U.S. patent application number 16/895498 was filed with the patent office on 2020-11-26 for fast multiphoton microscope.
The applicant listed for this patent is APPLIKATE TECHNOLOGIES LLC. Invention is credited to Michael Levene, Richard Torres.
Application Number | 20200371033 16/895498 |
Document ID | / |
Family ID | 1000005008360 |
Filed Date | 2020-11-26 |
![](/patent/app/20200371033/US20200371033A1-20201126-D00000.png)
![](/patent/app/20200371033/US20200371033A1-20201126-D00001.png)
![](/patent/app/20200371033/US20200371033A1-20201126-D00002.png)
United States Patent
Application |
20200371033 |
Kind Code |
A1 |
Levene; Michael ; et
al. |
November 26, 2020 |
FAST MULTIPHOTON MICROSCOPE
Abstract
The invention provides improved systems and methods for
multiphoton microscopy including pixel clocking techniques for
minimizing pixel integration time and providing consistent signal
intensity with maximized imaging speeds. Various systems and method
are described for optimizing laser repetition rate based on dye
lifetime, combining polygonal mirror scanning and stage
translation, using the laser pulse signal to time pixel collection,
and minimizing laser pulses and dye usage based on signal to
background ratios.
Inventors: |
Levene; Michael;
(Washington, DC) ; Torres; Richard; (East Haven,
CT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
APPLIKATE TECHNOLOGIES LLC |
Washington |
DC |
US |
|
|
Family ID: |
1000005008360 |
Appl. No.: |
16/895498 |
Filed: |
June 8, 2020 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
16523698 |
Jul 26, 2019 |
10677730 |
|
|
16895498 |
|
|
|
|
62800161 |
Feb 1, 2019 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 21/6458 20130101;
G01N 2201/06113 20130101; G01N 21/6486 20130101; G01N 2021/6463
20130101 |
International
Class: |
G01N 21/64 20060101
G01N021/64 |
Claims
1. A multiphoton microscope comprising: a pulsed light source; a
focusing system operable to focus light pulses from the pulsed
light source onto a sample on a sample stage; a scanning system
operable to move a focal point of the light pulses relative to the
sample; and a pixel clock operable to assign fluorescence signal to
each pixel in a recorded image wherein each pixel integrates signal
over a fixed integral number of light pulse interpulse
intervals.
2. The multiphoton microscope of claim 1 wherein the scanning
system comprises a spinning polygon mirror.
3. The multiphoton microscope of claim 1 wherein the integral
number of light pulses is between 1 and 100.
4. The multiphoton microscope of claim 1 wherein the pulsed light
source comprises a pulse repetition rate of about 70 MHz to about 1
GHz.
5. The multiphoton microscope of claim 4 wherein the pulsed light
source is a laser.
6. The multiphoton microscope of claim 4 wherein the pulsed light
source comprises an ultrafast laser, a beamsplitter, and a delay
line.
7. The multiphoton microscope of claim 1 wherein the light source
interpulse interval is between about 1 and about 3 times a
fluorescent lifetime of a fluorescent dye in the sample.
8. The multiphoton microscope of claim 2 wherein the scanning
system is operable to perform a translation of the sample stage in
a direction perpendicular to a scanning direction of the spinning
polygon mirror to image a strip of sample having a width defined by
the spinning polygon mirror scan and a length defined by the sample
stage translation.
9. A method for imaging a sample using a multiphoton microscope,
the method comprising: loading a sample that has been exposed to a
fluorescent dye into the multiphoton microscope, the multiphoton
microscope comprising: a pulsed light source; a focusing system
operable to focus light pulses from the pulsed light source onto a
sample on a sample stage; a scanning system operable to move a
focal point of the light pulses relative to the sample; and a pixel
clock operable to assign fluorescence signal to each pixel in a
recorded image over a pixel dwell time; imaging the sample using
the multiphoton microscope wherein the pixel dwell time is a fixed
integral number of light pulse interpulse intervals.
10. The method of claim 9 wherein the scanning system comprises a
spinning polygon mirror.
11. The method of claim 9 wherein the integral number of light
pulses is between 1 and 100.
12. The method of claim 9 wherein the pulsed light source is pulsed
at a rate of about 70 MHz to about 1 GHz.
