of the imaging optics;
a container holding a quantity of fluid and disposed on a movable
first stage, movable in the z-direction, wherein the z-direction is
orthogonal to the focal plane; a sample holder holding the
biological sample, wherein the biological sample is immersed in the
fluid and the biological sample is in the focal plane; wherein the
first stage has a range of motion such that the sample can be both
immersed in the fluid and in the focal plane and then withdrawn
from the fluid by the motion of the first stage, wherein the first
stage moves the container independently of the sample holder, the
imaging optics and the detector.
2. The light sheet microscope of claim 1, further comprising: a
movable optical subassembly configured to focus a plurality of
light beams into a single straight line that defines a light sheet,
and wherein the light sheet lies in the focal plane of the imaging
optics and therefore also in the biological sample; and a movable
second stage that holds the movable optical subassembly, wherein
the second movable stage moves laterally and parallel to the focal
plane, and perpendicularly to the z-direction, thereby moving the
light sheet to a different laterally adjacent region of the
biological sample,
3. The light sheet microscope as in claim 2, wherein the plurality
of light beams comprises at least three light beams, each of which
is separated by an angle, wherein the at least three light beams
are focused by the movable optical subassembly all to the same
straight line defining the light sheet, and wherein the light sheet
lies in the focal plane.
4. The light sheet microscope as in claim 2, wherein the plurality
of light beams comprises six light beams, with three of the six
light beams on either of two adjacent sides of the biological
sample and wherein each of the three light beams is separated by an
angle, and with each set of three beams focused to a single
straight line by two movable optical subassemblies disposed on
either side of the biological sample.
5. The light sheet microscope as in claim 2, wherein the container,
the sample holder, the biological sample, and first and second
stages are all movable without changing the focal plane of the
imaging optics and a location of the light sheet.
6. The light sheet microscope as in claim 2, wherein the different
laterally adjacent region comprises at least 5 regions spaced
laterally adjacent within the biological sample but within the
focal plane of the imaging optics.
7. The light sheet microscope as in claim 2, wherein by moving the
container holding the fluid in the z-direction, the sample holder
and biological sample may be held aloft of the container and fluid,
such that the biological ample can be manipulated without affecting
any one of the focal plane, the detector, the imaging optics, the
light sheet or touching the fluid.
8. The light sheet microscope as in claim 2, wherein the sample
holder is moved without changing a relative orientation of the
imaging optics or the focal plane or the light sheet.
9. The light sheet microscope as in claim 2, wherein the sample
holder is configured to allow the biological sample to be
manipulated without changing a relative orientation of the imaging
optics or the focal plane or the light sheet.
10. The light sheet microscope as in claim 2, further comprising an
imaging lens structure upon which the imaging optics is mounted,
wherein the imaging lens structure is also movable in the
z-direction and wherein the imaging optics are configured to form
the image on the detector, wherein the lens structure, the
container, the sample holder, the biological sample, and first and
second stages are all movable without changing the focal plane.
11. The light sheet microscope as in claim 2. wherein the detector
comprises: a pixelated detector wherein the imaging optics form a
pixelated image of the focal plane on the pixelated detector, and
wherein the imaging optics may be moved without moving the
pixelated detector, the biological sample or the container.
12. The light sheet microscope as in claim 2, wherein the imaging
optics may be submerged in the fluid by moving the lens structure
downward in the z-direction on the movable second stage or by
moving the first stage and container upward in the z-direction on
the movable first stage, and wherein the fluid is a clearing fluid
that renders the biological sample more transparent to
radiation.
13. The light sheet microscope as in claim 2, wherein the sample
can be removed and replaced by another sample without moving the
focal plane or the lens structure.
14. The light sheet microscope as in claim 2, wherein at least a
portion of the imaging optics is submerged in the fluid above the
sample and sample stage to image the irradiated sample.
15. The light sheet microscope as in claim 2, further comprising: a
movable third stage coupled to the sample holder, configured to
move the biological sample in the sample holder in the z-direction
to illuminate multiple depths in the biological sample with the
plurality of light beams.
16. The light sheet microscope of claim 15, wherein the light sheet
moves in the z-direction to irradiate multiple areas at different
depths within the sample by moving the third stage in the
z-direction, without affecting the position of the first stage or
second stage.
17. The light sheet microscope of claim 16, wherein the different
depths comprise at least 5 different depths.
18. The light sheet microscope of claim 15, wherein the sample
holder on the movable third stage, the lens structure on the
movable first stage, and the container on the movable second stage
can all move independently of, and without disturbing, the focal
plane.
19. The light sheet microscope of claim 15, wherein the movable
third stage has a throw of about 20-40 mm in the z-direction, and
70-90 mm laterally, orthogonal to the z-direction, and the movable
first stage has a throw of about 40-50 mm in the z-direction.
20. The light sheet microscope as in claim 1, wherein the
biological sample is replaced with a new biological sample, without
disturbing the sample stage, the lens structure, or the focal
plane, by moving the container on the movable first stage to expose
the biological sample.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The PCT application claims priority to U.S. Provisional
Application Ser. No. 62/783,231, filed Dec. 21, 2018 and U.S.
Provisional Application Ser. No. 62/7868917, filed Jun. 30, 2018.
Each of these prior applications is incorporated by reference in
its entirety.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] Not applicable.
STATEMENT REGARDING MICROFICHE APPENDIX
[0003] Not applicable.
BACKGROUND
[0004] This invention relates to a system for imaging biological
samples.
[0005] Imaging systems exist that use laser induced fluorescence to
image biological samples.
[0006] Some of these systems can generate images of subsurface
structures with impressive clarity, precision and resolution.
[0007] Some of these systems employ F-theta scanning mechanisms,
which scan a single focused laser into the sample using a rapidly
moving mirror. Because the speed of the mirror movement directly
determines the image acquisition speed, these systems are limited
in the speed with which data can be acquired, which limits the time
scale of effects they are able to capture. Also, the moving mirror
has significant mechanical complexity, which adds cost and makes
and makes high resolution problematic.
[0008] Accordingly, what is needed is a microscope which images
biological samples in three dimensions with excellent resolution,
contrast and accuracy with few moving parts, and no high speed
moving parts, that is cost effective and easy to use.
SUMMARY
[0009] An object of this invention is a microscope which has an
enhanced ability to precisely image internal, sub-surface
structures, or structures disposed in the bulk of a sample.
[0010] An object of this invention is a microscope which is able to
remove noise in a measured image to obtain a corrected image by
error correction.
[0011] An object of this invention is to create three-dimensional
images by scanning a light sheet laterally across a sample, and
then orthogonally through the depth.
[0012] An object of this invention is a microscope which can have
the sample changed without affecting the excitation or detection
optics.
[0013] An object of this invention is a microscope which can have
the objective lens change without affecting the excitation
optics.
[0014] An object of this invention is a microscope wherein the
sample can be withdrawn from the clearing fluid without touching
the sample or affecting the excitation or detection optics.
[0015] An object of this invention is a microscope which images
biological samples in three dimensions with excellent resolution,
contrast and accuracy with few moving parts, and no high speed
moving parts.
[0016] A light sheet microscope for imaging biological samples is
described. In a first embodiment, the microscope may include at
least two collimated light sources each emitting a beam of light
along at least two different propagation axes, at least two optical
subassemblies which focus the at least two beams of light into at
least two straight lines, wherein at least one of the two straight
lines defines a non-orthogonal angle with respect to its
propagation axis and wherein the at least two straight lines are
substantially overlapping, and wherein the straight lines and the
propagation axes define an excitation plane of the light sheet
microscope.
[0017] In another embodiment, a light sheet microscope for imaging
a biological sample is described, which may include at least one
light source focused by an optical assembly to a single line focus
illuminating the biological sample, defining an excitation with an
intensity distribution function .LAMBDA..sub.1. The excitation may
cause the biological sample to emit fluorescence. The microscope
may also include a means to move the single line focus, wherein the
single line focus remains in the object sided focal plane of the
microscope, and thereby illuminating a plurality of laterally
adjacent positions in the biological sample. The microscope may
further include an imaging system that generates a two-dimensional
image of the three-dimensional biological sample employing a
mapping function .LAMBDA..sub.2, and a pixelated detector. The
pixelated detector may convert the two-dimensional images of the
microscope of the fluorescence emitted by the sample to at least
two raw images, wherein the pixelated detector has a point spread
function .LAMBDA..sub.3, and a computer that can store and
manipulate the signals produced by the pixelated detector and is
programmed to produce a restored pixelated image F from the raw
images I by removing degradations associated with the functions
.LAMBDA..sub.1, .LAMBDA..sub.2 and .LAMBDA..sub.3.
[0018] In other embodiments, a light sheet microscope for imaging a
first biological sample disposed on a sample holder is disclosed.
In this embodiment, the microscope may include an imaging lens
structure including an operative objective lens having a focal
plane and at least one inoperative lens, and wherein the imaging
lens structure is movable in a z-direction orthogonal to the focal
plane by a movable first stage. The microscope may further include
a container holding a quantity of fluid, wherein the sample holder
is immersible in the fluid, and a detector which forms an image of
the focal plane, wherein the image includes at least a portion of
the first biological sample. The movable first stage supporting the
lens structure may have sufficient range of motion to submerge the
operative objective lens into the fluid held in the container,
thereby forming an image of the first biological sample on the
detector, and wherein the sample holder. The container may have a
shape which admits a movement of the imaging lens structure, when
the imaging lens structure is submerged.
[0019] In yet other embodiments, a light sheet microscope for
imaging a biological sample, located on a sample holder is
disclosed. The microscope may include a detector which forms an
image of the biological sample through imaging optics, wherein the
biological sample disposed in a focal plane of the imaging optics.
The microscope may further include a container holding a quantity
of fluid and disposed on a movable first stage, movable in the
z-direction, wherein the z-direction is orthogonal to the focal
plane, a sample holder holding the biological sample. Within this
embodiment, the biological sample may be immersed in the fluid and
the biological sample may be in the focal plane, wherein the first
stage has a range of motion such that the sample can be both
immersed in the fluid and in the focal plane and then withdrawn
from the fluid by the motion of the first stage, wherein the first
stage moves the container independently of the sample holder, the
imaging optics and the detector.
[0020] These and other features and advantages are described in, or
are apparent from, the following detailed description and
accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Various exemplary details are described with reference to
the following figures, wherein:
[0022] FIG. 1 is an illustration of a line focus in a biological
imaging device, wherein the line focus is tilted with respect to an
axis of propagation of radiation;
[0023] FIG. 2 is an illustration of a two overlapping line foci in
a biological imaging device with two axes of propagation of
radiation;
[0024] FIG. 3 is an illustration of a three overlapping line foci
in a biological imaging device with three axes of propagation of
radiation;
[0025] FIG. 4 is an illustration of a light sheet microscope with
three axes of propagation of radiation, showing the lateral
capability;
[0026] FIG. 5 is an illustration of a three overlapping line foci
in a biological imaging device with three axes of propagation of
radiation, having a rotatable slot structure that may define a
numerical aperture of the imaging system;
[0027] FIG. 6a is a perspective view of the rotatable slot
structure that may define a numerical aperture of the imaging
system; FIG. 6b is a cross sectional view of the rotatable slot
structure;
[0028] FIG. 7 is an illustration of the intensity of the line focus
as a function of the lateral distance showing two successive scans,
and demonstrating Gaussian propagation of the beam;
[0029] FIG. 8a is an illustration of the intensity of the line
focus as a function of lateral distance, and an illustration of the
beam waist and Rayleigh distance for a first numerical aperture
NA.sub.1; FIG. 8b is an illustration of the intensity of the line
focus as a function of lateral distance, and an illustration of the
beam waist and Rayleigh distance for a second numerical aperture
NA.sub.2, wherein NA.sub.1<NA.sub.2;
[0030] FIG. 9 is an illustration showing the lateral horizontal
movement of the light sheet within the biological sample;
[0031] FIG. 10a, FIG. 10b are two alternative arrangements of the
optical turning elements that can generate the lateral horizontal
movement of the light sheet within the biological sample for the
imaging device;
[0032] FIG. 11 is an illustration showing the left side and right
side lateral horizontal movement of the light sheet within the
biological sample, which may image the sample from the left and
from the right, respectively;
[0033] FIG. 12 is an illustration describing the correction for
refraction of the light beams at the boundary of the glass liquid
vessel;
[0034] FIG. 13 is an illustration of a light sheet microscope with
a pixelated detector imaging a sample within its field of view of
the detector;
[0035] FIG. 14 is an illustration showing the point spread
functions of the excitation .LAMBDA..sub.1 and the detection
.LAMBDA..sub.2, .LAMBDA..sub.3 produced by the light sheet
microscope from the biological sample;
[0036] FIG. 15 portrays the different steps (FIG. 15a, FIG. 15b,
FIG. 15c, FIG. 15d) of image formation;
[0037] FIG. 16 is an illustration describing how the left side of
the image is meshed with the right side image using the device
shown in FIG. 11;
[0038] FIG. 17 is an illustration showing the lateral horizontal
scanning of the light sheet through the biological sample in the
x-direction;
[0039] FIG. 18 is an illustration showing horizontal scans (FIG.
