Note: Descriptions are shown in the official language in which they were submitted.
Multichannel Line Scan STED Microscopy, Method and Device
FIELD OF THE INVENTION
The present invention relates to an optical novel microscopy method of
stimulated emission depletion
(STED),imaging fluorophore-labeled samples and a corresponding device for
performing the microscopy method.
More particular, the present invention relates to a multichannel line scan
STED microscopy imaging method and
devices providing high imaging frame rates and wide field of view super-
resolution microscopy methods capable
of resolving details below the Abbe diffraction limit.
BACKGROUND OF THE INVENTION
Fluorescence confocal microscopy is one of the most extensively used tools for
our understanding of how cells
function. Its popularity has steadily grown despite the fact that it
notoriously fails to image structures smaller than
about half the wavelength of light (-200 nnn), i. e. that it is limited by the
so called diffraction barrier. Development
of STED microscopy, which is one of the techniques that make up so-called
super-resolution microscopy,
highlighted the fact that the diffraction barrier to the spatial resolution
can be effectively overcome in a regular far-
field visible light microscope [1]. STED microscopy currently allows the
nanometer scale imaging of the structures
of a sample, which are marked with fluorescent labels, with a spatial
resolution below the diffraction limit, while
retaining most of the advantages of far-field optical operation, such as the
ability to non-invasively image cells in
3D [2]. From the beginning of the process, STED has allowed fluorescence
microscopy to perform tasks that had
been only possible using electron microscopy.
In a STED microscope, the excitation beam is spatially overplayed by a second
de- excitation or depletion
doughnut-shaped beam (also called a STED beam) which can de-excite, by
stimulated emission, the fluorescent
markers previously excited by the excitation beam. Since the doughnut-shaped
STED beam has at least one zero
intensity point only the fluorescent labels in the proximity of the zero
intensity points can actually emit
fluorescence when they return to the ground state.
The lateral FWHM of the STED focal volume¨and, therefore, spatial resolution¨
is well approximated by a
modified version of Abbe's equation (Eq. 1):
ArSTED ___________________________________
2NAV1+ ID IS (1)
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Here, /D is the peak intensity of the depletion focus and Is is the
characteristic saturation intensity (the intensity at
which 50% of fluorescence is quenched) specific to the fluorophore being used.
Given that ArsTED scales inversely
with approximately the square root of the depletion intensity, the resolution
of a STED microscope is considered
to be diffraction-unlimited in principle (Hell 2007).
The concept of enhancing resolution through the targeted switching of
fluorophores has also been generalized to
include switching mechanisms other than stimulated emission. Collectively,
this family of techniques has been
termed RESOLFT microscopy (Hell et at. 2003; Hell 2009). Recently, the use of
reversibly-switching fluorescent
proteins (FPs) in RESOLFT microscopy has come to fruition in biological
imaging (Brakemann et al. 2011;
Grotjohann et al. 2011; Grotjohann et al. 2012; Testa et al. 2012).
The main disadvantage of single point scan STED microscopy, which has
prevented its widespread use, is that
the image acquisition speed is relatively slow for large fields of view
because of the need to scan the sample in
order to retrieve an image. Thus, a frame rate of 200 frames per second (FPS)
was achieved [3], but for a very
small image size of (1.8 1.5) prn2 or 50 x 60 pixels only. Due to the
sinusoidal movement of the resonant scanner,
corrections to the image brightness and dwell times were necessary.
Furthermore super-resolution requires small
pixels, which leads to a longer acquisition time and multiple exposures of
specimen. This is particularly critical in
living biological tissue, which is easily damaged by excessive excitation
light, and with fast bleaching fluorescence
dyes.
The concept of RESOLFT inevitably requires scanning (with a zero), but not
necessarily with a single beam or a
point-like zero. Multiple zeros or dark lines produced by the interference of
counter-propagating waves [2] in
conjunction with conventional digital camera detection can also be used,
provided the zeros or the dark lines are
further apart than about the distance required by the diffraction resolution
limit of conventional CCD camera
imaging. Dark lines increase the resolution in a single direction only, but
stepwise rotation of the pattern plus
interleaved scanning of the minima (e.g. by shifting the phase in the
interference pattern) and subsequent
computational reassignment may provide, under some conditions, similar
transverse resolution as with points and
do so at higher recording speed.