13. The method of claim 12 wherein the pulsed light source is a
laser.
14. The method of claim 12 wherein the pulsed light source
comprises an ultrafast laser, a beamsplitter, and a delay line.
15. The method of claim 9 wherein the light source interpulse
interval is between about 1 and about 3 times a fluorescent
lifetime of the fluorescent dye.
16. The method of claim 10 wherein imaging the sample comprises the
scanning system performing a translation of the sample stage in a
direction largely perpendicular to a scanning direction of the
spinning polygon mirror to image a strip of sample having a width
defined by the spinning polygon mirror scan and a length defined by
the sample stage translation.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Non-Provisional
application Ser. No. 16/523,698, filed Jul. 26, 2019, which claims
priority to and the benefit of U.S. Provisional Application No.
62/800,161, filed on Feb. 1, 2019, the content of each of which is
hereby incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0002] The invention relates to systems and methods for multiphoton
microscopy.
BACKGROUND
[0003] Achieving high imaging speed is critical to enabling the
incorporation of digital imaging in a clinical workflow. Digital
slide scanning, or whole slide imaging (WSI), is increasingly used
in clinical environments owing to the benefits it provides in terms
of remote interpretation by experts, security and longevity for
archiving, potentially higher review efficiency, and amenability to
evolving tools for computer aided diagnosis. An important measure
of functionality is the throughput of the digitalization
instrument, most often quoted in terms of time required to scan a
15 mm.times.15 mm area and typically on the order of one to six
minutes. Since slides are usually imaged serially on a given
instrument, high throughput is desirable not only for ensuring
pathologists have access to images as quickly as possible, but
(perhaps most importantly) for reducing the cost-per-sample of the
scanning instrument which may otherwise be prohibitively high.
[0004] Multiphoton microscopes (MPM) (e.g., as described in U.S.
Pat. No. 5,034,613 to Denk et al.) have proven to be a valuable
tool for biological research and offer a potential alternative for
histologic analysis (and WSI) in clinical and research settings. In
order to allow future clinical implementation of MPM in histologic
analysis, existing speed limitations must be critically
addressed.
[0005] Various aspects contribute to achievable image acquisition
speeds in MPM. Systems typically comprise a laser source that
produces ultrashort laser pulses of about 50 fs-2 ps, with a
repetition rate of .about.80 MHz. The light from the laser is
focused by a microscope objective to a point inside the sample.
This point is scanned across the sample by a system of mirrors
placed upstream of the objective that may be mounted on
galvanometers. The generated fluorescence is typically collected
back through the objective lens and directed to one or more
detectors by a series of dichroic and emission filters, or by
collection optics positioned on the opposite side of the sample
from the objective lens and directed to one or more detectors by a
series of dichroic and emission filters. The generation of
fluorescence in the sample occurs by the simultaneous absorption of
two or more photons from the laser. The use of short pulses leads
to high peak intensities for more efficient multiphoton absorption
without requiring excessive average laser powers.
[0006] Galvanometers used for point scanning are typically
comprised of mirrors mounted on shafts that rotate in either a
linear or sinusoidal fashion, deflecting the beam in a line
pattern. The typical multiphoton microscope scans one line of the
sample in .about.1 ms, with pixel dwell times on the order of
.about.1 us. The pixel clock, which determines when signal coming
from the detectors gets assigned to a new pixel, is synced to the
position of the scanning optics in order to create pixels of
uniform size. Because the pixel dwell time is long compared to the
time between laser pulses (typically 12 ns), many pulses will
strike the sample during a given typical pixel dwell. It is
therefore unimportant to count the number of pulses arriving per
pixel. However, when imaging at much higher scan rates than the
typical system, pixel dwell times may shorten to the point that the
variation in the number of pulses per pixel leads to unwanted
variation in the amount of fluorescence collected from pixel to
pixel.
[0007] For microscopes that incorporate at least one resonant
galvanometer for high-speed scanning, the non-linear, sinusoidal
scan pattern of the resonant galvanometer results in a very large
variation in the number of pulses per pixel. This creates
pronounced inhomogeneity in image intensity across the
field-of-view and limits the maximum rate that can be achieved for
a given minimum number of pulses per pixel. The latter occurs
because the sinusoidal movement of the mirror results in pixel
collection that is slower at the edges of the field of view than at
the midpoint of rotation where the speed is at its maximum. The
inhomogeneity in image intensity translates to image quality
limitations that limit the applicability of resonant galvanometer
based MPM systems to diagnostic interpretation of tissue
histology.