18a, x-dimensions) and scanning in depth (FIG. 18b, z-dimension) to
produce a three dimensional image by the light sheet microscope
from the biological sample
[0040] FIG. 19 is an illustration showing the filter selection to
choose a working wavelength for the light sheet microscope
excitation and detection;
[0041] FIG. 20 is an illustration showing the plurality of optical
sources (FIG. 20a and FIG. 20b) for the light sheet microscope
excitation and detection;
[0042] FIG. 21 is an illustration showing the components of the
detector for the light sheet microscope;
[0043] FIG. 22a is an illustration showing the ability to rotate a
turret holding a plurality of objective lenses; FIG. 22b is a
perspective illustration of the sample stage and turreted lenses,
accommodating the turreted lenses; FIG. 22c is a perspective
illustration of the turreted lenses;
[0044] FIG. 23 is an illustration showing how the light sheet may
be moved laterally independently of the turreted objective
microscope;
[0045] FIG. 24 is an illustration showing a view of how the cuvette
may be immersed by vertical movement, (FIG. 24a) independently of
the sample and the optical system (FIG. 24b);
[0046] FIG. 25a plan view showing the movable cuvette; FIG. 25b is
a cross sectional view, and FIG. 25c is an end-on view how the
cuvette and sample holder may be moved vertically independently of
the sample and the optical system;
[0047] FIG. 26a is a side view illustration showing how the sample
may be accessed without disturbing the rest of the optical system;
FIG. 26b shows the sample completely withdrawn from the clearing
fluid;
[0048] FIG. 27 is an illustration showing how multiple samples may
be handled using the novel sample holder.
[0049] It should be understood that the drawings are not
necessarily to scale, and that like numbers may refer to like
features.
DETAILED DESCRIPTION
[0050] The first portion of this description is directed to the
optical details of the novel optical imaging device for biological
samples using light sheets, and error correction. The device also
has some design features that make it remarkably simple and easy to
use. The novel imaging device may reduce the uncertainly deriving
from the shadows cast by opaque structures, and also has no high
speed moving parts and so may be considerably simpler than other
scanning methodologies. The second portion discusses the error
correction methodology used to improve the image data in terms of
accuracy, contrast and resolution. The third portion describes some
of the novel mechanical features of the light sheet microscope that
make it particularly advantageous and easy to use.
[0051] In the following description of the preferred embodiment,
reference is made to the accompanying drawings which form a part
hereof, and in which is shown by way of illustration a specific
embodiment in which the invention may be practiced. It is to be
understood that other embodiments may be utilized and structural
changes may be made without departing from the scope of the present
invention. The following reference numbers are used to refer to the
following features. It should be understood that this list is
provided as a convenience, and may not be exhaustive of the
reference numbers used in the text that follows. [0052] 10 Lens
turret [0053] 60 Turning mirrors including 61, 62 and 63 [0054] 61,
62, 63 Turning mirrors embodiment 100 [0055] 68, 64, 66, 74 Turning
mirrors in alternative embodiment [0056] 65, 67, 69 Turning mirrors
embodiment 101 [0057] 74 First turning mirrors embodiment 100, 101
[0058] 51 rotating aperture including slot 73 and aperture 52
[0059] 68, 69 turning mirrors [0060] 70 telescoping lens [0061] 73
slot [0062] 100 Movable optical assembly [0063] 101 Alternative
embodiment of optical system [0064] 100' Adjacent side movable
optical assembly [0065] 120 Line focusing optical subassembly #1
[0066] 140 Line focusing optical subassembly #2 [0067] 160 Line
focusing optical subassembly #3 [0068] 200 Biological sample [0069]
210 Line focus/focal plane [0070] 300 Cuvette [0071] 310 Field of
view of detector [0072] 350 Top surface opening for objective lens
[0073] 351, 352 Cutouts from cuvette 300 [0074] 360, 361
Transparent windows for admitting radiation to the cuvette and
sample [0075] 400 Controller [0076] 500 light source [0077] 521-526
Laser sources [0078] 530 Collimating lens [0079] 550 Filter wheel
excitation [0080] 560 collimator [0081] 600 Detector [0082] 660
Filter wheel detector [0083] 620 imaging lens [0084] 660 detection
filter [0085] 700 objective lens [0086] 750 particle [0087] 670
objective lens [0088] 800 sample holder [0089] 810 sample stage
[0090] 820 sample stage support point [0091] 930 movable cuvette
stage [0092] 900 stage for movable optical assembly [0093] 951, 952
Cutouts from cuvette 300 [0094] 1000 clearing fluid
[0095] A coordinate system applies to the figures in general. The
x-axis is generally the scanning dimension, that is, it is the axis
along which the line focus will be scanned. The y-axis is generally
the direction of the line focus, that is, it is the direction that
the focus lies along, with relatively uniform intensity within and
along the focal line. The radiation beams entering the sample are
traveling along an x-axis in the x, y plane. The z-axis is
generally the viewing direction. That is, the optical axis of the
camera and/or detector will lie above and orthogonal to the x- y-
plane, along the z-axis.
[0096] In one aspect, the light sheet microscope may make use of
Scheimpflug optics, which is the phenomenon whereby tilting an
optical element with respect to its optical axis, the focusing
properties of that element may be tilted as well. By careful
placement and relative orientations, three excitation light sources
may be arranged to create a single line focus that can then be
scanned across the sample.
[0097] Accordingly, at least one optical element may be tilted with
respect to propagation direction by an angle of < >90 degrees
(non-orthogonal). When the angle .alpha..sub.1 is defined relative
to the line orthogonal to the propagation axis (see FIG. 1), the
angle .alpha..sub.1 may be less than 30 degrees preferentially,
more preferably less than 20 degrees and more preferably about 16
degrees. As discussed below, this angle may be corrected for
refraction effects occurring at the liquid cuvette holder boundary.
This results in a tilt of the line focus with respect to the axis
of propagation through the optical system by and angle
.alpha..sub.1. .alpha..sub.2 may be similar in magnitude but in the
opposite sense as .alpha..sub.1.
[0098] According to one aspect of the invention, using the light
sheet microscope as described herein, shadows cast by a particle
are reduced or at least rendered unambiguous. Even more precision
may be obtained using the error correction process described
herein. In this process, a number of distinct shadows, which are
switchable in this case by switching illumination sources, can be
easily calculated out compared to a scanned beam, which creates one
smooth shadow.
[0099] According to another aspect of the invention, a column of
pixels can be treated in generally the same way in the image
processing algorithm, because illumination in this dimension is
uniform because of the properties of the line focus.
[0100] According to another aspect of the invention, very high
precision three dimensional images may be produced using very few
moving parts, and no high speed moving parts. This greatly improves
repeatability, cost and reliability.
[0101] Light Sheet Microscope
[0102] FIG. 1 is an illustration of a line focus used in a
biological imaging device, wherein the imaging device uses a single
collimated light source 500. Radiation from the source 500 is
focused into a line focus that is tilted with respect to an axis of
propagation of radiation. Optical radiation from a source 500 is
shaped by an optical subassembly 120, and focused into a line 210
that falls within a biological sample 200, but is tilted with
respect to the axis of propagation through the optical subassembly
120.
[0103] The optical subassembly 120 may contain a plurality of
optical elements, including for example two confocal or spherical
lenses and a cylindrical lens. At least one of the lens elements
may be tilted with respect to the axis of propagation of the
radiation. The tilt angle is denoted by .alpha..sub.1 wherein
.alpha..sub.1 may be in the range 0<.alpha..sub.1<40.degree.,
and more typically in the range 5<.alpha..sub.1<25.degree.,
and more preferably about 16.degree.. This tilt may result in the
line focus being tilted by a similar angle .alpha..sub.2 with
respect to the propagation axis of the radiation. The propagation
axis or the axis of propagation is the direction traveled by
photons at or near the center of the beam of collimated light. The
optical axis is often the same or parallel to the propagation axis
but is defined as the neutral axis of optical elements: the line
passing through the center of curvature of lens and parallel to the
axis of symmetry is the optical axis. The optical axis of the
pixelated detector, for example, is the line from a first lens or
transparent window on the front of the camera or detector, to the
pixelated detector array at the rear of the camera or detector.
These angles are defined relative to the axis orthogonal to the
axis of propagation as shown in FIG. 1.
[0104] In the embodiment shown in FIG. 1, the second confocal lens
(the third optical element in optical subassembly 120) may be
tilted by the angle with respect to axis of propagation. The angle
it forms, .alpha..sub.1, is measured relative to the orthogonal
direction with respect to this propagation axis. As a result of
this tilt, the line focus 210 is also tilted by an angle
.alpha..sub.2 with respect to this orthogonal axis. Based on the
geometry shown in FIG. 1, .alpha..sub.1 may be similar in magnitude
but opposite in sign with respect to this orthogonal axis. This is
the basis of Scheimpflug optics. The Scheimpflug principle is a
geometric rule that describes the orientation of the plane of focus
of an optical system (such as a camera) when the lens plane is not
parallel to the image plane. This principle is used here to form
overlapping lines in an biological sample, as described in detail
below.
[0105] The elements of the optical subassembly 120 may be arranged
in a number of different ways, such as
cylindrical/spherical/spherical or spherical/spherical/cylindrical.
However, the configuration of spherical/cylindrical/spherical
(shown in FIG. 1) may result in the most compact arrangement.
[0106] Within the optical subassembly 120, two lenses may be glued
together. Scheimpflug conditions can also be realized with more and
glued elements. However the configuration shown in FIG. 1 may be
most advantageous for both Scheimpflug and curvature correction.
Separate lenses also give an additional degree of freedom, because
the distance of separation may affect the shape of the line focus
and its curvature.
[0107] FIG. 2 is an illustration of another embodiment of two
optical subassemblies 120 and 140. The components of the optical
subassemblies may be similar or identical, each containing for
example, two confocal lenses and a cylindrical lens. The
arrangement of these components may be similar to that illustrated
in the embodiment shown in FIG. 1. However this is exemplary only
and the optical assembly may have different components or may they
may be in a different arrangement or order.
[0108] The second optical subassembly 140 may also create a line
focus, however this optical assembly may not have a tilted element.
Accordingly, the line focus may not be tilted with respect to the
image plane, but may instead lie nearly exactly along the
orthogonal direction, that is, it may lie in the image plane.
[0109] It should be noted that optical subassembly 120 may be
tilted with respect to optical subassembly 140. In other words, the
optical axis and of optical subassembly 120 may form an angle with
respect to the optical subassembly 140, and the propagation axis of
the light traveling through it. Additionally, optical subassembly
120 may form a line focus at an angle with respect to its optical
axis, and the propagation axis of the light traveling through it.
By proper arrangement of the components of optical subassembly 120
and optical subassembly 140, the line focus caused by optical
subassembly 120 may fall substantially exactly over the line focus
resulting from optical subassembly 140. These overlapping line foci
may be applied to a biological sample within a biological imaging
device with two axes of propagation of radiation.
[0110] By "substantially overlapping" or "substantially exactly
overlapping", it should be understood that the line foci deviate in
overlap from one another by less than a defined amount. This amount
may be in lateral misregistration or angular deviation. That is,
the line foci although substantially exactly overlapping, may
nonetheless deviate a finite amount laterally and angularly. The
amount of lateral deviation allowed while still "substantially
overlapping" or "substantially exactly overlapping", may be defined
in terms of Rayleigh length. The amount of angular deviation may be
defined in terms of degrees of angular deviation. For the purposes
of this description, "substantially overlapping," the overlapping
line foci will deviate from one another by less than 4 Rayleigh
lengths laterally and less than 5 degrees angularly. More
preferably, the "substantially overlapping" line foci will deviate
from one another by less than 2 Rayleigh lengths and 3 degrees. Yet
more preferably, "substantially overlapping" or "substantially
exactly overlapping", overlapping foci may deviate from one another
by less than 1 Rayleigh length and less than 2 degrees.
[0111] "Partially overlapping" may be understood to mean the
placement of one line focus within 5 Rayleigh lengths of the
adjacent line focus.
[0112] The biological sample 200 may have biological structures
which are tagged with a fluorescent moiety. Accordingly, biological
sample 200 may fluoresce when radiation from source 500 having the
proper wavelength excites these fluorescent moieties. This
fluorescence may be detected by an appropriate detector and used to
gain information about the biological sample. The device is
described in detail below.
[0113] Importantly, therefore, the device shown in FIG. 2 may use
light 500 coming from multiple directions. The different directions
may each derive from an independent light source, or they may use
the same light source but split off the single source by partially
transmitting and partially reflecting surfaces. These
configurations are also described in further detail below.
[0114] Having radiation coming from different directions but with
overlapping line foci may have several advantages. One advantage
may be that shadows cast by an opaque structure intercepting light
from one source may be distinguished from other sources of
contrast. Accordingly, the contrast seen in an image may be
attributed to an obscuring structure, and the detailed morphology
of that structure may be ascertained by comparing the images
collected using the radiation coming from multiple directions.
Accordingly, successive scanning with the off-center optics 120 and
160 may also reveal the depth and extent of shadowing from a single
opaque structure in the biological sample, Accordingly, this
technique can be used to ascertain what features in a scan are due
to shadowing effects, an what features are related to real, new or
discrete structures within the sample.