The US Pat. No. 2016/0363751 discloses a Multipoint STED microscope, which by
means of two optical gratings
and an objective lens, forms two crossing line gratings of the luminescence
depletion light, and two crossing line
gratings of the excitation light so that local intensity minima of an overall
intensity distribution of the luminescence
depletion light are delimited in at least two directions, and that local
intensity maxima or local intensity minima of
an overall intensity distribution of the excitation light coincide with the
local intensity minima of the depletion light.
Further, the device moves the overall intensity distributions of the further
light and the luminescence inhibition
light to scan the structure. One of major problems of such a microscopy system
a very high laser power required
for getting s significant increase of the imaging resolution. Another problem
is absence of local circular symmetry
of the depletion light in the vicinities of the excitation light maxima.
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Axial resolution is very important in light microscopy since it enables 3D
sectioning of the sample providing in-
depth information of biological systems. Therefore there have been numerous
studies where investigators have
tried to enhance the resolution of the STED microscopy in z plane by combining
STED and 4pi microscopy [??].
Another simpler approach in extending the power of STED microscopy to the
third dimension has been the
introduction of an axial-doughnut. This was achieved by using a phase mask
pair, one of which acts for lateral
and the other for axial resolution enhancement.[ Javad N. Farahani1 , M,
2010].
SUMMARY OF THE INVENTION
In the prior art a single point scanning STED system is made to scan the
sample a region of interest (ROI) point
by point with increments smaller than the optical resolution limit of the
optical system, and an image is captured at
each stationary beam scan position producing an array of pixel intensities.
Multichannel STED microscopy systems may employ a principle similar to one of
single point scanning STED
systems, but image multiple points in the sample plane simultaneously. While
the principles of scanning STED
microscopy do not rest on those of the confocal microscope, multiple
illumination and multipoint scanning
methods, developed for confocal microscopy, may be applied for implementation
of a multichannel scanning
STED microscope to great effect. Multichannel linear scanning systems offer
several useful advantages over
single spot scanners, for example, faster scanning of the sample ROI enabling
higher image capture rates and/or
a large sample ROI to be achieved. As compared to conventional single point
scanning, multibeam scanning
requires a significantly lower level of light intensity per unit area, which
results in significantly reduced photo
bleaching and photo toxicity effects, especially when applied to the 2D and 3D
imaging of living cells in life
science research.
Line scanning STED microscopy system provides much higher imaging signal
level, which decreases
proportionally a STED resolution (i.e. ¨Ax), unlike point scan STED systems,
whose signal levels drop down
proportionally to a square of its resolution (i.e. ¨ Ax2). Consequently
improved spatial image resolution in these
systems is also highly desirable. Multifocal specimen scanning makes it
possible to employ longer excitation laser
pulses of few nanosecond duration against femto- and picosecond pulse
durations in conventional single-point
scanning STED microscopes, thus making it possible to get significantly higher
fluorescence signals and signal-
to-noise ratios when applying excitation light pulses of lower pulse power.
Resolution of the line scan STED microscopy system may be described in terms
of point spread function (PSF).
The PSF of the line scan microscope in XY-plane has an elliptical shape with
its semi-major axis, oriented along
the focal line of beamlet projection (X-axis), equal to Airy function width
characterizing a diffraction-limited
resolution and its semi-minor axis, oriented across the beamlet projection
line (Y-axis), defined by the STED
resolution.
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The concept of RESOLFT inevitably requires scanning (with a zero), but not
necessarily with a single beam or a
point-like zero. Multiple zeros or dark lines produced by the interference of
counter-propagating waves (US Pat.
2016/0363751) in conjunction with conventional digital camera detection can
also be used, provided the zeros or
the dark lines are further apart than about the distance required by the
diffraction resolution limit of conventional
CCD camera imaging. Dark lines increase the resolution in a single direction
only, but stepwise rotation of the
pattern plus interleaved scanning of the minima (e.g. by shifting the phase in
the interference pattern) and
subsequent computational reassignment (Heintzmann and Cremer, 1998; Heintzmann
etal., 2002) may provide,
under some conditions, similar transverse resolution as with points and do so
at higher recording speed.