[0008] Spinning polygons have also been used to increase the rate
of line scanning in point-scanning systems and do not suffer from
the variable speed issues of standard or resonant galvanometers.
Shack et al. (1979) described theoretical speed optimization using
continuous wave lasers and spinning polygons for point scan imaging
of biological samples by coupling it with continuous motion of
perpendicularly oriented stage movement. The description refers to
the potential for fast imaging, but beyond being theoretical, the
description predates the invention of the multiphoton microscope
and thus does not address aspects specific to the coupling of
spinning polygons to multiphoton excitation.
[0009] Some multiphoton microscopes have used high-repetition-rate
laser pulse trains in order to increase the speed of imaging or to
lower the peak power in order to reduce photobleaching and
photodamage. Amir et al. 2007 used a beamsplitter and a delay line
to double the effective repetition rate of 23 MHz laser pulses,
while Cheng et al. 2011 used multiple beamsplitters, resulting in a
4-fold increase in the 80 MHz pulse rate of the source laser.
However, both Amir and Cheng focused the outputs of the
beamsplitters onto different spots within the sample, such that the
effective pulse rate for a single spot was unchanged from that of
the source laser. Chu et al. 2003 used an ultrafast laser with a 2
GHz repetition rate for second harmonic imaging, but did not use
this laser for multiphoton fluorescence imaging. Ni et al. 2008
used a series of beamsplitters and delay lines to image with a
single scanning spot with an effective pulse repetition rate of 640
MHz-10.24 GHz, much faster than the lifetime of the fluorescent
protein they were imaging. While that approach worked for the
specific samples used, its general applicability with other samples
and dyes is questionable due to excited-state absorption resulting
in increased dark state population and photobleaching. Thus, no
solution presented to date has been able to achieve a MPM with
maximized speed that ensures maximal quality for detailed
histologic evaluation in a timely fashion.
SUMMARY
[0010] The invention provides a multiphoton microscope with various
features that enable efficient, high quality imaging at high speed
of fluorescently labeled tissue samples. Systems and methods of the
invention are of particular interest for diagnostic interpretation
of tissue specimens. In some aspects, the design is specifically
geared toward the fast multiphoton imaging of tissue specimens that
have been processed with a method described in a prior patent
application. The multiple components work in concert and all
contribute to achieving high speed imaging using multiphoton
microscopy with quality amenable to primary diagnostic
interpretation. The combined features allow for image quality
heretofore not achieved at the rates described herein. Accordingly,
the systems and methods of the invention allow for practical
histologic analysis from MPM with the quality and speed required
for clinical use and large volume imaging for investigative
purposes.
[0011] In certain aspects, systems and methods of the invention
include a pixel clock configured such that each pixel integrates
fluorescent photons generated by a fixed integer number of laser
pulses, ranging from 1-20. In some aspects, methods of the
invention include the use of a pulsed laser with a repetition rate
that is similar to the lifetime of the dyes used in the object
being imaged. Certain systems and methods of the invention include
the use of a polygonal mirror for rapid scanning in one direction
and a translational stage for moving the specimen in a direction
perpendicular to that of the scan line generated by the rotating
polygonal mirror. Some systems and methods of the invention include
a pixel clock that is coordinated to the laser pulses such that the
start of each pixel has a fixed time delay, which may be zero, with
respect to the timing of the most recent laser pulse in order to
minimize cross-talk between pixels due to the long exponential tail
typical of fluorescence decays.
[0012] Systems and methods of the invention contemplate the
minimization of pulses per pixel while maintaining the quality of
images based on signal to background ratio and the use of
sufficient fluorescent dye concentrations in a specimen to achieve
a threshold of signal to background with a limited number of pulses
but not more than needed so as to control cost of dyes and avoid
fluorophore self-quenching and absorption of fluorescent
photons.