[0115] FIG. 3 is an illustration of a three overlapping line foci
in a biological imaging device with three axes of propagation of
radiation. FIG. 3 shows three optical subassemblies, 120, 140 and
160. The components of the three optical subassemblies 120, 140 and
160 may be similar or identical, each containing for example, two
confocal lenses and a cylindrical lens. The arrangement of these
components may be the same as illustrated in the embodiment shown
in FIG. 1. However this is exemplary only and each optical
subassembly may have different components or may they may be in a
different arrangement or order.
[0116] Once again, the second optical subassembly 140 may create a
line focus, however this optical assembly may not have a tilted
element. Accordingly, the line focus may not be tilted with respect
to the image plane, but may instead lie nearly exactly along the
orthogonal direction, that is, it may lie in the image plane.
[0117] It should be noted that third optical subassembly 160 may be
tilted with respect to optical assemblies 120 and 140. In other
words, the optical axis of optical subassembly 160 may form an
angle with respect to the optical axis of optical assemblies 120
and 140, and the propagation axis of the light traveling through
it. Additionally, optical subassembly 160 may form a line focus at
an angle with respect to its optical axis. By proper arrangement of
the components of optical subassembly 160 and optical assemblies
120 and 140, the line focus caused by optical subassembly 160 may
fall substantially exactly over the line foci resulting from
optical assemblies 120 and 140. Accordingly, in FIG. 3, there may
be three substantially overlapping line foci, all designated by
reference number 210, because they are substantially
overlapping.
[0118] Accordingly, the third optical subassembly 160 creates
another line focus that falls exactly on the first two line foci
from optical subassembly 120 and 140. This third optical
subassembly 160 may be the mirror image of optical subassembly 120,
with its tilted optical element tilted in the mirror image sense,
as shown in FIG. 3. Accordingly, there may be a symmetry axis as
depicted in FIG. 3, with the optical top half 120 mirrored by the
optical bottom half 160.
[0119] The overlapping line foci may be applied to a biological
sample 200 within a biological imaging device with three axes of
propagation of radiation. As before, the biological sample 200 may
have biological structures which are tagged with a fluorescent
moiety. Accordingly, biological sample 200 may fluoresce when
radiation from source 500 having the proper wavelength excites
these fluorescent moieties. This fluorescence may be detected by an
appropriate detector and used to gain information about the
biological sample. By applying the radiation from three different
directions, the shadowing effects of opaque structures can be
effectively measured, such that detailed information about the
morphology of the structure may be ascertained. If this line focus
is then scanned laterally through the sample, detailed information
about structures contained in the sample, but separated laterally,
may be obtained. The means and methods for moving the line focus
through the sample are described in considerable detail below.
[0120] If the light sources coming from the three directions 120,
140 and 160 are activated sequentially rather than in unison, the
shadows may be unambiguously detected. This is because the shadow
as cast from an opaque object illuminated by a light source
impinging from one direction, will obscure a different area
directly behind the opaque object than a shadow cast by a light
source coning from another direction. Accordingly, in some
embodiments, the light sources 500 may be energized sequentially,
at least during a portion of the data collection.
[0121] Finally, by energizing the three beams sequentially, a
shadow cast by a structure embedded in the sample can easily be
distinguished from a shadow cast by, for example, a lens or mirror
defect. Furthermore, this structure can also be measured in extent
by sequential irradiation from the three beams. Because of the
different trajectories of the three beams, shadows cast by a
particle, by using three beams may be reduced or separated. Even
further, by deconvolving a number of distinct shadows, which are
switchable in this case, the effect of the shadows can be more
easily calculated compared to a scanned beam, which creates one
smooth shadow.
[0122] FIG. 4 is an illustration of a three overlapping line foci
in a biological imaging device with three axes of propagation of
radiation, wherein the biological sample is contained in a fluid
vessel and imaged by a pixelated detector. The biological sample
200 may be submerged in a cuvette or other vessel 300 containing a
fluid. "Cuvette" is a term used here to refer to a vessel
containing sample embedded in various optical liquids. The cuvette
may have at least one window which is optically transparent with
low distortion to allow for diffraction limited light sheet
generation. Thus the terms "cuvette", "vessel" and "container" are
used interchangeably to refer to a holder or receptacle that
contains a quantity of fluid. The fluid may be a clearing fluid in
which a biological sample is immersed. The cuvette, vessel or
container may also be open on one side for immersion of the
biological sample 200 and for dipping of the objective lenses into
the fluid, as will be described further below. A "clearing fluid"
is a fluid that renders a biological sample relatively transparent
to a probing radiation.
[0123] "Lateral" or "laterally adjacent" to a point should be
understood to be defined with respect to a plane through a sample,
wherein a second point laterally adjacent generally is in the same
plane but offset from a first point also in the plane. An
"anamorphic lens" may be a lens whose focal distance in one
dimension is different that its focal distance in the other
dimension. A cylindrical (line focusing) lens is one example of an
anamorphic lens, having a first and a second the focal distance,
wherein the first focal distance is finite, and the second focal
distance is essentially infinite. Such a lens will produce a line
focus at the first focal distance.
[0124] A "clearing fluid" is a biological fluid containing
compounds designed to minimize non-uniform light absorption or
scattering by biological structures in the sample. The use of
clearing fluids is important in imaging into the depths of thicker
biological sample, such that the probing radiation is able to
penetrate into the depth. Hydrogen peroxide, for example, can be
used as a clearing fluid to de-color hemoglobin and myoglobin, two
of the primary molecules responsible for light absorption in
biological tissue.
[0125] The fluid in the cuvette 300 may be a clearing fluid 1000,
which may render the biological tissue transparent to radiation.
The cuvette 300 may also be transparent, or may at least have at
least one transparent window or opening on its side, allowing the
optical radiation from the source 500 to pass into the cuvette 300,
into the fluid and into the biological sample. 200. The lateral
distance "A" to "B" may indicate the thickness of the transparent
walls of the cuvette. Refraction effects may occur at this
boundary, and the treatment of this refraction is discussed below
with respect to FIG. 12.
[0126] As mentioned previously, the plurality of optical assemblies
120 and 140 (and 160 if present) may be configured such that each
focuses incoming radiation into a line. The line focus due to
optical subassembly 120 and 160 may substantially overlap the line
focus due to optical subassembly 140. Accordingly, all optical
assemblies 120, 140 and 160 may focus radiation into the same line
focus 210 that falls within the biological sample 200.
[0127] Above the cuvette 300 (and not shown in FIG. 4), a detector
may image the sample 200 in order to detect fluorescence emitted by
the fluorescent tags, for example, when irradiated by the line
focus 210. The biological sample 200 may be placed in the
horizontal plane (plane of the paper) and the detector may be
placed above this plane. The line focus 210 may also fall
substantially in this plane. The detector may have field of view
310 that includes the line focus 210 in the sample, and may also
include laterally adjacent areas. That is, the field of view of the
detector may include the line 210 in its first position as well as
subsequent adjacent positions, as the line focus is scanned
laterally. The lateral movement of the line focus is described
further below with respect to FIG. 9.
[0128] FIG. 5 is an illustration of a three overlapping line foci
in a biological imaging device with three axes of propagation of
radiation, wherein the illustration includes additional optical
elements that direct the radiation onto the sample 200. In one
embodiment shown in FIG. 5, the radiation may come from a single
source 500, and may be directed off three partially reflecting and
partially transmitting mirrors 61, 62 and 63. Each of these mirrors
may direct a portion of the radiation into parallel lines. These
parallel lines may pass through an aperture 51 described further
below and then proceed in a parallel fashion to a movable optical
assembly 100. FIG. 5 does not show the components of subassemblies
120, 140 and 160. The important feature is that light entering
movable optical assembly 100 containing subassemblies 120, 140 and
160 enters the movable assembly 100 in a parallel fashion, such
that movable assembly 100 may be moved laterally without affecting
the optical paths within movable assembly 100. This feature is
important in moving the line focus laterally through the sample,
and allows the high resolution, highly repeatable scanning
capabilities of the light sheet microscope.
[0129] Accordingly, movable optical assembly 100 may contain
optical subassemblies 120, 140 and 160 as was shown in FIG. 3. In
addition, movable optical assembly 100 may also contain two pairs
of turning mirrors 53, 54, 55 and 56. The first pair of turning
mirrors 53 and 54 may redirect the parallel incoming light and
direct it into optical subassembly 120 along its optical axis. The
second set of turning mirrors 55 and 56 may redirect the lower leg
of incoming parallel light into optical subassembly 160 along its
optical axis. Accordingly, once again, the lower legs are the
mirror-image of the upper legs, reflected across the symmetry axis
shown.
[0130] Because the light incoming to movable optical assembly 100
is parallel, the movable optical assembly 100 can be moved
laterally without changing the angles of beam propagation within
the movable optical assembly 100, or the focusing properties
thereof. The lateral motion will, as a result, move the line focus
laterally, such that the line may be scanned left and right to move
the line foci laterally within the sample. Thus, the scanning
direction may be lateral, in the plane of the paper. That is,
movable optical assembly 100 may be scanned horizontally (in the
plane of the paper) in order to shift the line focus 210 laterally
through the sample. This function is described more thoroughly with
respect to FIG. 9.
[0131] An optical element 51 is also shown in FIG. 5. This
component may be a rotatable component 51 which may be equipped
with a number of apertures having a certain shape that pass the
incoming radiation and define its optical properties. The slotted
aperture 51 is shown in more detail in FIG. 6. For example, the
slotted aperture 51 may define the optical numerical aperture of
the system, as described in detail below with respect to FIGS. 6, 7
and 8. As indicated, the slotted aperture 51 may be rotated quickly
to adapt the numerical aperture to different values within the
device.
[0132] The rotatable aperture 51 may be used to select the Gaussian
beam properties. In particular, as the rotatable aperture is
rotated to intercept a larger portion of the beam and allow a
smaller portion to pass, has the effect of defining a smaller
numerical aperture to the system.
[0133] The rotatable aperture 51 may also be able to block or
disable one, two or all three of the beams of light being reflected
from any of the turning mirrors 61, 62 or 63. This selection may be
performed by rotating the rotatable member 51 to a position where
one, two or three of these parallel light beams is blocked. The
rotatable member 51 may generally be oriented in a direction
perpendicular to optical axes of the beams reflected off turning
mirrors 61, 62 and 63 and thus can be used to select (or turn off)
any of 61, 62 or 63.
[0134] Also shown in FIG. 5 is a coordinate system that applies to
this figure and the figures in general. The axes in the coordinate
system, x, y and z, are orthogonal with respect to one another. The
x-axis is generally the scanning dimension, that is, it is the axis
along which the line focus will be scanned. The y-axis is generally
the direction of the line focus, that is, it is the direction that
the focus lies along, and along which the radiation intensity is
relatively uniform. However, it should also be understood that the
scanning of the line focus may also be performed in the
y-direction, that is, in the same direction as the length or extent
of the line, rather than perpendicular to this dimension. The
z-axis is generally the viewing direction. That is, the camera
and/or detector will lie above the x- y-plane, along the z-axis. It
should be understood that these orientations are arbitrary, as are
designations such as "left," right," "up," "down," and "top,` and
"bottom" and refer only to opposing or obverse sides. The device
may be held in any orientation without loss of generality.
[0135] FIG. 6 shows the rotating slotted aperture 51 in greater
detail. As shown in FIG. 6, the rotating member 51 may be formed
with a plurality of rectangular through holes 52 formed therein.
The rotating member 51 may further be equipped with a plurality of
intersecting slots 73. By rotating these through holes 52 and slots
73, the clearance available to a ray of light is expanded or
diminished. In other words, a shadow is effectively cast by the
incoming aperture which serves to stop down the light transmitted
through the structure. This effect is shown in the cross section of
FIG. 6b. Because of the blocking effects of the apertures in the
rotating slotted aperture 51, The radiation may be restricted in
the z-direction, thus defining the numerical aperture of the system
when the beam is focused into a line. These effects are described
further below.
[0136] The rotating member 51 may be adjusted quickly by a single
motorized axle, which may rotate the rotatable member 51. Because
the plurality of apertures may be provided for each of the
plurality of beams, the optical properties of the overall system
may be defined quickly and inexpensively using a single actuator or
a rotating stepper motor, for example. The turning mirrors 61, 62
and 63 may direct the radiation into the appropriate aperture 52 or
slot 73.
[0137] The cross section shown in FIG. 6b shows the effect of
rotating the rotatable component 51. As the member 51 is rotated,
the leading edge of the aperture 52 obscures the beam path,
blocking the upper portion of the beam from being transmitted
through the aperture 52. This then truncates the downstream portion
of the beam to a narrower portion than was incident on the
rotatable member 51. The effect may be to decrease the numerical
aperture of the optical system.
[0138] As illustrated in FIG. 6, the rotating member 51 may be a
cylinder with cutouts for slots, and the cylinder may be rotated to
define numerical aperture of the beam. By rotating the slotted post
51 as a unitary body, the beams sizes for all the three of the
laser beams coming off turning mirror 61, 62 and 63 may be defined
with one action. In other words, a single motorized stage or
stepper motor (not shown) may be required to define the numerical
aperture for all three beams. Because of its cylindrical geometry,
the rotating member 51 may have little rotational inertia so beam
switching can be very rapid. The slots that obscure parts of beam
may also eliminate non-uniform fringes. A telescoping cylindrical
lens 70 may determine the image size and illumination area, as will
be described below with respect to FIG. 9. As with many of the
optical holders and stages, the rotating member may be formed of
anodized aluminum, by machining for example.