The major objective of the present invention is a provision of a method
providing high frame rate wide field ROI
imaging attractive and affordable for 2D and 3D super-resolution STED imaging
biological samples and
particularly in-vivo super resolution imaging.
Another objective of the present invention is a provision of a multichannel
line scan STED microscopy system
capable to provide high STED imaging rate desired for 2D and 3D imaging live
micro-objects and/or other
biological specimens.
Yet another objective of the present invention is provision of implementations
of the multichannel STED
microscopy system showing vide field of view.
The present invention describes methods of realization of a multichannel line
scanning STED microscopy, and
examples of implementation of a wide-field multichannel line scan STED
microscope to achieve this requirement
Furthermore, a number of exemplary embodiments of the respective multichannel
line scan STED microscopy
systems, which realize the method is provided. They are seen as simple, cost
effective instruments that acquire
images faster as and with less damaging effects on samples than the STED point
scanning systems.
Even if the invention will be described by particular reference to a STED
microscope in which the STED beam is
the beam of depletion light in the following, the invention is not limited to
STED microscopes but also relates to all
other fluorescence light scanning microscopes using a beam of suppression
light in addition to a beam of
excitation light, like, for example GSD-microscopy, up-conversion-depletion
microscopy [5], other RESOLFT
techniques [6].
Other features and advantages of the present invention will become apparent to
those skilled in the art upon
examination of the following drawings and the detailed description. It is
intended that all such additional features
and advantages be included herein within the scope of the present invention,
as defined by the claims.
SHORT DESCRIPTION OF THE DRAWINGS
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The invention will be better understood with reference to the following
drawings. The components in the drawings
are not necessarily to scale, emphasis instead being placed upon clearly
illustrating the principles of the present
invention. In the drawings, like reference numerals designate corresponding
parts throughout the several views.
FIG. 1 depicts a schematic perspective view of a first exemplary embodiment of
a multichannel line scan STED
microscopy system in accordance with the present invention. In addition to the
major components and units there
are shown in FIG. 1 conjugate image planes, as well as chief rays of
aggregated excitation light beam, illustrated
by outlined arrows, aggregated depletion light beam, illustrated by bold
arrows, and an imaging light beam,
illustrated by stripy arrows. Insert: optional astigmatic imaging optics
providing changed aspect ratio images.
FIG. 2 presents perspective diagrams of 2D intensity profiles of excitation
and depletion light probe beamlet
involved in the line scan STED microscope. Panels (a, b) depict the lateral XY
intensity distributions of the
excitation and depletion light beam profiles. Panel (c) depicts the
corresponding resulting XY intensity distribution
of the detection light. Comparison of (a) and (c) exhibits the effect of the
STED technique.
FIG. 3 is pictorial schematic views of the phase modulation plate designs
according the present invention with
corresponding laser intensity YZ distributions in the sample plane of the
microscopy system and XY intensity
distributions of depletion light providing lateral (Y ¨axis) fluorescence
sample STED confinement.
FIG. 4 depicts a simplified schematic diagram of the second exemplary
embodiment of the multichannel line scan
STED microscopy system in accordance with the present invention. There are
schematically shown major optical,
electronic and mechanical components, conjugate image planes of the optical
setup, as well as chief rays of
aggregated excitation light beam, illustrated by outlined arrows, aggregated
depletion light beam, illustrated by
bold arrows, and an imaging light beam, illustrated by stripy arrows.
FIG. 5 depicts a simplified schematic diagram of the third exemplary
embodiment of the multichannel line scan
STED microscopy system in accordance with the present invention. Multifocal
STED microscopy systems may
employ a similar principle, but image multiple points in the sample plane
simultaneously. These fast scan
multichannel STED systems preferably use an array of ... illumination
apertures that simultaneously capture
multiple points of the image. Multifocal confocal systems may be important
microscopy tools, for example, in life
science research.
FIG. 6 depicts a simplified schematic diagram of one possible implementation
of the pulsed laser light sources.
DETAILED DESCRIPTION
Embodiments according to the present invention will now be described
hereinafter with reference to the
accompanying drawings, where similar reference numbers indicate similar or
identical features throughout the
drawings.