[0013] Aspects of the invention include a variety of microscope
configurations to increase efficiency, image quality, and speed in
MPM and other imaging techniques. For example, a second laser may
be employed incident upon the polygonal mirror along with a
detector that collects the reflected signal which can then be used
for tracking the mirror position for coordinating the pixel clock
with scanning of the excitation spot in the sample. Group velocity
dispersion in the microscope optics can be pre-compensated for to
minimize the laser pulse width at the sample. Detector amplifiers
that are balanced for speed and amplification and matched to the
rate of pixel acquisition can be used in devices of the
invention.
[0014] An appropriate wavelength can be selected for the
simultaneous excitation (and detection) of both nuclear and protein
fluorescent dyes to allow for combined imaging thereof. Laser power
levels can be optimized to maximize optical resolution and minimize
optical section thickness while maintaining desired signal to
background levels. Systems and methods of the invention can include
using a microscope objective that has a combination of high
numerical aperture, large field-of-view, and very long working
distance, and is optimized for imaging of specimens with normalized
high refractive index such as by clearing techniques.
[0015] A detection filter combination can be used that can separate
the emission of green-fluorescent protein dyes such as eosin from
the emission of hematoxylin-like blue fluorescent nucleic acid dyes
such as DAPI and Hoechst, so as to enable reproduction of the
standard histologic stain of hematoxylin and eosin (H&E).
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1A shows a 12 ns pixel clock selected as an integral
multiple of a laser 4 ns interpulse interval.
[0017] FIG. 1B shows an 11 ns pixel clock that is a non-integer
multiple of the laser interpulse interval.
[0018] FIG. 2 shows a schematic of a microscope according to
certain embodiments.
DETAILED DESCRIPTION
[0019] For a given image quality, determinants of maximum
achievable speed include fluorescence lifetime of dyes, multiphoton
pulse rate, beam movement efficiency including duty cycles and
constancy of dwell-times, timing efficiency for coordinating laser
beam movement and data acquisition, non-imaging time (e.g. sample
positioning time), light collection efficiency (including objective
lens numerical aperture and signal collection pathway), and related
signal-to-noise aspects such as dye efficiency, dye concentration,
detector efficiency, and detector and other noise sources. The
various features employed in the invention have been devised to
address these determinants in order to guarantee high image quality
at high speed.
Pixel Dwell Times that are Integer Multiples of the Laser
Interpulse Interval
[0020] Maximizing imaging speed requires minimizing the integration
time per pixel known as the pixel dwell time. Decreasing the pixel
dwell time results in fewer laser pulses striking the sample pixel.
If the pixel dwell time is not an integer number times the
interpulse interval of the excitation laser, the number of laser
pulses striking the sample will vary from pixel to pixel. Shorter
pixel dwell times result in fewer laser pulses striking the sample
per pixel on average, leading to larger variation in signal
intensity from pixel to pixel, even for a uniform sample. One
aspect of the invention is the use of a pixel clock that results in
uniform pixel dwell times that are a fixed integer number times the
interpulse interval of the excitation laser.
[0021] The number of pulses at which the effect of non-fixed
integer number of pulses results in variability with visually
detectable image degradation is dependent on the specific
application. For visual analysis of biologically derived images
such as for histologic interpretation, pixel-to-pixel variability
of 5% or more stemming from interpulse integer variability may
produce detectable image degradation. As such, when pixel dwell
times are in the range of 20 interpulse intervals, pulse counting
becomes increasingly relevant for image quality. In a preferred
embodiment, the pixel dwell time is 1-12 integer times the length
of the interpulse interval. In various embodiments, the pixel dwell
time can be about 2 to about 10 times, about 2 to about 100 times,
about 2 to about 6 times, about 2 times, about 4 times, about 6
times, or about 8 times the length of the interpulse interval.
Where an integer number is referred to herein with regard to the
relationship between pixel dwell time and interpulse interval, one
of ordinary skill in the art would understand that practically
achievable precision must be taken into account. Accordingly, pixel
dwell times of 2 to 100 times the interpulse interval, for example,
should be understood to include each integer between 2 and 100 to a
degree of decimal places required to achieve the desired effect of
equally distributing pulses among the imaged pixels. As such the
precision of the dwell time to interpulse interval relationship
should be defined such that any deviations (e.g., imprecision at
several decimal places) from a strict integer relationship do not
result in significant pulse variation among the imaged pixels.