[0139] It should be noted that the beam path lengths from turning
mirrors 61, 62 and 63 are all different, as shown in FIG. 5.
Accordingly, the light source 500 may need a limited coherence
length to avoid interferometric effects such as constructive or
destructive interference. Within the rotatable aperture 51, wedged
plates (not shown) may be moved to reduce coherence time below
camera exposure.
[0140] In another embodiments, a set of simple adjustable slits may
be used which may be independently adjustable. Use of the slits or
some other selectable aperture may also reduce the amount of
radiation applied to the sample so reduces heating and bleaching
but primarily determines the numerical aperture and thus the
quality of the imaging (resolution, step size, etc.). However, by
putting the features all on a single post makes the performance and
optical attributes selectable by a single actuator or motor.
[0141] Many beams such as laser beams emitting in the TEM 00 mode
have beam profiles that have a Gaussian intensity distribution.
This may be the case with the optical system described here. FIG. 7
illustrates the beam intensity versus lateral position within the
beam. The shape of the intensity curve shown in FIG. 7 is intended
to show a generally Gaussian curve, wherein the width of the
profile is characterized by the parameter b. The parameter b on the
curve indicates the point at which the intensity drops to 1/sqrt(2)
of the peak intensity. As is usual, the beam reaches peak intensity
near the center of the distribution, and drops off with some
characteristic shape to a value 1/sqrt(2) at a distance b from the
center. b is referred to as the confocal parameter.
[0142] The geometric dependence of the fields of a Gaussian beam
are governed by the wavelength .lamda. of the radiation (in the
dielectric medium, not free space) and the following beam
parameters, all of which are connected as detailed in the following
sections. The Gaussian beam width w(x) is a function of the
distance along the x-direction in which the beam propagates.
W.sub.0 is the beam waist and b is the depth of focus. The
properties of Gaussian beams are well known, and these properties
are summarized here in order to introduce parameters that will be
referred to in the discussion of the design and operation of the
system, which follows.
[0143] The shape of a Gaussian beam of a given wavelength .lamda.
is governed solely by one parameter, the beam waist w.sub.0. This
is a measure of the beam size at the point of its focus (x=0 in the
above equations) where the beam width w(x) (as defined above) is
the smallest (and likewise where the intensity on-axis (r=0) is the
largest). From this parameter the other parameters describing the
beam geometry are determined. This includes the Rayleigh range
x.sub.R and asymptotic beam divergence .theta.. These quantities
are illustrated in FIGS. 8a and 8b.
[0144] The Rayleigh distance or Rayleigh range x.sub.R is
determined given a Gaussian beam's waist size. Here .lamda. is the
wavelength of the light in the medium of propagation. At a distance
from the waist equal to the Rayleigh range x.sub.R, the width w of
the beam is larger than it is at the focus where w=w.sub.0, the
beam waist. That also implies that the on-axis (r=0) intensity
there is one half of the peak intensity (at x=0). This point along
the beam also happens to be where the wavefront curvature (1/R) is
greatest. The numerical aperture of a Gaussian beam is defined to
be NA=nsin .theta., where n is the index of refraction of the
medium through which the beam propagates and .theta. is the
divergence angle. This means that the Rayleigh range is related to
the numerical aperture by x.sub.R=w.sub.0/NA. The distance between
the two points x=.+-.x.sub.R is called the confocal parameter b, or
depth of focus of the beam. Gaussian beams are described in, for
example, https://en.wikipedia.org/wiki/Gaussian_beam.
[0145] Accordingly, the numerical aperture NA.about.2w.sub.0/b. The
smaller the NA, the larger the confocal parameter for a given beam
waist. If more resolution is needed, the confocal parameter b must
be smaller so the NA must be larger and the depth of field shorter.
These properties may be defined by the position of the rotatable
member 51 in selecting a numerical aperture. If instead, better
contrast is needed and extended focal length, to get better
contrast over field of view, a lower magnification and larger
confocal parameter b may be selected (smaller NA). The NA aperture
may also be tailored to increase the useful area of the detector.
Accordingly, the rotatable member 51 may be used to extend focal
length, to get better contrast over field of view by lower
magnification (lower NA), and to increase the useful area of the
detector. The rotatable member 51 may be used to rapidly adjust the
width and numerical aperture of the system.
[0146] Accordingly, the effect of a tighter focus is a shorter beam
waist and larger divergence angle .theta., in other words, a
shorter depth of focus and higher resolution. Tighter focusing is
associated with a larger numerical aperture NA. Accordingly, a
larger numerical aperture NA implies a smaller confocal parameter
b. Thus a more tightly focused beam can give better resolution but
over a shorter distance.
[0147] FIG. 7 is an illustration of the intensity of the line focus
as a function of the lateral distance from the center of the focus.
A typical light intensity profile for successive scans, may
generally have a Gaussian shape and follow Gaussian optics as
discussed above. However, other functions may also be used, such as
for example, Bessel and Lagrangian shapes.
[0148] The width of the line focus b and separation .DELTA.x
between scans may be related and chosen based on attributes of the
sample (size, thickness, density, etc.), and based on performance
considerations. In general, the higher the resolution, the smaller
the step size and the longer the time required to complete a scan.
In addition, the sample will be subjected to higher excitation
intensities and thus higher temperatures, bleaching and
photodamage. In other words, these variables may be related, such
that design tradeoffs may need to be made. A given choice of
numerical aperture may determine the step size and resolution of
the scanned image. These parameters and design tradeoffs are
discussed further below with respect to FIGS. 12 and 13. The choice
of NA which determines the focusing attributes may also determine
step size, acquisition speed, and other important system level
operational attributes.
[0149] FIG. 8a is an illustration of the intensity of the line
focus as a function of lateral distance, and an illustration of the
beam waist and Rayleigh distance for a first numerical aperture
NA.sub.1. FIG. 8b is an illustration of the intensity of the line
focus as a function of lateral distance, and an illustration of the
beam waist and Rayleigh distance for a second numerical aperture
NA.sub.2, wherein NA.sub.2>NA.sub.1. As mentioned previously,
the Rayleigh length is determined by the waist radius wo and the
wavelength .lamda..
[0150] There can thus be a trade-off between a more strongly
focused beam with higher optical intensity in the focus, and a less
strongly focused beam with longer Rayleigh length, i.e. larger
depth of focus. The reduced NA may be perpendicular to light sheet.
Choosing a wider slot increases the numerical aperture of the beam,
which decreases the confocal parameter b and increases the
resolution. Conversely, choosing a narrower slot may decrease the
confocal parameter b, and reduce the resolution. As mentioned
previously therefore, the choice of numerical aperture may drive
the spacing between scans .DELTA.x and thus the speed of image
acquisition and maximum sample irradiation intensity. In view of
this, the choice of numerical aperture chosen with the rotating
member 51 may be a central design choice.
[0151] FIGS. 9 and 10 are embodiments exemplifying an important
aspect of the light sheet microscope described here. This aspect is
the ability of the movable optical assembly 100 and 101 to move
laterally with respect to the sample. Because of the line focusing
ability of optical assemblies 120, 140 and 160, all of which focus
the radiation into substantially a single line focus, this line
focus may be moved laterally back and forth by moving the movable
optical assembly 100 and 101 back and forth. It should be
understood that a wide variety of arrangements of optical
components may exist that can render this feature, of which movable
optical assemblies 100 and 101 are but two examples. These
embodiments and many others are contemplated, which have this
capability and a plurality of optical arrangements that accomplish
this purpose are encompassed by the appended claims.
[0152] FIG. 9 is an illustration showing the lateral horizontal
movement of the light sheet within the biological sample. Beam
shaping may also be done by means of slits or telescopes,
independent of the z-direction, to adjust the y-extent of the beam.
A telescoping lens 70 shown in FIG. 9 may be used to magnify or
expand the illumination area, by expanding the line focus 210 in
the y-direction. In other words, magnification of the line focus,
or rather expanding its lateral extent, may be adjusted by moving
an upstream cylindrical telescoping lens 70 back and forth.
[0153] In other embodiments, the magnification may be determined
not only by one cylindrical lens, but by a proper optical
telescoping lensing system. However, the cylindrical lens 70 may
have the advantage that it is simple to implement, and spherical
errors in y may not impact performance substantially.
[0154] It should be understood that beams using different laser
wavelengths may still be imaged using this optical system because
the components are all achromatic. Accordingly, the line foci will
still overlap because the focus is not a property of wavelength.
The foci will all still overlap along a substantial portion of the
length of the line, to approximately the diffraction limit of the
focus. In optical assembly 100, the material of lenses may also be
chosen (and different) to reduce chromatic aberration. Excitation
using different color lasers is discussed further below with
respect to FIG. 20. Detection colors may also be selected with the
appropriate choice of optical filters in front of the detector, as
discussed below with respect to FIG. 19.
[0155] The whole movable optical assembly 100 (or all the elements
included in movable assembly 100) can be moved back and forth along
the axis shown in the x-direction, to scan the light sheet through
the sample. This scanning direction effectively defines the x-axis.
Because of the need to move this assembly laterally without
altering the focal conditions within the sample, the turning
mirrors that direct the light into the movable assembly 100 may be
configured so as to deliver the light in a direction parallel with
the movement direction of movable assembly 100. This situation is
illustrated in FIGS. 9 and 10.
[0156] It should be understood that any and all optical elements
mentioned here, including the telescoping lens 70, the rotatable
structure 51, turning mirrors 61, 62 and 63, and optical assemblies
120, 140 and 160 that appear in FIG. 9 on the left hand side of the
biological sample 200 may also be duplicated and disposed on the
right hand side of the sample 200. Accordingly, the sample may be
illuminated from the left, from the right or from both sides. This
system is described below with respect to FIG. 11. The components
on the left hand side of light sheet system are denoted by 100. The
components on the right hand side of light sheet system are denoted
by 100'.
[0157] Accordingly, as shown in FIG. 9, the movable optical
assembly 100 (or 100') may be moved by a distance .DELTA.x. This
results in the movement of the line focus 210 by an amount similar
or identical to .DELTA.x, as shown.
[0158] FIG. 10 illustrates a two embodiments 100 of a movable
assembly, 100 and 101. In the first embodiment 100, similar to
movable optical assembly 100 of FIG. 9, optical turning elements
are included that can enable the entire optical assembly to be
moved laterally toward and away from the biological sample. The
effect of this movement of imaging optics is to move the line focus
210 laterally but in a plane defined by the movement of the optical
assembly 100. The movable stage therefore generates the lateral
horizontal movement of the light sheet within the biological sample
for the imaging device.
[0159] Embodiment 100 shown in FIG. 10a uses three turning mirrors
61, 62 and 63 to deliver three parallel beams into movable assembly
100. The top beam is redirected by other turning mirrors 74 and 68
and into the optical subassembly 120. Similarly, the lower beam is
redirected by two other turning mirrors 64 to 66 and into the lower
optical assembly 160. The third beam may enter optical subassembly
140 straight on.
[0160] In the first embodiment 100 of movable optical assembly
shown in FIG. 10a, turning mirrors 61, 62 and 63, are used to
direct the radiation from source 500 into the optical subassemblies
120, 140 and 160. Mirror 61 turns the radiation about 90 degrees
and into the movable assembly 120. Mirror 62 turns the radiation
about 90 degrees and into the movable assembly 140. Mirror 63 turns
the radiation about 90 degrees and into the movable assembly 160.
With the movable assembly 100, turning mirrors 74, 68, 64 and 66,
direct the beams into optical subassemblies 120 and 160,
respectively. For optical subassembly 140, the radiation may enter
directly from turning mirror 62. Mirrors 74, 68, 64 and 66, may be
analogous or identical to mirrors 53, 54, 56 and 55 in FIG. 5.
[0161] In another embodiment 101 of movable optical assembly shown
in FIG. 10b, turning mirrors a single turning mirror 61 may be used
to direct a single beam into the moving optical subassembly 101.
Mirror 61 turns the radiation about 90 degrees and into the movable
assembly 100. Within the movable optical assembly 101, partially
reflective/partially transmitting mirrors 74, 65, 67 and 69 may be
used to direct the radiation from source 500 into the optical
subassemblies 120, 140 and 160. More specifically, movable assembly
then uses turning mirror 74 inside movable optical assembly 101 to
direct the beam onto the partially reflecting and partially
transmitting mirrors 65, 67 and 69. Each of these partially
reflecting and partially transmitting mirrors 65, 67 and 69 then
direct the radiation into the optical subassemblies 120, 140 and
160. Because these subassemblies 120, 140 and 160 are disposed at
angles with respect to the symmetry axis and center optical
subassembly 140, the angles of the three partially reflecting and
partially transmitting mirrors 64, 65 and 66 are all different, as
shown in FIG. 10b.
[0162] As mentioned, the second embodiment of movable optical
assembly 101 may use the single turning mirror 61 to deflect the
radiation from the source 500 into the movable assembly 101. This
embodiment may have fewer turning mirrors than the embodiment shown
in FIG. 9, and may thus be cheaper or easier to align. However, as
with embodiment 100 above, optical turning elements are included
that can enable the entire optical assembly to be moved laterally
toward and away from the biological sample. The effect of this
movement of imaging optics is to move the line focus 210 laterally
but in a plane defined by the movement of the optical assembly 101.