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In describing preferred embodiments of the present invention illustrated in
the drawings, specific terminology is
employed for the sake of clarity. However, the invention is not intended to be
limited to the specific terminology so
selected, and it is to be understood that each specific element includes all
technical equivalents that operate in a
similar manner to accomplish a similar purpose.
As used herein, "multichannel line scan STED microscopy" refers to the
confocal line-scanning microscopy
technique whereby observations of fluorophore-labeled samples can be made
using a plurality of astigmatic probe
beamlets comprising spatially and temporally overlapped pulses of excitation
and of phase-modulated double-line
shaped de-excitation or depletion light of different wavelengths, which are
focused into a sample ROI. Since
each double-line shaped depletion beamlet has at least one zero intensity
line, only the fluorescent labels in the
proximity of the zero intensity line can actually emit fluorescence when they
return to the ground state.
A "beamlet", as used herein, refers to any one of plurality wide thin beams
provided by a lenslet array. An
"individual beamlet (spot, axis)" refers to one of plurality of corresponding
beamlets (spots, axes).
A "probe beamlet" or "STED beamlet", as used therein may be used
interchangeably and refer to a two
wavelengths beamlet, which being focused provides a diffraction limited
excitation light strip overlaid with a
double-line shaped strip of depletion light, in other words, each probe
beamlet presents a single channel of the
multichannel line scan STED microscope.
An "aggregated excitation (depletion, probe, and imaging) beam", as used
herein, refers to any beam comprised
of a plurality of corresponding beamlets, i.e. of excitation, depletion,
probe, and imaging beamlets.
A "compound beam", "compound beamlet" as used herein, refers to any aggregated
beam and/or beamlet
comprised of two co-aligned beams of pulsed laser radiation of different
excitation and depletion wavelengths.
NA ¨ "numerical aperture", is the sine of the maximum angle of a light beam in
the range of angles over which an
optical system can accept or emit light. NA of a converging and/or diverging
light beam is the sine of the half-
angle of the beam cone multiplied by a refractive index of the medium. An
astigmatic beam having a rectangular
cross-section may have two different NAs characterizing the beam
divergence/convergence in two orthogonal
planes.
FOV ¨ "field of view" is an area of the analyzed object captured on the
system's imager;
ROI ¨ "region of interest" is an observable part of an object to be imaged,
sometimes used as a synonym of FOV;
FWHM -- "full width at half maximum" is a parameter commonly used to describe
the width of a "bump" or "dip" on
a curve or function. FWHM is applied to such phenomena as the duration of
pulse waveforms and the spectral
width of filters.
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FPS ¨ "frames per second" is an imaging frame rate.
In the discussion that follows, several scanning mirrors are described as
"galvanometer-controlled." This is for
sake of brevity and clarity only. There are many different means to deflect a
light path, such as piezo-controlled
scanning mirror, parallel glass plate image shifting scanner, and the like.
All of these devices are encompassed
by the present invention and can be used in place of the galvanometer-
controlled scanning mirrors explicitly
depicted in the drawing figures. Collectively, these devices or any
combination of these devices is referred to
herein as "means for scanning the light path."
Referring now to FIG. 1, which is a simplified schematic diagram of the first
exemplary embodiment of the
multichannel line scan STED microscopy system in accordance with the present
invention. In addition to the
major optical components and units there are shown in FIG. 1, chief rays of
aggregated excitation light beam,
illustrated by outlined arrows, aggregated depletion light beam, illustrated
by bold arrows, and aggregated
imaging light beam, illustrated by stripy arrows, sample plane marked SP, as
well as conjugate image planes
marked IP1, IP2, and IP3.
The entire assembly of the proposed embodiment is preferably comprising: a
pulsed excitation laser light source 1
that provides excitation pulsed laser radiation a pulsed depletion laser light
source 2 that provides depletion
pulsed laser radiation; a phase modulation and light beam segmentation unit 3
incorporating a phase modulation
mask 31, beam combining means 32, light beam segmentation means 33, and a
projection lens 34, an optical
microscope 4 incorporating a high magnification objective lens 41, a tube lens
42, a XYZ scanning microscope
stage 43, and a microscopy sample 44 placed on said microscope stage 43, a
multi-beam scanner module 5
incorporating a galvo-scanner 51 with a scanning mirror 52, relay optics 53,
54, and an image rotator 55, and a
light detection and imaging module 6 incorporating a 2-dimensional imaging
detector 61, a beam sampling optical
element 62, an imaging lens 63, and a blocking filter 64.