[0022] FIG. 1A shows the result of a uniform pixel dwell time that
is an integer multiple of the laser interpulse interval. The blue
line shows the fluorescence signal level with time from a uniform
sample as a result of excitation by laser pulses with an interpulse
interval of 4 ns. The black vertical lines represent the start of
each pixel dwell time of 12 ns, 3 times the interpulse interval.
Each pixel dwell integrates over a time period with a fixed
relation to the laser pulse train, resulting in uniform pixel
intensity as shown by the black horizontal line at the top of the
figure. Even if the pixel clock is shifted slightly in time
relative to the laser pulse train, the signal will still be
uniform.
[0023] FIGS. 1A and 1B show the effect of non-integer pixel dwell
times. In FIG. 1A, a 12 ns pixel clock (vertical lines touching the
X-axis) is an integral multiple of the laser 4 ns interpulse
interval. The integration of the fluorescence signal (wave lines on
graph) results in uniform pixel intensities (line along top of the
graph) for a uniform sample. In FIG. 1B, a 11 ns pixel clock is a
non-integer multiple of the laser interpulse interval, resulting in
non-uniform pixel intensities FIG. 1B shows the results of a
uniform pixel dwell time that is not an integer multiple of the
laser interpulse interval. The signal wave line on the graph shows
the fluorescence signal level with time from a uniform sample as a
result of excitation by laser pulses with an interpulse interval of
4 ns. The vertical lines touching the x-axis of the graph represent
the start of each pixel dwell time of 11 ns, or 2.75 times the
interpulse interval. The resulting pixels shift their integration
window with respect to the laser pulse train, resulting in
non-uniform pixel intensity as shown in the black line across the
top of the figure.
Optimizing Laser Repetition Rate Based on Lifetime of Fluorescent
Dyes being Imaged
[0024] Choosing laser repetition rates that result in inter-pulse
intervals that are much shorter than the fluorescence lifetime can
lead to unwanted effects, such as excited state absorption and
increased photobleaching and/or photodamage, and result in
inefficient dye excitation with consequent reductions in quality.
Choosing laser repetition rates with inter-pulse intervals that are
much longer than the fluorescence lifetime results in slower
imaging speeds. With the number of pulses per pixel fixed according
to the minimum required total collected signal per pixel, higher
repetition rates lead to faster imaging. If the number of pulses
per pixel is not fixed, the variation in the number of laser pulses
from pixel to pixel may be minimized by increasing the number of
pulses per pixel through the use of a higher repetition rate laser.
The repetition rate of the laser can be chosen such that the
interpulse interval, the time between laser pulses, is similar to
the lifetime of the fluorescence dyes. In order to avoid one laser
pulse exciting dye molecules that are already in the excited state
due to the previous laser pulse, the inter-pulse interval should be
on the order of the dye lifetime. In a preferred embodiment, the
interpulse interval should be somewhat longer than the dye lifetime
because the exponential decay of the fluorescence lifetime has a
long tail to its distribution. For example, the inter-pulse
interval can be 1-3 times the lifetime of the longest lifetime dye
present in the sample. The nuclear fluorescent dye DAPI has a
fluorescence lifetime of 1.6 ns, while the fluorescent protein dye
Eosin has a lifetime of 2.4 ns. Applying the above embodiments in
such a case would result in selecting a laser with a repetition
rate of 250 MHz which allows sufficient time for the vast majority
of eosin molecules to return to ground state, but minimizes the
collection time during which there is no effective signal
acquisition.
Combination of Polygonal Mirror and Translating Stage for Rapid
Point Scanning of Multiphoton Laser
[0025] In microscopy techniques discussed herein, the laser is
scanned across a sample in order to form an image. That scanning
can be done by mirror(s) mounted to one or more galvanometers, by
one or more mirrors mounted in a piezo-driven mirror mount, by
translation of the stage in one or more directions, or by any other
method or combination of methods capable of moving the focused
laser spot across the sample. In a preferred embodiment, the pulsed
laser beam is rapidly scanned in angle by a spinning polygon with
mirrored facets, such as those currently available from Lincoln
Laser Company, Phoenix, Ariz.
[0026] The scanned beam can be imaged to the entrance pupil of a
microscope objective using a scan lens and a tube lens. In some
embodiments, the scan lens is a telecentric F-theta lens.