The movable stage therefore generates the lateral horizontal
movement of the light sheet within the biological sample for the
imaging device.
[0163] FIG. 11 is an illustration of a larger portion of a light
sheet microscope according to this invention. Illustrated in FIG.
11 is a first movable optical assembly 100 on the left hand side
and a second movable optical assembly 100' on the right hand side.
It should be understood that this movable optical assembly 100 may
be that illustrated in FIG. 9, or it may be that illustrated in
FIG. 10a or 10b, or it may be another embodiment capable of moving
the substantially single line focus laterally within the biological
sample. Also shown in FIG. 11 is a second movable optical assembly
100' disposed on the opposite side of the biological sample 200 and
cuvette 300. Accordingly, the biological sample may be illuminated
from the left by movable optical assembly 100 and from the right by
movable optical assembly 100'.
[0164] As will be explained in greater detail below, an image may
be created by collecting a plurality of camera images each with the
line focus 210 in a different location within the sample. At the
beginning of image acquisition, a partially transmitting turning
mirror 57 may direct radiation from a source 500 to a mirrored
shutter 58, which may redirect the source radiation 500 into the
first movable optical assembly 100. Movable optical assembly 100
may focus the radiation in a substantially single line focus. Then,
by moving optical assembly 100 from left to right, the far left
edge of the biological sample may first be illuminated, and then
the line focus moved successively rightward until the line focus
reaches the middle of the sample. At this point, a shutter 58 may
be moved or retracted to allow the radiation to pass over to the
right hand movable optical assembly 100'. The sample may from that
point onward be illuminated by the right hand movable optical
assembly 100'. After collecting each of these successive images, a
single image may be constructed from these individual scans.
[0165] Accordingly, illumination may come from either side to
minimize the amount of sample material the light must penetrate.
Which of the two sides 100 or 100' is operative may be selected by
a shutter, or a flip mirror 58. Preferably, the shutter or flip
mirror 58 is located on the symmetry axis, as shown, so that the
path lengths on the right and left sides are similar or
identical.
[0166] It should be understood that right hand movable optical
assembly 100' is largely the mirror image of left hand movable
optical assembly 100. That is, the angles of the turning mirrors
60' (in 100') may be the same as turning mirror 60 but reflected
across the symmetry axis shown in FIG. 11. The components belonging
to mirror image optical assembly 100' are designated by the prime
(') to distinguish them from the left hand movable optical assembly
100. It should be understood that many other switching arrangements
may be employed to send the radiation from source 500 to either the
left hand side 100 or the right hand side 100'. These alternative
optical arrangements are a design choice, and a plurality of
optical arrangements may exist that accomplish this purpose of
routing the beams in the desired directions. These alternative
arrangements fall within the scope of the appended claims.
[0167] FIG. 12 is an illustration describing the correction for
refraction of the light beams at the boundary of the transparent
liquid vessel 300. The transparent liquid vessel 300 may be glass
or quartz, for example. Because of the architecture of the light
sheet microscope system as described here, some of the beams of
light must necessarily enter the cuvette 300 via the transparent
window 360 and 361, with a somewhat oblique angle of incidence
.beta.. Accordingly, passing into the cuvette 300, the radiation
must pass several boundaries, and so is refracted at a different
angle than was incident. It should be understood that the largest
refractive effect may occur at the outside air/glass boundary, as
this boundary separates materials with the most dissimilar
refractive indices.
[0168] As is well known from Snell's law, refraction of the light
will then occur at the boundary between materials, such that the
oblique light enters the cuvette with a somewhat shallower angle
than it enters. Accordingly, because of refraction at glass and
liquid boundaries, the focus of inner, central beam 140 will occur
at a different spot in x-direction relative to outer beams 120 and
160. In other words, if the angle between the upper leg and the
lower leg is 2.beta., with which the radiation enters the
transparent window 360 and cuvette 300, the radiation may exit with
a different angle between the upper and lower legs. In general, the
exit angle is somewhat smaller than the entrance angle, 2.beta.-.
Accordingly, the overlapping line foci will take place at a
slightly longer distance D from the transparent window as it would
have had refraction not occurred.
[0169] For this reason, it may be advantageous to displace or shift
the optical assembly 100 and 100' by an amount to accommodate this
change in focal length. The amount of the shift D can readily be
calculated using basic optical principles such as Snell's Law. For
example, N=1 outside cuvette, n=1.5 inside. Sin a/sin b=1.5. So if
the original angle .alpha..sub.1 is 16 degrees, the exit angle may
be closer to 11 degrees, and focus may occurs at about n.times.d
away from nominal focus. Accordingly, it may be important to retard
the placement of the movable optical assembly 100 by this amount,
to assure that the line focus falls within the biological sample
200 as intended.
[0170] Alternatively, the components 120 and 160 may be staggered
with respect to component 140 in order to accommodate the change in
angle of their obliquely incident light. Accordingly, it may be
possible to advance middle optics 140 relative to outer optics 120
and 160 by an amount to account for refraction at the material
boundaries.
[0171] These same operations may be performed on the components in
the right hand side optical assembly 100'.
[0172] Alternatively, the tilt of the elements 120 and 160 may be
adjusted to compensate for this offset, such that lines overlap
perfectly at the line focus 210, or substantially to the
diffraction limit, which is to say within about 5.times. of the
diffraction limit.
[0173] In some embodiments, the software running the controller may
be told what the index of the transparent window 360 and/or the
fluid 1000 is, in order to shift image by an appropriate amount.
This may be especially important in blending of the left hand side
and right hand side illuminated scans.
[0174] Because the biological sample 200 may be immersed in a
clearing fluid 1000, the sample 200 and clearing fluid 1000 may be
contained in a cuvette 300 as mentioned previously. Since the
radiation being must pass into the cuvette, at least a portion of
the cuvette may be made from a transparent material such as glass
or quartz. The transparent material will have a different index of
refraction as compared with air. Glass for example has an index of
refraction of about 1.5, as compared to the index of refraction of
air, which is about 1.
[0175] Because of the difference in refractive index between the
two materials, refraction of the light may occur, resulting in
changes to the focal characteristics of the light beam. A
correction for this effect may be made, as described above.
[0176] The discussion now turns to the computational aspects of the
light sheet microscope.
[0177] Image Formation and Restoration
[0178] The discussion now turns to image processing techniques
which may be used in combination with the light sheet microscope
described above to obtain high resolution, three dimensional images
of a biological structure.
[0179] FIG. 13 is an illustration showing the pixelated detector
which captures the image produced by the light sheet microscope
from the biological sample. FIG. 13 shows the relationship between
the line focus 210 and the detector field of view 310. As shown,
the line focus may be generated at an angle with respect to the
optical axis. The detector may be a pixelated detector such as a
CMOS or CCD camera. The figure also shows the relationship between
the pixelated detector, the line focus and the coordinate system
used in the figures to follow.
[0180] FIG. 13 shows a line focus generated at an angle with
respect to the optical axis of optical assembly 120 or 160. The
y-dimension is generally in the direction of the line focus, and
along that line. The x-direction which is the direction of the
lateral scanning, is orthogonal to the y-direction. The camera is
generally positioned such that the sample image falls generally
near the center of the field of view of the detector, in order to
efficiently use the pixel area within the detector. The optical
axis of the objective lens of the imaging microscope is aligned
along the z-axis and orthogonal to both x- and y-directions.
[0181] The data acquired by pixelated detector 600 may include
blur, distortion, and/or optical aberrations, and the blur,
distortion, and/or optical aberrations may be a repeatable
characteristic of the complete optical system. The excitation
(lasers) may exhibit its own unique and characteristic distortions,
such that a part of the recorded image's blur is caused by the
intensity distribution of the collimated laser sheets
.LAMBDA..sub.1 which is not completely flat but a 3D-intensity
distribution, see FIG. 15a. Further the x/y-plane defined by the
geometry of the excitation lasers may not be exactly perpendicular
to the microscope turret's z-axis and consequently does not align
to the microscope's focal plane (the term focal plane means the
focal plane of the microscope on the side of the sample to be
magnified), see FIG. 15c for perfect alignment. Further unique and
characteristic distortions of the recorded image are caused by the
microscope, which exhibits in general a function .LAMBDA..sub.2
that maps the observed 3D-fluorescence distribution traveling
towards the microscope to a 2D image on the surface of the
pixelated detector (see FIG. 15d: "microscope image"), whereat that
mapping function .LAMBDA..sub.2 may change its shape or orientation
across the x/y-plane (as shown in FIG. 15b) which means that it is
spatially varying and as such it rules out a simplistic numerical
deconvolution. Finally this 2D microscope image is sampled and
converted by the pixelated detector 600 which may come with its own
610 characteristic point spread function (PSF) .LAMBDA..sub.3.
[0182] It should be understood that the term "point spread
function" is used interchangeably herein with the term "intensity
distribution function" and "excitation distribution function". Each
of these terms should be understood to refer to a measurable and
repeatable noise source within the imaging system. Furthermore, the
term "imaging system" should be understood to may an optical system
that can form a two dimensional image of an object. Examples of
imaging systems are optical lensing system that form an image, or a
microscope for example.
[0183] Knowledge of the functions .LAMBDA..sub.1, .LAMBDA..sub.2,
.LAMBDA..sub.3 can be very useful because these reproducible
distortions may be removed from the final raw image I recorded by
the pixelated detector using a numerical 3D restoration process
resulting in the restored image F, see FIG. 14. This goal may be
pursued by employing e.g. an iterative maximum-likelihood
optimization process. Alternatively, artificial intelligence driven
processes may eliminate blur, distortion, and/or optical
aberrations from recorded a raw image I merely on a data driven
approach that may not need specific knowledge the of the functions
.LAMBDA..sub.1, .LAMBDA..sub.2, .LAMBDA..sub.3. Also a mixture of
both approaches may be beneficial, especially when the functions
.LAMBDA..sub.1, .LAMBDA..sub.2, .LAMBDA..sub.3 are known only
approximately. As the functions .LAMBDA..sub.1, .LAMBDA..sub.2,
.LAMBDA..sub.3 constitute a part of the total system matrix A the
system matrix itself may also be determined by a maximum likelihood
optimization of A parallel to the restoration of the raw image I. A
simplistic approach to correct the raw image I is a deconvolution
process, which may be carried out in the frequency domain.
Deconvolutions and the more sophisticated methods from above are
well known, and the specific details will not be set forth here.
The discussion to follow is directed primarily to the acquisition
of the functions .LAMBDA..sub.1, .LAMBDA..sub.2,
.LAMBDA..sub.3.
[0184] FIG. 14 shows a schematic illustration of the excitation
intensity distribution .LAMBDA..sub.1, the microscope mapping
function .LAMBDA..sub.2 and the pixelated detector's PSF
.LAMBDA..sub.3. The blur, distortion, and/or optical aberrations
are shown schematically simply as boxes, to indicate that an
incoming signal is altered or transformed in a predictable and
reproducible way as effect of the functions .LAMBDA..sub.1,
.LAMBDA..sub.2, .LAMBDA..sub.3. To the extent that .LAMBDA..sub.1,
.LAMBDA..sub.2, .LAMBDA..sub.3 can be ascertained or measured, as
described next, these predictable alterations can be removed from
the raw image I by the controller 400 by one of the aforementioned
processes. The functions .LAMBDA..sub.1, .LAMBDA..sub.2,
.LAMBDA..sub.3 may be calculated numerically by controller 400
based on the data acquired through the lenses and detector.
Controller 400 may also perform the restoration process employing
these functions .LAMBDA..sub.1, .LAMBDA..sub.2, .LAMBDA..sub.3. The
result will yield the corrected image F, as shown in FIG. 14.
Because .LAMBDA..sub.3 generally occurs in combination with
.LAMBDA..sub.2, in some instances only .LAMBDA..sub.2, will be
mentioned. However, depending on the context, .LAMBDA..sub.2, may
be understood to also include .LAMBDA..sub.3.
[0185] As shown in FIG. 14, the functions .LAMBDA..sub.1,
.LAMBDA..sub.2, .LAMBDA..sub.3 are produced by the light sheet
microscope (laser optics, microscope optics, pixelated detector)
and contribute to the data collected and displayed as the raw image
I from the biological sample. FIG. 14 illustrates the controller
400 which may control image acquisition, image processing, and
movement of the movable optical assemblies 100 and 100'.
[0186] FIG. 15 portrays the different steps of image formation.
FIG. 15a shows a cross section in the x/z plane that shows two
iso-intensity lines of a laser sheet. This laser sheet will give
rise for fluorescence when hitting an appropriate sample. The
movable optical assemblies 100 and 100' however will produce
several light sheets and each of them may initially not be oriented
perfectly horizontally, but may be tilted along the x-axis or
y-axis or both. Further, the initial alignment of the individual
light sheets may be suboptimal. FIG. 15b shows an example of the
microscope's mapping function .LAMBDA..sub.2, here depicted as a
series of double-cones whose tails, depicted as the points where
the double-cones are joined together, in general define the
microscope's focal plane. In FIG. 15b the focal plane really is
depicted flat, generally it may however deviate from a plane and
exhibit some curvature. The focal plane defines the working
distance for sample structures to be imaged with the microscope's
highest resolution. FIG. 15b also shows an example for the spatial
variation of the mapping function by different orientations of the
double-cones. In general, also variations of the cone-shape may
occur. FIG. 15c shows how the microscope's focal plane and the mean
layer of the light sheet are aligned in order to match the plane of
highest microscopic resolution with the plane of highest
irradiation intensity.