The pulsed excitation laser light sources 1 provides a preferably collimated
or nearly collimated highly uniform
("top-hat") millimeter-scaled light beam of pulsed excitation laser radiation
of a first wavelength A,, and preferable
pulse duration Ti = 0.5 ... 10 ns or more for exciting, in single- or multi-
photon excitation mode, a fluorophore in a
sample to be imaged. The pulsed depletion laser light sources 2 provides a
similar preferably collimated or nearly
collimated top-hat millimeter-scaled light beam of pulsed depletion laser
radiation of a second wavelength A2 for
depopulation of excited states of fluorophore molecules and so suppressing
spontaneous emission of
fluorescence light by the fluorophore in the sample. A preferable duration T2
of depletion light pulses mast satisfy
the condition: 12
+ 0.2...2 ns, so that temporal FWHM of the depletion light pulse exceeds a
full duration of the
excitation light pulse, and temporally overlays sad excitation light pulse.
The depletion light pulses preferably may,
for example, arrive at the sample plane synchronously with the excitation
light pulses and proceed for at least 0.2
ns longer than the excitation pulses. The excitation and depletion laser pulse
energies and/or pulse powers are
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functions of a number of the probe beamlets, physical properties of samples
and fluorophore labels used, field of
view (FOV) and optical properties of the STED microscopy system.
The pulsed laser light sources 1, 2 may be built in a variety of forms,
previously developed for illumination in
confocal microscopy. Each pulsed laser light sources may comprise one or more
lasers, each laser generating
light of a different wavelength and additional optical elements, including,
for example, lenses, dichroic and turning
mirrors, one or more optical fibers, one or more beam shaping elements,
diaphragms, and mechanical
components. One possible implementation of the pulsed laser light sources 1, 2
is schematically depicted in FIG.
6. For example, said pulsed laser light source 1 preferably comprising at
least one pulse laser 11 for providing a
beam of excitation light of an excitation wavelength Al, (or a beam of
stimulated emission depletion light of a
depletion wavelength A2), light delivery optics, schematically presented in
the form of fiber coupling lens 12,
providing coupling said excitation or depletion light beam into a single mode
fiber 13, and beam conditioning
means schematically presented in the form a collimation lens 14, beam shaping
means 15, and a projection lens
16. Said beam conditioning means are providing a collimation and shaping of
the corresponding delivered laser
beam, in other words, said means transform the diverging light beam outgoing
from the distal end of said fiber 13
into collimated top-hat light beam of the laser radiation having millimeter-
scale cross section and exhibiting high
evenness of the transverse intensity distribution. Said excitation and
depletion pulsed laser light sources 1, 2 are
synchronized with and their repetition rate and laser pulse powers depend on
specifications of said multi-beam
scanner 5 and of said multi-channel light detection and imaging module 6, as
it is illustrated by an example of
realization hereinafter.
Said light delivery fiber 13 may be implemented in the form of a single-mode
fiber patch cable and/or a single-
mode polarizing fiber patch cable. Said beam shaping means 15 may be built in
a variety of forms, previously
developed for illumination in confocal microscopy, for example, those based on
diffractive beam shaper elements,
refracting optical components, such as crossed Powell lenses, filter means, or
beam shaping lens (for example
GTH-5-250-4, by TOPAG), and comprising some additional optical components,
such as imaging and projection
lenses, mirrors and similar.
Substantially uniform transverse distributions of the composed light beams
exiting the pulsed laser light sources
1, 2 may result in substantially equal intensities of the individual
astigmatic probe beamlets that results in uniform
excitation and following uniform selective depletion of fluorophore molecules
in the sample, which, after a
complete scan across a sample to be probed by a microscope, may result in a
substantially uniform illumination
thus making it possible to achieve a substantially uniform spatial resolution
and brightness of a resulting STED
image of the sample FOV and to avoid providing undesired artifacts. Note that
the power density of excitation light
on sample plane is responsible for STED image brightness, while the power
density of depletion light on the
sample plane is responsible for STED image resolution, and strongly affects on
its brightness.