Alternatively the scan lens may be an F-theta lens, or any suitable
lens system that, when used together with the tube lens,
effectively images the angle-scanned beam from the surface of the
spinning polygon mirror to the entrance pupil of the microscope
objective. The microscope objective focuses the beam to a spot in
the sample contained within the sample cartridge. The angle
scanning of the beam by the spinning polygon results in the spot
scanning in a line across the sample.
[0027] A stage may be designed to hold a sample in place and to
perform a translation of the sample in a direction largely
perpendicular to the direction the laser spot is scanning due to
the motion of the spinning polygon mirror. Thus, as the laser spot
is scanned across the sample by the polygon mirror, the stage moves
the sample in the orthogonal direction to the scan line, resulting
in the imaging of a strip of sample, the width of which is
determined by the extent of the laser scan and the length of which
is determined by the length of the stage scan. The stage may also
be capable of fine scanning in the direction of focus of the
microscope objective in order to change the focal plane.
Alternatively, or in addition, the objective lens may be mounted on
a focusing apparatus. The fine scanning in the direction of focus
can occur between sessions of image acquisition or during an image
acquisition session, the latter resulting in collection of image
data that is in a line that is not perpendicular to the either the
sample holding stage or the objective. The stage can also be
capable of scanning in largely the same direction as the moving
laser focus in order to image multiple strips of the object being
imaged (e.g., a tissue sample), the resulting images of which may
be assembled into a mosaic using software during or after image
acquisition. A scan pattern with partial overlap between successive
strips may be chosen in certain embodiments to aid in the later
assembly of the mosaic from the scan images. The stage translation
and the polygon rotational speeds may be coordinated such that the
distance travelled by the stage during the period the laser is
reflected off a given polygon facet is equal to the desired length
of one dimension of a pixel.
[0028] In some embodiments the desired pixel dimension in the
translated stage direction can be the same as the pixel dimension
in the polygon rotation direction. In other embodiments the pixel
dimension in the translated stage dimension can be longer or
shorter than in the rotating mirror scanned dimension. That may be
done so as to reduce noise by averaging adjacent pixels along the
polygon scanning direction. This may reduce noise differently than
averaging of signal across multiple axes simultaneously, which
would allow independent optimization of imaging speed in different
axes. In some embodiments the pixel dimension in the translated
stage dimension can be an integer multiple of the dimension in the
rotating mirror scanned direction.
Using a Laser Pulse Signal to Time Pixel Collection
[0029] Each laser pulse excites a fluorescence signal that decays
exponentially in time. In some cases, the long tail of this decay
may result in some fluorescent photons generated by the last pulse
in a given pixel dwell arriving at the detector during the
subsequent pixel dwell. That cross-talk between pixels may act to
blur the signal from one pixel into the adjacent pixel. A computer
can be used to assign the signal from the detectors to pixels in
sequence. Timing the start of each pixel dwell time to coincide
with the peak fluorescence signal from the first laser pulse in
that pixel dwell time minimizes the cross-talk between pixels. As
such, in one embodiment of the invention a synchronization signal
from the laser can be used to trigger a detector signal
digitization board to assign collected fluorescence for a set
period of time to a particular pixel. Different embodiments may use
different polygon scanning speeds and different numbers of laser
pulses per pixel. For the same scanning speed, using larger numbers
of pulses per pixel, such as 5 or 10, would result in larger
pixels.
[0030] In certain embodiments, the relationship between the start
of pixel collection and the laser peak pulse can be manipulated to
help minimize pixel-to-pixel cross talk. As such, in some
embodiments the data acquisition board can be triggered to record
signal from the detectors by a separate clock with a rate that is
an integer multiple of the pixel rate. Such an arrangement requires
sufficient precision and accuracy in both the laser pulse timing
and the separate clock to ensure some pixels do not suffer from
artifacts of pulse number variation.
Minimization of Number of Pulses Based on Signal to Background
[0031] In certain aspects systems and methods of the invention
relate to the determination and use of a minimal number of pulses
that preserve a desired degree of contrast and clarity for the
fluorescent images derived. That determination can be based on the
subjective visualization of acquired images such that images
collected with many pulses per pixel may be compared to images with
progressively smaller number of pulses per pixel and judged as to
overall ability to discern features of potential interpretive
importance for say pathologic diagnosis. In certain embodiments,
that subjective visualization can be translated to a measured
signal to background (SBR) as follows: For the background, areas
devoid of staining tissue are collected using MPM and the standard
deviation of the signal, or other similar measure of variation such
as variance, in the otherwise dark regions may be used as a measure
of background noise. That measure of background noise is compared
to the average maximum intensity of multiple areas that contain
known bright features. For a nucleic acid stain, this may be
lymphocyte nuclei or other portions of nuclei. For a protein stain,
this may be red blood cells.