[0187] FIG. 15d sketches the image formation employing some
pointlike particles 750 at different positions that are irradiated
by the laser and that consequently emit fluorescence light. It is
useful to understand the microscope's imaging process as if it maps
the fluorescence light originating from a specific point-like
particle 750 in the 3D-sample space to a blob or to a ring-like
area in its 2D-image plane (the term image plane means the focal
plane of the microscope on the side of the image generated by the
microscope). If the particle lies in the focal plane, the sharpest
part of the mapping function .LAMBDA..sub.2 will give rise to a
mapped blob, whereas a point-like structure away from the focal
plane of the microscope will be sampled by the cone-wall-like part
of the mapping function resulting in a mapped ring in the
microscope's image plane. For point-like structures at the side of
the optical axis of the microscope the mapping function may be
tilted and consequently a particle outside the microscope's focal
plane will be mapped to an ellipse. In order to generate digital
images a pixelated detector may be used to detect the image formed
by the microscope on the surface of the pixelated detector. With
some simplifications a constant PSF .LAMBDA..sub.3 may be
attributed to the pixelated detector resulting in some spatially
invariant blur in the resulting recorded final raw image I.
[0188] Points are not imaged to points because of optical
imperfections, scattering and finite resolution (diffraction
limit). Furthermore, beyond the line focus of the laser, the laser
light diverges and excites fluorescence in an increasingly broader
z-range of the sample, which is imaged by the microscope and
introduces additional blur due to superimposing adjacent z-layers
of the sample to the microscope's image which is recognized as a
loss of resolution.
[0189] Accordingly, the system may measure point spread function of
excitation by moving excitation laterally and vertically. Each of
these individual scans may be used to 1) contribute a subset of
pixels to the composite total raw image I.sub.C, and 2) be used for
computation of the PSF. These two techniques may be applied to each
lateral scan (x-direction), and may also be applied to each scan
taken step-wise through the depth (z-direction) of the sample.
[0190] FIG. 16 is a schematic diagram illustrating how an image is
assembled from a plurality of scans taken with different
illumination conditions. Starting to move the movable optical
assembly 100 on the left hand side, a scan is collected digitally
by a pixelated camera with the line focus in a first position. The
line focus is then moved laterally, and another scan is taken. The
data handling of each of these lateral scans is discussed below
with respect to FIG. 17. This continues until approximately the
middle of the sample is reached. At this point, the right hand
movable optical assembly is used to illuminate the sample from the
opposite side. In the middle of the image where the illumination
shifts from the left hand side movable optical assembly 100 to the
right hand side movable optical assembly 100', the scans are
blended electronically using a software algorithm.
[0191] This software algorithm may identify noteworthy pixels or
sets of pixels that provide an identifiable feature in both the
right hand scan and the left hand scan. The scans are then aligned
to this point or to a set of points. The scanned images are then
blended to assemble what will be referred to hereafter as the
"composite raw Image I.sub.C" wherein I.sub.C can be understood to
be a digital file having the blended data associated with each
pixel in the pixelated detector. A blending and offset function may
be applied to the data to improve the smoothness and accuracy of
the match. In the middle transition region from left side optical
assembly 100 to right side optical assembly 100', the contribution
from the side which has been active up until the transition may be
given a gradually reduced weight, whereas the contribution from the
side coming on line is given a gradually increasing weight. This
transition and the functions used are shown in FIG. 16.
[0192] FIG. 17 is an illustration showing the step-wise lateral
horizontal scanning of the light sheet through the biological
sample. For each image, 1, 2, 3, 4 and 5, the line focus 210 is
shifted laterally by translating the movable optical assembly 100.
An image is taken with the line focus as shown in each image.
Although five line foci are shown in FIG. 17, it should be
understood that there may be many more images and many more line
focus positions than just five. The lateral separation (i.e. the
distance between the scans) may be a performance choice that can be
made, based on the numerical aperture selection made as described
above with respect to FIGS. 8 and 9.
[0193] The detector and computer may capture a sequence of images
1-5 as shown in FIG. 17. These raw images I may be used as input
for image restoration and may also be used to calculate the
functions .LAMBDA..sub.1 and .LAMBDA..sub.2. On the other hand,
they may be used as input for assembly of the composite raw image
I.sub.C that is composed of not image-restored stripes of raw image
I sequences. As the image restoration process takes into account,
if available, all sequences of scanned line foci and z-positions
(scanned through the depth of the sample), the assembly of
composite restored images F.sub.C may be performed by the
restoration process itself. These techniques are described
below.
[0194] Firstly, the portions of the image which include the line
focus 210 which is depicted as the cross-hatched portions, may be
assembled strip by strip into the final composite raw image I.sub.C
shown at the bottom of FIG. 17. The composite raw image may
therefore have subsets of each of scans 1-5 included therein. That
portion of the scans 1-5 may be selected whose width is
approximately within the Rayleigh length z.sub.R (see FIG. 16) of
the beam waist. Data outside that range may be ignored in the
formation of the composite image I.sub.C, however this date may
still contribute to the calculation of the functions .LAMBDA..sub.1
and .LAMBDA..sub.2, as described below, or for the image
restoration process. Accordingly, the Rayleigh length may determine
the step size of the sequential scans. The blending of the
transitions may be handled as was the left/right transition shown
in FIG. 16. Performing the blending algorithm over these scanned
areas may result in superior homogeneity. It may be important to
avoid adjusting any parameters during any scan, such that pixels
can be compared and areas matched.
[0195] The width of strip along x (i.e. the width of the
illumination) may depend on the tightness of focus w.sub.0 (see
FIG. 16) and thus on the NA of the optical system. By increasing
resolution (increasing NA and thus increasing the tightness of
focus wo) one narrows the strip and decreases the number of useful
pixels for the composite image. Typical is 10 steps per image with
about 200 pixels per step, resulting in usually 2160 useful pixels
in x-direction (scanning direction). AR is 1/10 or 1/15 or so. If
the aspect ratio 10, there may be only 200 useful pixels/image, for
example.
[0196] In some embodiments, it is advantageous to use a relatively
large numerical aperture and perform a large number of steps. In
other embodiments, a larger Rayleigh length z.sub.R and fewer steps
are more appropriate. Such details will depend on the application
and the type and quantity of data being sought.
[0197] Accordingly, the composite raw image I.sub.C may be
assembled from the individual scans 1-5. As with the center
blending algorithm the composited image may be assembled by
identifying noteworthy features in a scan sequence, and fitting the
data based on the characteristics of the noteworthy feature. Using
this method, a composite raw image I.sub.C may be reconstructed
from a series of sequential, overlapping light sheet scans.
However, each of scans 1-5 may also be used in the computation of
the functions .LAMBDA..sub.1 and .LAMBDA..sub.2, as described
below, or for the image restoration process.
[0198] However, in addition, the entirety of the data from each of
scan 1-5 may also be fed to the computer or controller, because
these multiple images of the same field of view contain information
on the functions .LAMBDA..sub.1 and .LAMBDA..sub.2, namely the
intensity distribution of the laser and the mapping function of the
microscope. In other words, as the line focus 210 is swept over a
field of view, different portions of the Gaussian beam illuminate
different portions of the sample. Accordingly and importantly, ALL
the data collected in EVERY scan may contribute to the calculation
of the function .LAMBDA..sub.2. This data may then be used for the
restoration process that forms from the raw images I the restored
images F.
[0199] In other words, each of the images 1-5 may contain, both
within the cross hatched regions and elsewhere, information about
the functions .LAMBDA..sub.1 and .LAMBDA..sub.2. This includes
portions that are not being significantly illuminated by the line
focus 210. Accordingly, the function .LAMBDA..sub.2 may have a
contributions from the entire first image I.sub.11. It may have
another contribution from the entire second image 112. It may have
yet another contribution from the entire third image 113 and so on.
The total function .LAMBDA..sub.1 that covers the complete spatial
extent of the applied laser intensity distribution from the far
left part of the line focus to the far right part of the line
focus, and the total function .LAMBDA..sub.2 of the microscope
mapping functions over the complete spatial extent of the
microscope's FOV may be derived from all the individual scans. This
technique is then also applied to scans done through the depth of
the sample, as described next.
[0200] FIG. 18 is an illustration showing scanning in the lateral
dimension (x-dimension, FIG. 18a) and scanning in depth
(z-dimension, FIG. 18b) to produce a three dimensional image by the
light sheet microscope from the biological sample. The x-dimension
scanning may be produced by moving the movable optical assembly 100
and 100' laterally in the x-dimension, which has the effect of
moving the line focus 210 laterally across the biological sample
200. The separation between successive scans may be di Furthermore,
di may be related to the beam waist and thus to numerical aperture
and Rayleigh length z.sub.R of the light sheet. Similarly, the
width of the line focus d.sub.2 may also be related to the beam
waist and thus to numerical aperture and Rayleigh length z.sub.R of
the light sheet.
[0201] The sample may then be scanned through its depth in the
z-direction, orthogonal to the focal plane of the microscope. The
depth scanning shown in FIG. 18b may be accomplished by moving the
biological sample up and down in the z-dimension, and then
repeating the lateral scanning just described.
[0202] As was done with the lateral (x-direction) scans, the
composite raw three dimensional image I.sub.C may have subsets of
each of the depth scans included therein. That portion of the scans
may be selected whose width is approximately within the Rayleigh
length of the beam waist. The blending of the transitions may be
handled as was done with the left/right transition shown in FIG.
16. Performing the blending algorithm over these scanned areas may
result in superior homogeneity. Accordingly, all the data from all
the scans is used either in the generation of the composite image
I.sub.C. Alternatively all the data from all the scans,
specifically of the depth-scans could be used for the calculation
of the z-variation of the functions .LAMBDA..sub.1 and
.LAMBDA..sub.2, namely the z-variation of the intensity
distribution of the laser and the mapping function of the
microscope; having accomplished the measurement the functions
.LAMBDA..sub.1, .LAMBDA..sub.2 altogether with .LAMBDA..sub.3, all
the data from all the scans may be used as input for the
restoration process.
[0203] To give an example, an acceptable acquisition speed and
resolution may require on the order of 20 individual scans. If B is
the total width of the image plane, as shown in FIG. 18, then
d.sub.1=B/N, n=1 to N, typically less than 20 individual scans n. N
can be as much as 100 but typically less than 20. sheets should
ideally overlap. d.sub.1 should be chosen keeping in mind the size
of the structures being imaged.
[0204] In some embodiments, the same plane may be addressed several
times. The rest of the sample does not experience sharp focused
light. If one uses a sampling which is too high, photo bleaching
may get relevant because the energy transfer of excitation lasers
light to the sample grows with smaller sampling steps and tighter
laser focus. If one uses a very low number of samples, it becomes
tantamount to using a confocal microscope.
[0205] If a standard confocal microscope is used to address all
layers, the whole sample is illuminated during image acquisition.
As a result, the whole sample is bleached repeatedly during
scanning, generating destructive heat as well. Using the scanning
method of the presented invention however, the sample is
illuminated by the laser specifically only in the plane of interest
which may be moved vertically to the next plane of interest.
[0206] When an image is collected using several scans with the line
focus moving between successive scans, the line foci may be
overlapping. This may give improved performance and accuracy,
because identifiable features within the image can be used to align
the scans accurately before merging them into the final image.
[0207] Finally, one can combine all the data to obtain an overall
description of the optical degradation processes by the functions
.LAMBDA..sub.1 and .LAMBDA..sub.2, namely the z-variation of the
intensity distribution of the laser and the mapping function of the
microscope. It should be noted that each of the three beams in the
movable optical assemblies 120, 140 and 160 exhibits a similar
function .LAMBDA..sub.1 because they have similar optical
properties, but it may be noteworthy to point out the benefit when
the geometric orientation of the three movable optical assemblies
120, 140 and 160 is meticulously aligned.
[0208] The restoration process may yield better a signal-to-noise
ratio (SNR) and fewer blurring and deformation artifacts because
the measured input to the restoration addresses each point in the
sample volume with different excitations in x and z. Accordingly,
the plurality of scans can be seen as a system of coupled linear
transformations of the true unblurred and undeformed
3D-representation of the sample that can be used to set up an
objective function that reaches its minimum for this true
3D-representation of the sample--which describes the usual approach
of minimizing an objective function for getting a restorated image
of a sample.
[0209] For a measurement with a specific sample one may separate
the effect on the optical degradation imposed by the functions
.LAMBDA..sub.1 and .LAMBDA..sub.2, namely the intensity
distribution of the lasers and the mapping function of the
microscope, between the part that originates from the optical
system only and the part that originates due to the influence of
the current sample. In order to do so, it is beneficial to perform
measurements with ideal sample structures as for example glass
beads (nano beads) that are embedded in a preferentially
mechanically stable environment of similar refractive index as in
the measurement of the specific sample, as for example agarose. As
these measurements are performed on point-like objects in a
surrounding environment of constant refractive index, the
excitation light may be regarded as travelling through the probe
without being deformed. As such the functions .LAMBDA..sub.1 and
.LAMBDA..sub.2 resulting out of these measurements plus the
function .LAMBDA..sub.3 (describing the PSF of the pixelated
detector) represent altogether the characteristics of the image
forming process of the laser microscope, however without additional
degradations induced by the biological sample.