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The pulsed laser light sources may be alternatively implemented in the form of
a single unit providing both the
beam of excitation light and the overlaying beam of depletion light and
provided with a dichroic beam splitting
means separating and properly directing excitation and depletion light beams.
Such a common laser source may
be implemented, for example, by coupling two separate lasers into common
optical fiber, by using a multi-
wavelength laser, or by using a super-continuum light source or another light
source providing both the excitation
light and suppression light (see for example WO 2009/047189 Al). Preferably,
depletion and excitation light
pulses are delivered by a single two wavelengths (compound) light beam by the
same fiber optic patch cord13,
which delivers the single two wavelengths beam of pulsed laser radiation to a
common beam conditioning means,
identical to said collimation lens 14, beam shaping means 15, and a projection
lens 16.
According the present invention, said phase modulation and light beam
segmentation unit 3 comprises a phase
modulation mask 31 provided with phase step bars 311 oriented along, for
example, X-axis, and providing phase
modulation of said collimated top-hat light beam of the depletion pulsed laser
radiation, a beam combining means
32, which merges said top-hat excitation light beam and the phase modulated
top-hat depletion light beam and
provides compound beam of two wavelengths pulsed laser radiation, and light
beam segmentation means 33
comprising an array of cylindrical focusing elements 331 splitting said
collimated compound beam, comprising
excitation and phase modulated depletion pulsed laser radiation, into an array
of astigmatic focused probe
beam lets.
Said phase modulation mask 31, said beam combining means 32, and said light
beam segmentation means 33
are arranged so that said phase modulation mask is disposed in a vicinity of
the back focal plane of said beam
segmentation means 33, with its phase step bars 311 parallel to axes of its
cylindrical focusing elements 331
oriented along X-axis, and a common focal plane of said lenslets 331
coinciding with an intermediate image plane
IP2 of the proposed line scan multichannel STED microscopy system.
Said phase modulation mask 31 is a square wave phase modulator meant to impart
phase retardation into at
least one part of the collimated depletion beam along, for example, Y-axis and
leaving it undisturbed in an
orthogonal X-direction. In this regard, the phase modulation mask 31 may be a
phase ruling with phase step bars
oriented along X-axis. Said phase ruling, a generalized design of different
implementations of which is presented
in FIG. 3a, is preferably built in the form of an optical substrate provided
with one or more equidistant parallel Tr-
step retarder bars 311 (colored in gray) of optical material having the step
height h and the step width wi, and
spaces 312 having the same equal widths w2 = wl . The step height h is adopted
to provide difference between
optical path lengths for rays, travelling through bars and through spaces,
equal to X2 /2, thus providing -rr - phase
retardation for these rays:
h =pn ___________________________ (2k+1)
2(n-1)
(2)
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Here n is a refractive index of the retarder bar material, k = 0, 1, 2... Said
phase ruling may be manufactured by
chemical or plasma etching, by plastic or glass molding, or rather by
deposition of optical materials, such as
magnesium fluoride or silicon dioxide onto a glass substrate. Heights of said
retarder bars depend on a material
of the bars and a wavelength of light to be retarded, and varies, for example,
in the range 0.86 pm to 1.37 pm for
MgF2 and in the range 0.75 pm to 1.2 pm for 5102 for the wavelength range 473
nm to 750 nm.
Schematic views of alternative example implementations of the phase ruling 31
providing one or more Tr-step
phase modulation of said top-hat depletion light beam with wavelength A2, and
their superposition with said beam
segmentation means 33 are schematically depicted in FIGS. 3b, 3d ¨ 3e being
accompanied with corresponding
light intensity YZ distributions of the depletion light, providing the lateral
(3b, 3d) and axial (3e) fluorescence -
STED confinement in the image and/or sample planes of the microscopy system;
FIG. 3c additionally presents an
XY intensity distribution (not in a scale) of depletion light providing
lateral (Y ¨axis) fluorescence sample STED
confinement. The phase ruling of the first design (FIG. 3b) has retarder and
space widths w1 = w2 = a, where a is
a single lenslet dimension along Y-axis; the phase ruling of the second design
has retarder and space widths w1
= w2 = a/2 (FIGS. 3d, 3e).