[0032] Images with SBR lower than about 20 appear undesirable for
analysis whereas those with SBR greater than about 50 do not
typically show additional improvement from standard histologic
stains. Thus, target SBR for optimization using systems and methods
described herein can be between 20 and 50. In some cases a SBR of
50 may be desirable. In other cases a SBR of 40 may be desirable.
In other cases a SBR of 30 may be desirable. In other cases a SBR
of 20 may be sufficient.
[0033] Once the threshold is established, then it is possible to
analyze the same tissue sample in the same manner while varying the
number of pulses per pixel. With the preparation method and
instrument implementation configuration described herein, it has
been determined that between 2 and 8 pulses are the required
minimum for achieving the desired SBR.
Use of Minimal Amount of Dyes that Yield Threshold of Signal to
Background with Few Pulses
[0034] A principal concern in diagnostic imaging is cost. Some dyes
are expensive and can form a large part of the overall processing
cost which could affect the usability in high volume environments
such as for clinical imaging use. Determination of the optimal dye
concentration to use for high speed, high quality imaging can be
based on the measurement of SBR at a given pulses per pixel as a
function of dye concentration. By optimizing dye concentration,
waste can be avoided and costs reduced using the methods described
herein.
[0035] FIG. 2 shows a schematic of a preferred embodiment of a
microscope of the invention. The microscope consists of a laser
source suitable for multiphoton excitation of the dyes used for
labeling the sample. The laser may be a Ti:Sapphire laser, or a
fiber laser, or any other laser capable of producing ultrashort
laser pulses. In various embodiments, pulses ranging from several
picoseconds to less than 150 fs may be used. In some embodiments,
the light from the laser passes through a beam shaping system that
consists of a series of lenses and expands the beam to the required
beam width to effectively illuminate the back aperture of the
microscope objective that focuses the laser to a spot on the
sample. The laser may have a fixed center wavelength, as is common
with fiber lasers, or it may be broadly tunable, as is common with
some Ti:Sapphire lasers. In a preferred embodiment, the laser would
generate laser pulses with pulse widths <150 fs with center
wavelength chosen for optimum excitation of dyes. In a preferred
embodiment, the center wavelength is selected to optimally
simultaneously excite both 4',6-diamidino-2-phenylindole (DAPI) and
eosin, which has been determined to be between 740 and 820 nm, and
more preferably the wavelength can be selected to reduce excitation
of unbound dye, determined to be between 760 and 800. In certain
embodiments the wavelength may be selected to be between 760 and
780. In some embodiments the wavelength may be 765 nm. In other
embodiments the wavelength may be 780 nm, 770 nm, or 800 nm.
[0036] As shown in FIG. 2, the sample can be illuminated from below
in what is commonly referred to as an "inverted microscope."
Alternatively, the sample may be illuminated from above in what is
commonly known as an "upright microscope."
[0037] Fluorescent light can be collected by the objective lens,
reflected from a dichroic mirror and sent to one or more detectors.
In some cases, additional lenses or mirrors may be used to
efficiently collect the fluorescent light to the detectors. A
second dichroic mirror may be used to send different color
fluorescence to a different channel. For example, in a preferred
embodiment for imaging Eosin and DAPI, Dichroic A is a 735 long
pass, Dichroic B is a 550 short pass, Emission filter 1 is a 550
short pass, and Emission filter 2 is a bandpass for wavelengths 550
nm-665 nm. Other filters may be chosen as appropriate by one
skilled in the art.