[0210] The influence of the sample itself may deviate the exciting
lasers from their primary paths whilst travelling deeper and deeper
into the sample. Consequently portions of the sample may get
illuminated that would stay unexposed if the lasers have travelled
without being interacting with the sample. This deviation of
function .LAMBDA..sub.1 may be disentangled for example by
techniques that compare exposures coming from focal line positions
of the left hand movable optical assembly 100 and the right hand
movable optical assembly 100' that both should have exposed the
same part of the sample, given that there is no influence and
deviation by the sample itself. Concerning .LAMBDA..sub.1, this
deviation may not only be a geometric deformation of the laser
intensity distribution but also an overall attenuation and also so
called shadows caused by an attenuation of certain sample
substructures that appear opaque to the laser light due to
insufficient or impossible probe clearing.
[0211] The influence of the sample itself may change also the
microscope's mapping function .LAMBDA..sub.2 and this deviation in
general will change whilst observing z-planes of the sample that
lie, from the microscope's perspective, deeper and deeper inside
the sample. This is a consequence of the increasing path length
that the emission light has to travel through the sample towards
the microscope. Consequently the mapping function .LAMBDA..sub.2
may be distorted in several ways; firstly the geometry of the
mapping, depicted for example as double-cones in FIG. 15 may be
deformed in a way specific to the sample. Secondly, the
microscope's focal plane may be distorted and a curvature may be
introduced, which means that the positions in x/y/z where the local
mapping function has its tail, depicted as the points where the
double-cones in FIG. 15 are joined together, may change. This
deviation of function .LAMBDA..sub.2 may, alongside with for
example artificial intelligence based approaches, be calculated
during the restorations process itself.
[0212] A way to calculate the sample-induced deviation of function
.LAMBDA..sub.2, (and also the deviation of function .LAMBDA..sub.2)
may be performed by a semi-blind restoration process that starts to
restore the image of the sample by initially using the undisturbed
functions .LAMBDA..sub.1 and .LAMBDA..sub.2, (see previous
paragraphs) for an explanation of how these undistorted functions
.LAMBDA..sub.1 and .LAMBDA..sub.2 could be measured and calculated.
After some iterations, given the numerical method operates
iteratively, the functions .LAMBDA..sub.1 and .LAMBDA..sub.2 may be
themselves be optimized in a way that minimizes another objective
function where the currently achieved image of the sample enters as
a constant input entity. From this stage on, both the image of the
sample and the sample distorted functions .DELTA..sub.1 and
.DELTA..sub.2 may by optimized by an alternating approach. It
should be noted that this description is one among others that can
be used to optimize both the image of sample and the sample
distorted functions .LAMBDA..sub.1 and .LAMBDA..sub.2. In yet
another alternative, totally blind restoration may be performed
without measuring any functions .LAMBDA..sub.1, .LAMBDA..sub.2 and
.LAMBDA..sub.3 at all. However this is very time consuming and may
not deliver a meaningful result at all, as it is in general a
non-convex optimization problem that may end up in a local minimum
rather than the global minimum. Therefore also in blind restoration
processes one usually makes some appropriate assumptions of how the
functions .LAMBDA..sub.1, .LAMBDA..sub.2 and .LAMBDA..sub.3 may be
structured. If these assumptions are derived from measurements and
calculation of undisturbed functions .LAMBDA..sub.1 and
.LAMBDA..sub.2 according to previous paragraphs (a starting
assumption for function .LAMBDA..sub.3 of the pixelated detector
may also be given), one ends up for example in the semi-blind
restoration process described above.
[0213] The discussion now turns to the mechanical and optical
aspects of the light sheet microscope.
[0214] Optical Sources and Alternative Optical Paths
[0215] FIG. 19 is an illustration showing additional optical
components which may be advantageous for the functioning of the
light sheet microscope system. The optical path may include at
least two wavelength or color selectors. First wavelength selector
may be a filter wheel 550 on the source of the radiation 500. The
second wavelength selector may be another filter wheel 660. on the
detection path. The first filter wheel 550 may select or choose the
working wavelength for the light sheet microscope excitation and
detection. Ordinarily detection is perpendicular (along vertical
axis) to excitation but not necessarily.
[0216] A filter wheel may be an assembly of apertures arranged in a
circle and covered by a material that filters some wavelengths of
light while allowing other wavelengths to pass. Different apertures
may be covered by different materials, and therefor transmit
different wavelengths. Accordingly, the transmitted wavelength may
be selected by rotating the filter wheel, to place different
apertures in the beam path. One filter wheel 550 may be used after
the source 500 and another filter wheel 660 may be used in front of
the detector 600.
[0217] FIG. 20 is an illustration showing some exemplary optical
sources for the light sheet microscope excitation and detection. In
one embodiment, a white light source 500 (FIG. 20a) can be used,
and filter wheel 550 that then selects, for example, any of eight
or so constituent wavelengths. Filter wheel 560 may have 8 colored
filters, and may be used to serially select which wavelength to
view by rotating filter wheel 550. The filter wheel 550 may have
settings capable of passing important radiation lines, well known
in fluorescence microscopy. These lines may include the six
excitation lines at 488, 515, 553, 591, 640, 785 nm, for
example.
[0218] In other embodiments (FIG. 20b), separate light sources may
be used, for example laser light source 521, laser light source
522, laser light source 523, laser light source 524, and laser
light source 525. Each light source may have an associated turning
mirror which deflects the emitted radiation into an imaging lens
530 which may collimate or focus the radiation and input the
radiation into an optical fiber 510. The optical fiber 510 may
transmit the collimated radiation to another component 540 which
may further modify the radiation and finally into a collimating
lens 580. From collimating lens 580, the radiation may follow the
path shown in FIG. 19. In other words, in the embodiment shown in
FIG. 20, the single source 500 may be substituted with a plurality
of discrete light sources emitting at different wavelengths
521-525. There may be 5 lasers as shown in FIG. 20. The five laser
sources may be coupled by dichroic elements as shown. The laser
outputs may all then be focused by a lens 530 into a fiber optic
cable 510. The diverging fiber optic output may then be collimated
by collimating lens 580. Of course, there may be fewer or more
discrete light sources other than those shown as 521-525.
[0219] Alternatively, the light source may use a laser combiner or
a supercontinuous laser emitting in a very broad band 450-2000 nm.
This range may cover four or more excitation lines. The laser
combiner may be software controlled to add continuously varying
quantity of any color laser.
[0220] In operation, each of the five lasers may be switched on and
off. An acousto-optical modulator may be convenient for this
switching. The five lasers may be high speed, femtosecond pulse
emitters, so as to capture very short events.
[0221] FIG. 21 is an illustration showing the components of a
detection path for the light sheet microscope. The detector may
include an imaging objective lens 620 and another filter wheel 660.
Like filter wheel 550, filter wheel 660 may only pass certain
wavelengths of light. For example, it may be desirable to image all
structures which are emitting at 515 nm for example. In this
situation, the filter wheel 660 may be rotated until the filter
element that passes 515 is placed into the path of the radiation.
The selected wavelength may then be imaged onto the pixelated
detector 600 by a number of other optical components including a
microscope objective lens 670, depending on the application.
Finally, the pixelated detector 600 may measure the light intensity
at the selected wavelength and in the particular pixel element. As
shown in FIG. 21, there may also be a magnifying/demagnifying
element 59, which changes the magnification of the imaged sample
without changing the objective lens 670. It should be understood
that pixelated detector 600 depicted in FIG. 21 may be a similar or
identical to pixelated detector 600 depicted in FIG. 19 and
referred to in FIG. 14.
[0222] Alternatively, the detector may use two or more cameras
which respond to different colors. The detector may alternatively
use a color camera.
[0223] Independent Motions
[0224] One of the major advantages of the light sheet microscope
described here, is that the components can be moved independently
of one another. For example the line foci may always fall in a
single plane defined by the motion of 100 and placement of its
components 120, 140 and 160. This configuration of parts defines
the image plane in which the sample is placed, and the focal plane
defined by the line foci. This plane may remain fixed while many
other components are moved with respect to this plane. Accordingly,
once the plane is established and detection is focused or adjusted
with respect to it, these adjustments may not need to be made
again. This may be true even if the sample is changed, or the
imaging lens is changed, or the clearing fluid is changed. The
movable sample stage may be configured to always hold the sample in
this plane. These advantageous features may be accomplished by
using stages with independently movable stages with respect to the
image plane and arranged as described next.
[0225] FIGS. 22 and 22a, which includes FIGS. 22a, 22b, and 22c,
are illustrations showing a first important mechanical/optical
aspect of the light sheet microscope. A turret 10 may be used which
holds a plurality of objective lenses. The optical axis of each of
these lenses may be parallel. Using the design shown in FIG. 22,
the turret 10 may be rotated and a different objective lens may be
selected, without moving the sample 200 or any of the excitation or
other detection optics. The rotation may be accomplished without
moving the sample or the focal plane of the line focuses. This may
be important for obtaining reliable, reproducible images at a
varying magnification. The movement is accommodated by having the
cuvette 300 shaped with recesses that allow the objective lenses to
penetrate the plane of the biological sample, even when these
lenses are not in use. This allows the imaging of different areas,
with different magnifications, in a manner that is exceedingly easy
and fast.
[0226] For example, a microscope is described which has the three
objective lenses 1, 2 and 3 mounted on a rotating turret 10. The
optical axes of the lenses 1, 2 and 3 may be parallel, as mounted
in the turret 10. One of the plurality of lenses, say lens number
3, may be the imaging lens which is actually in use. The others 1
and 2 of the plurality allow different magnifications and fields of
view to be chosen, but are not in use at the present time. When an
image is taken, the operative lens 3 is lowered into position just
above the biological sample, such that the lens is submerged in the
clearing fluid 1000 and contained within the perimeter of the
cuvette 300. Since the non-operative lenses 1 and 2 are mounted
also to the turret 10 and thus coupled to the same mechanism, these
lenses 1 and 2 are lowered as well. Two cutouts or relieved areas
or curved surfaces or voids 350 and 351 are designed into the
cuvette to allow these lenses 1 and 2 to be lowered as well, but
not to interfere with any other structures in the microscope.
Accordingly, when the operative lens is submerged, the at least one
inoperative lens is not submerged (i.e. is located beside the
container)
[0227] The other cut outs 950 and 951 may be made in the movable
stage 900 which supports the movable optical assembly 100 or 101.
Theses cutouts may also be dimensioned to admit any one of the
lenses 1, 2 or 3. Together, cutout 352 and 952 may admit lens 2 for
example, while cutout 351 along with another cutout in stage 900
(not shown for convenience of rendering) may admit lens 1. The
movable sample stage 800 may be movably placed within the cuvette
300 as shown.
[0228] When a different magnification or field of view is desired,
the turret 10 may be raised until it clears the cuvette 300 and the
turret 10 is then rotated to select a different operative lens.
With the new lens in position, the turret is again lowered into the
cuvette 300 to a position just above the sample. Because of the
relieved areas 350, 351 for lenses 1 and 2, these lenses can be
lowered as well without mechanical interference, because of the
cutouts or relieved areas or curved surfaces or voids 350 and
351.
[0229] FIG. 22a is a plan view illustration showing the ability to
rotate a turret holding a plurality of objective lenses. FIG. 22b
is a perspective illustration of the sample stage 800 and cuvette
300, accommodating the turreted lenses 1, 2 and 3. FIG. 22c is a
perspective illustration of the turreted lenses 1, 2 and 3. The
turret 10 is shown schematically in FIG. 22c, as a rotatable
mechanism to which three lenses, 1, 2 and 3 are attached. As shown
in FIG. 22c, the different lenses may have different physical
dimensions. In particular, one of the lenses 1, 2 or 3 may be
substantially longer than the other two. One may also be wider in
diameter than the others. The cutouts or relieved areas 350 in the
cuvette 300 may be shaped so as to accommodate this dimensions.
Cutout 350 may be circular and may admit the operative lens 3. Cut
outs 351 and 352 may be portions of a circle, and shaped to admit
the otherwise interfering portion of lenses 1, 2 or 3 as was shown
in FIG. 22a. Accordingly, lens selection can be changed without
moving anything but the lens carriage. i.e., without moving sample
200, cuvette 300 or light sheet plane or line focus plane 210. As
mentioned, the other cut outs 950 and 951 may be made in the
movable stage 900 which supports the movable optical assembly 100
or 101. Theses cutouts may also be dimensioned to admit any one of
the lenses 1, 2 or 3. Together, cutout 352 and 952 may admit lens 2
for example, while cutout 351 along with another cutout in stage
900 (not shown for convenience of rendering) may admit lens 1. The
movable sample stage 800 may be movably placed within the cuvette
300 as shown.
[0230] FIG. 23 is another plan view illustration showing how the
light sheet or line focal plane 210 may be moved laterally
independently of the turreted objective microscope. FIG. 23 is
similar to FIG. 22 except that the movable optical assemblies 100
and 100' are also shown in relation to the cuvette 300. The sample
holder 800 is also shown in FIG. 23 and its motion is also shown
diagrammatically. Cut outs 351, 352, 951 and 952 described above
are also illustrated in FIG. 23, as are cuvette 300 and sample
stage 800.