Said phase modulation mask 31 is described herein as a "phase ruling". This is
for sake of brevity and clarity only;
it may refer to any plurality of optical elements that can be used to provide
desired phase modulation of a
collimated top-hat light beam of the depletion pulsed laser radiation. The
optical elements may be, for example,
transmitting and/or reflecting phase modulating masks providing stepwise or
smooth phase modulation functions,
phase masks having finer modulation structures, or any other periodic or
aperiodic phase modulation optical
element, as it would be apparent to someone skilled in the art.
Said beam combining means 32, which merges said collimated excitation light
beam and the phase modulated
collimated depletion light beam and provides compound beam of two wavelengths
pulsed laser radiation, may be
implemented in the form of a dichroic mirror or, alternatively, in the form of
other beam coupling components,
such as, for example, polarizing beam splitters, diffraction gratings, or
other preferable optical components, as
would be apparent for someone skilled in the art.
Said light beam segmentation means 33 comprising one or more cylindrical
focusing elements 331 is preferably
implemented in the form of a lenticular lens, i.e. an array of identical
positive cylinder or acylinder lenslets 331
with equal focal distances, equal Y-axis dimensions denoted as a, and equal Y-
axis numerical apertures NAT.
The numerical apertures NA y of the cylindrical lenslets 331 are preferably
equal to the NA.41 of the objective lens
41 divided by magnification ratio of lenticular focal plane IP2 to SP, which
is defined in the present first
embodiment by focal distances of said lenses 53, 54, and by the microscope
magnification equal to the ratio of
focal distances of the microscope tube lens 42 and of said objective lens 41:
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F54
NA21 = NA" F41
F42 F5 3 (3)
The light beam segmentation means 33, are meant to provide one or more probe
beamlets, each of which is
collimated in X-direction along lenslet axes, is focused in Y-direction,
orthogonal to the lenslet axes, travels in Z-
direction, and comprises excitation light pulses and spatially and temporally
overlaying Tr-step phase-modulated
depletion light pulses. Said probe beamlets have equal Y-axis numerical
apertures NAT, while their X-axis values
NA x= 0, and are focused into a common focal plane, which is an illumination
aperture of the proposed STED
microscopy system, coinciding with an intermediate image plane IP2 conjugate
to an image plane IP1 and to a
sample plane SP of the microscope 4, and is to form in said image plane IP2
corresponding one or more regular
nearly diffraction limited co-axial focal lines of excitation light overlaid
with double-line shaped depletion light
projections.
Said light beam segmentation means 33 is described herein as a "lenticular
lens" or "cylinder lenslet array". This
is for sake of brevity and clarity only; it may refer to any plurality of
optical elements that can be used to focus light
onto corresponding focal lines in said illumination aperture IP2. The optical
elements may be cylindrical
microlenses, cylindrical micromirrors, or any other cylindrical focusing
elements, as it would be apparent to
someone skilled in the art.
In one exemplary implementation of said phase modulation and light beam
segmentation unit 3, depicted in FIGS.
3b, 3d, said phase modulation masks of one of two alternative designs, is
mounted close to the back focal plane
of said lenticular lens 33, so that the bars 311 of said phase ruling 31
provide Tr-step retardation of one half of
each depletion light beannlet in a line, projected onto axis and/or vertex of
a corresponding cylinder lenslet, thus
providing multiple similar linear laser intensity YZ distributions of
depletion light in the image plane IP2 and the
conjugate sample plane SP of the microscopy system, schematically presented in
FIGS. 3b, 3d (not in a scale); a
corresponding XY intensity distribution is depicted in FIG. 3c. These
distributions provide lateral sample
fluorescence confinement along one coordinate axis (Y ¨axis) desired for
providing a line scan lateral STED
image of a sample FOV.
In another exemplary implementation of said phase modulation and light beam
segmentation unit 3, depicted in
FIG. 3e, said phase modulation mask is mounted close to the back focal plane
of said lenticular lens 33, so that
the bars 311 of said phase ruling 31, provide Tr-step retardation of the
depletion light beam over the central part
along each of said one or more probe beamlets and consequently provide
multiple linear laser intensity YZ
distributions of depletion light in the sample plane of the microscopy system,
schematically presented in FIG. 3e
(not in a scale). These distributions provide axial sample fluorescence
confinement along Z coordinate axis
desired for providing a line scan axial STED image of a sample FOV.