[0038] In some embodiments, an additional detector and associated
collection optics may be used on the opposite side of the sample
chamber from the objective lens to detect transmitted light signal
to increase the detection efficiency of the fluorescence signals or
of second harmonic generation signals by the use of additional
detectors. A lens or series of lenses may be used to focus the
transmitted light onto the detector(s), and appropriate dichroic
and emission filters may be used. The detectors may be any detector
sensitive to appropriate wavelengths of the fluorescence or second
harmonic signals, such as photomultiplier tubes. In some
embodiments the additional detector on the opposite side of the
sample from the objective lens is used to collect second harmonic
generation, the non-fluorescent signal generated by
non-centrosymmetric molecules in response to short pulsed laser
excitation and useful for characterizing collagen in tissue
specimens.
[0039] A computer can be used to send and receive control signals
to and from the scanning system. In a preferred embodiment, the
computer sends a signal to the polygon that controls the speed of
rotation. In a preferred embodiment, the computer also receives a
signal from the polygon each time a new facet passes the laser to
determine when a new line in the image has begun. In a preferred
embodiment this signal is generated by means of a low power laser
incident on the polygon mirror and reflected onto a diode or other
light detector, which in turn sends an electrical signal to the
computer and from which the polygon position can be determined. In
a preferred embodiment the incident laser is directed towards the
facet that is adjacent to the facet upon which the pulsed laser is
directed. This will ensure the most accurate timing by addressing
small potential differences in the facet dimensions. A fixed period
of time, which may be zero but is proportional to the rate of
polygon rotation and may include electric system delays, is set to
transpire before data collection begins.
[0040] As one skilled in the art would recognize as necessary or
best-suited for the systems and methods of the invention, systems
and methods of the invention include one or more computers that may
include one or more of processor (e.g., a central processing unit
(CPU), a graphics processing unit (GPU), etc.), computer-readable
storage device (e.g., main memory, static memory, etc.), or
combinations thereof which communicate with each other via a
bus.
[0041] A processor may include any suitable processor known in the
art, such as the processor sold under the trademark Core by Intel
(Santa Clara, Calif.) or the processor sold under the trademark
Ryzen by AMD (Sunnyvale, Calif.).
[0042] Memory preferably includes at least one tangible,
non-transitory medium capable of storing: one or more sets of
instructions executable to cause the system to perform functions
described herein (e.g., software embodying any methodology or
function found herein); data (e.g., portions of the tangible medium
newly re-arranged to represent real world physical objects of
interest accessible as, for example, content including images or
text for news articles); or both.
[0043] While the computer-readable storage device can in an
exemplary embodiment be a single medium, the term
"computer-readable storage device" should be taken to include a
single medium or multiple media (e.g., a centralized or distributed
database, and/or associated caches and servers) that store the
instructions or data. The term "computer-readable storage device"
shall accordingly be taken to include, without limit, solid-state
memories (e.g., subscriber identity module (SIM) card, secure
digital card (SD card), micro SD card, or solid-state drive (SSD)),
optical and magnetic media, hard drives, disk drives, and any other
tangible storage media.
[0044] Any suitable services can be used for storage such as, for
example, Amazon Web Services, memory of a server, cloud storage,
another server, or other computer-readable storage. Cloud storage
may refer to a data storage scheme wherein data is stored in
logical pools and the physical storage may span across multiple
servers and multiple locations. Storage may be owned and managed by
a hosting company. Preferably, storage is used to store records as
needed to perform and support operations described herein.
[0045] Input/output devices according to the invention may include
one or more of a video display unit (e.g., a liquid crystal display
(LCD) or a cathode ray tube (CRT) monitor), an alphanumeric input
device (e.g., a keyboard), a cursor control device (e.g., a mouse
or trackpad), a disk drive unit, a signal generation device (e.g.,
a speaker), a touchscreen, a button, an accelerometer, a
microphone, a cellular radio frequency antenna, a network interface
device, which can be, for example, a network interface card (NIC),
Wi-Fi card, or cellular modem, or any combination thereof.
INCORPORATION BY REFERENCE
[0046] References and citations to other documents, such as
patents, patent applications, patent publications, journals, books,
papers, web contents, have been made throughout this disclosure.
All such documents are hereby incorporated herein by reference in
their entirety for all purposes.
EQUIVALENTS
[0047] Various modifications of the invention and many further
embodiments thereof, in addition to those shown and described
herein, will become apparent to those skilled in the art from the
full contents of this document, including references to the
scientific and patent literature cited herein. The subject matter
herein contains important information, exemplification and guidance
that can be adapted to the practice of this invention in its
various embodiments and equivalents thereof.
* * * * *