[0231] It should be understood that although the scanning
direction, that is, the direction of lateral motion of the line
focus, is described as being generally in the x-direction, and
therefore orthogonal to the extent of the line focus, the scanning
could also be performed in the y-direction (i.e. parallel to the
extent of the line focus).
[0232] The software may know the focal distance of each lens so may
focus automatically. The focus may be obtained simply by moving the
operational objective up and down in the z-dimension. This is
possible because excitation plane and the sample have not moved
during this process. This capability offers substantial ease of use
for changing samples and ease of use changing lens/imaging. It also
lends itself to robotic, or automated functioning.
[0233] As shown in FIG. 23, the movable optical assemblies 100 and
100' may be moved laterally without moving or interfering with any
other features of the light sheet microscope. The turreted lenses
1, 2 and 3 on turret 10 may also be moved without affecting or
disturbing any other feature of the light sheet microscope. As
described previously, cutouts 350, 351, 352, 951 and 952 may serve
to admit each and any of objective lenses 1, 2 or 3. Lastly, the
motion of the sample holder 800 may also be moved independently,
and which movement is described further below with respect to FIG.
24.
[0234] Accordingly, for a turret with three objective lenses, the
lenses are all allowed to dip into the plane of the sample. The
lens may be raised by a lens raising mechanism, a new viewing lens
rotated into position, and finally lowered into the fluid.
Accordingly, the motion may be raise, then rotate, then lower. The
system therefore fulfils the object to keep the lenses within setup
while they are changed, and without moving any other components.
The turret 10 holds the optical axes of the lenses in parallel and
on its circular, rotating tray.
[0235] The light sheet microscope system may have yet other
independently movable features. It should be understood that the
openings 350, 351, 352, 951, 952, that admit the turreted objective
lenses may be made in the movable optical assemblies 100, 100'' or
they may be made in other solid surfaces of the microscope body,
such as optical platforms and stages. But in any case, theses
openings 350, 950 and 951 are formed in the solid material of the
light sheet microscope in order to allow the movements just
described.
[0236] The light sheet microscope system may have yet other
independently movable systems. The sample stage 800 and cuvette 300
may also be movable independently from the other systems.
Throughout these motions, the light sheet image plane 210 may
remain fixed. This configuration may be ideal for robotic handling
and trays. These capabilities are illustrated in FIGS. 24, 25 and
26.
[0237] FIG. 24 illustrates another independently movable feature of
the light sheet microscope. The cuvette stage 930 which supports
the cuvette 300 with clearing solution 1000 may also be raised and
lowered independently of the movable optical assemblies 100 and
100' the objective lenses 1, 2 and 3 on turret 10, and the sample
200 on sample holder 800. This capability is illustrated in FIG.
24a and FIG. 24b.
[0238] In FIG. 24a, the operative lens 700 is in position just
above the biological sample 200. In this position, the operative
lens 700 is submerged in the clearing fluid 1000. If the clearing
solution of the sample 200 needs to be manipulated or changed, the
stage 930 supporting the cuvette 300 may be lowered. This lowering
may elevate or withdraw the biological sample 200 from the clearing
fluid 1000. As shown in FIG. 24b, either the sample 200 or the
clearing fluid 1000 may be changed or manipulated without changing
or interfering with any other aspect of the light sheet microscope.
The cuvette can be lowered without affecting the sample or sample
holder. This implies that the user can take sample out without
dipping into solution. Focusing, e.g., may still be adjusted even
though sample has changed. The light sheet plane and detector may
stay in the same relative position. No other operations are needed
to move the liquid filled vessel out of the way, or to return it
into position with fresh fluid, submerging the sample for imaging.
As before, transparent windows 360 and 361 may admit the radiation
to the sample 200.
[0239] FIGS. 25a and 25b show further details of the sample holder
800, which also has some important features. The sample holder 800
may include a sample stage arm 810 which may extend laterally from
a supporting point 820. This supporting point 820 may be coupled to
a movable actuator (not shown) that can raise and lower the
supporting point 820. By moving the supporting point, the sample
200 may be raised or lowered into or out of the clearing solutions.
The side view of FIG. 25b illustrated the relationship of the
movable stage 930, sample stage 800, and cuvette 300. The sample
stage 800 coupled to a sample supporting point 820. This supporting
point 820 may be moved laterally or vertically by a thumbscrew or
rack and pinion, for example.
[0240] It should be understood that if the sample 200 is raised,
then the operative objective lens may also need to be raised to
allow clearance. However, with movable supporting point 820, the
sample may be removed from the clearing fluid and manipulated or
exchanged without disturbing any other aspects of the light sheet
microscope.
[0241] The two independent motions, of the cuvette stage 930, and
the sample supporting point 820 are shown in FIG. 25b. FIG. 25a
shows the components in their operative orientations in plan view.
The cuvette 300 may have a large circular hole 350 in the top
surface 350 that will admit the operational objective lens. This
aperture 350 may allow the lens to be lowered into the clearing
fluid 1000 until it is the proper distance from the biological
sample such that the structures are in focus. The operational lens
may then be raised until it clears the cuvette 300, and the lens
assembly and turret may be rotated to place another objective lens
into the operational position. The other non-operational lenses
will be lowered into the cutaway surfaces 351, 352, 951 and
952.
[0242] As shown in FIG. 25, the biological sample 200 may be
mounted on a movable sample stage 800. The sample stage 800 may be
a motorized x, y, z stage, movable in three dimensions.
[0243] Thus, the sample stage may be movable vertically with a
throw of, for example, about 30 mm. However the sample stage may
also be movable laterally, with a throw of about 80 mm. Thus, the
sample can be moved laterally with respect to the movable optical
assemblies but importantly, it may also be movable vertically. The
vertical movement may enable the three dimensional imaging
capability discussed earlier. The lateral scanning, as explained
before, is accomplished by moving the movable optical assemblies
100 and 100' laterally with respect to the sample, rather than by
moving the sample laterally. Accordingly the plane of the line
focus, that is, the plane within which the line focus moves, is
established by the motion of the movable optical assemblies 100 and
100' and may not change during operation in general.
[0244] The movable sample holder 800 allows for imaging large
samples e.g. up to 100.times.100 mm. Accordingly, the field of
view/magnification may accommodate a detector with an active area
on the order of 22 mm.
[0245] Accordingly, the sample holder may be manipulated
independently of optics, fluidics, excitation and detection. The
sample may be moved vertically in the plane for different depths
and laterally for a changed imaging location. The ability to change
samples without touching the excitation or detection optics offers
significant benefits in terms of ease of use. The fluid receptacle
or cuvette 300 can move vertically in the z-direction independently
of the sample, the detector and the excitation. Accordingly, it may
be possible to change samples without touching the optical system.
It is also possible to change samples 200 without touching the
sample holder 800.
[0246] It should also be understood that artificial intelligence
techniques, such as machine learning and deep learning, using for
example tensor flow records, may be used to improve the final image
quality.
[0247] FIG. 26a is a side view illustration of the structures shown
in plan view of FIG. 25. FIG. 26a shown the sample 200 submerged in
the cuvette 300 and clearing fluid 1000. FIG. 26b shows the sample
200 retracted from the cuvette 300 and clearing fluid 1000. The
light sheet plane 210 may stay in position relative to everything
but the cuvette 300.
[0248] FIG. 27 is an illustration showing how multiple samples may
be handled using the novel sample holder. Because of the large
throw (25 mm in this lateral dimension), multiple biological
samples may be loaded on the sample holder simultaneously. In FIG.
27, five samples 810 are shown loaded on to the sample holder 800.
Using the lateral actuator, each of these samples can successively
be brought in to the plane of the line foci and imaged.
Accordingly, Multiple biological samples may be imaged without
moving any other structure with in the light sheet microscope. No
optics, no detector, no fluidics, no imaging systems need to be
moved. As a result, the conditions under which successive images
are taken are as nearly as is possible, identical. This allows for
particularly easy and straightforward automated or robotic
operation.
[0249] The light sheet microscope may be made by machining and
anodizing aluminum, using a combination of publicly available
lenses, lasers, optical elements such as turning mirrors, movable
stages, stepper and continuous motors, for example, in addition to
custom parts. The detector may be a CCD camera readily available
from a variety of sources. The device may be calibrated and focused
using standard procedures in microscopy, and by imaging materials
with known attributes, such as glass beads. The images may be
displayed on monitors on the machine or remotely over the internet,
for example.
[0250] Among all the afore described independent motions, the light
sheet focal plane always stays in the same position however, within
the cuvette and with respect to the system. The detection locks to
this excitation focal plane.
[0251] Accordingly, a light sheet microscope is described. The
microscope may include a detector which forms an image of the
biological sample through imaging optics, wherein the biological
sample disposed in a focal plane of the imaging optics, and a
container holding a quantity of fluid and disposed on a movable
first stage, movable in the z-direction, wherein the z-direction is
orthogonal to the focal plane. The microscope may also include a
sample holder holding the biological sample, wherein the biological
sample is immersed in the fluid and the biological sample is in the
focal plane. The first stage may have a range of motion such that
the sample can be both immersed in the fluid and in the focal plane
and then withdrawn from the fluid by the motion of the first stage,
wherein the first stage moves the container independently of the
sample holder, the imaging optics and the detector.
[0252] The light sheet microscope may further comprise a movable
optical subassembly configured to focus a plurality of light beams
into a single straight line that defines a light sheet, and wherein
the light sheet lies in the focal plane of the imaging optics and
therefore also in the biological sample. The light sheet microscope
may also include a movable second stage that holds the movable
optical subassembly. The second movable stage may move laterally
and parallel to the focal plane, and perpendicularly to the
z-direction, thereby moving the light sheet to a different
laterally adjacent region of the biological sample,
[0253] The plurality of light beams may comprise at least three
light beams, each of which is separated by an angle, wherein the at
least three light beams are focused by the movable optical
subassembly all to the same straight line defining the light sheet,
and wherein the light sheet lies in the focal plane. The plurality
of light beams may further comprise six light beams, with three of
the six light beams on either of two adjacent sides of the
biological sample and wherein each of the three light beams is
separated by an angle, and with each set of three beams focused to
a single straight line by two movable optical subassemblies
disposed on either side of the biological sample. Within the light
sheet microscope, the container, the sample holder, the biological
sample, and first and second stages may all be movable without
changing the focal plane of the imaging optics and a location of
the light sheet.
[0254] The different laterally adjacent regions may comprise at
least 5 regions spaced laterally adjacent within the biological
sample but within the focal plane of the imaging optics.
Furthermore, by moving the container holding the fluid in the
z-direction, the sample holder and biological sample may be held
aloft of the container and fluid, such that the biological ample
can be manipulated without affecting any one of the focal plane,
the detector, the imaging optics, the light sheet or touching the
fluid. The sample holder may be moved without changing a relative
orientation of the imaging optics or the focal plane or the light
sheet. The sample holder may be configured to allow the biological
sample to be manipulated without changing a relative orientation of
the imaging optics or the focal plane or the light sheet.
[0255] The light sheet microscope may further comprise an imaging
lens structure upon which the imaging optics is mounted, wherein
the imaging lens structure is also movable in the z-direction and
wherein the imaging optics are configured to form the image on the
detector, wherein the lens structure, the container, the sample
holder, the biological sample, and first and second stages are all
movable without changing the focal plane.
[0256] Within the light sheet microscope, the detector may comprise
a pixelated detector wherein the imaging optics form a pixelated
image of the focal plane on the pixelated detector, and wherein the
imaging optics may be moved without moving the pixelated detector,
the biological sample or the container. The imaging optics may be
submerged in the fluid by moving the lens structure downward in the
z-direction on the movable second stage or by moving the first
stage and container upward in the z-direction on the movable first
stage, and wherein the fluid is a clearing fluid that renders the
biological sample more transparent to radiation. The sample can be
removed and replaced by another sample without moving the focal
plane or the lens structure. At least a portion of the imaging
optics may be submerged in the fluid above the sample and sample
stage to image the irradiated sample.
[0257] The light sheet microscope may further comprise a movable
third stage coupled to the sample holder, configured to move the
biological sample in the sample holder in the z-direction to
illuminate multiple depths in the biological sample with the
plurality of light beams. The light sheet may move in the
z-direction to irradiate multiple areas at different depths within
the sample by moving the third stage in the z-direction, without
affecting the position of the first stage or second stage.
[0258] The different depths may comprise at least 5 different
depths. The sample holder on the movable third stage, the lens
structure on the movable first stage, and the container on the
movable second stage can all move independently of, and without
disturbing, the focal plane. The movable third stage may have a
throw of about 20-40 mm in the z-direction, and 70-90 mm laterally,
orthogonal to the z-direction, and the movable first stage may have
a throw of about 40-50 mm in the z-direction.
[0259] The biological sample may be replaced with a new biological
sample, without disturbing the sample stage, the lens structure, or
the focal plane, by moving the container on the movable first stage
to expose the biological sample.
[0260] While various details have been described in conjunction
with the exemplary implementations outlined above, various
alternatives, modifications, variations, improvements, and/or
substantial equivalents, whether known or that are or may be
presently unforeseen, may become apparent upon reviewing the
foregoing disclosure. Accordingly, the exemplary implementations
set forth above, are intended to be illustrative, not limiting.
* * * * *
References