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Said phase modulation mask with w1 = w2 = a/2 presented in FIGS. 3d, 3e may be
universally used as for lateral
fluorescence confinement (Y-axis confinement, FIG. 3d), as for axial one (Z-
axis confinement, FIG. 3e) and a
corresponding STED imaging. For these purposes said phase modulation mask 31
(design d, e) is to be shifted
for a half of a retarder bar width, i.e. for a/4, providing Tr-step
retardation of one half of each depletion light
beamlet (FIG. 3d), or over the central part of each depletion light beam let.
Said optical microscope 4 having an image plane IP1 and a sample plane SP is
preferably implemented in the
form of fare field light microscope comprising a high magnification, high
numerical aperture (NA) preferably
immersion objective lens 41, a tube lens 42, a XYZ scanning microscope stage
43, and a microscopy sample 44
placed into the sample plane SP of the microscope 4 and fixed on said
microscope stage 43.
Said optical microscope 4, provides demagnified projection of said plurality
of astigmatic probe beamlets into a
corresponding plurality of regular nearly diffraction limited focal strips of
excitation light overlaid with a
corresponding plurality of co-axial and double-line shaped strips of depletion
light in the sample plane SP,
collecting fluorescence light spontaneously emitted by the fluorophore in said
plurality of excited areas of
overlapped co-axial strips of excitation light and double-line shaped strips
of depletion light in the sample plane
SP and magnified imaging the plurality of the fluorescence light emitting
lines on said image plane IP1 of the
optical microscope 4.
The diffraction limited focal lines of the excitation light onto the sample
plane have profiles described by the
function:
-2
= sl
(Y) = sin(y
y = sl
(4)
while profiles of double-line shaped projections of the depletion light are
described by the function:
(Y)
¨ COSO, = S2)] , 2 = /20 = y (5)
= s2
where /10 and /20 are maxima of the intensity distributions of the excitation
light and of the depletion light
respectively, and si = 2ir = NA I (where i = 1, 2) is the pattern steepness
across the excitation line (Si)
and across the depletion double-line (S2). FIG. 2 presents perspective views
of both XY profiles of the excitation
light intensity distribution (panel a) and the depletion light intensity
distribution (panel b) of probe beam lets
involved in the line scan STED microscope. Panel (c) depicts the resulting XY
intensity distribution of the
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fluorescence light emitted from the lines confined by the depletion light.
Comparison of (a) and (c) exhibits the
effect of the line scan STED technique providing, for example, four-fold
increase of the microscopy resolution.
It should be noted that a lateral offset of the central lines of the phase
modulation mask bars with regard to the
optical axes of the cylinder lenslets does not affect the intensity zero of
the intensity distribution of the depletion
light at the focal lines, but just tilts the double-line shaped intensity
distribution of the depletion light with regard to
the common optical axis and slightly shifts the intensity zeros laterally with
regard to the geometric focal lines.
However, the intensity zero lines remain essentially at the depletion light
strips..
Said multi-beam scanner module 5 comprising a single axes beam scanning head,
schematically presented an
the form of a galvo-scanner 51 and a galvanometer-controlled scanning mirror
52, relay optics, schematically
presented in the form of two lenses 53, 54, and an image rotator schematically
presented in the form of Dove
prism 55. Said multi-beam scanner module 5 provides a parallel multi-beam line-
scanning technique to increase
the speed of STED image capture over single beam point-scanners. Line scan
multichannel STED microscopy
systems proposed in the present invention employ a principle similar to the
single point scan STED systems, but
unlike the single point systems, images multiple STED lines in the sample
plane SP simultaneously. Said scanner
module 5 provides simultaneous scan of said sample 44 by multiple parallel
astigmatic probe beamlets formed by
said phase modulation and light beam segmentation module 3 and descan beam
lets of fluorescent light from the
focal lines on the sample plane SP field of view for being projected onto an
image sensor of a light detection and
imaging module 6.
Said single axes beam scanning head 51, 52 may be built as a single axis step-
by-step, continuous, or random
access mode scanner in a variety of forms, for example, those employing single-
axis angle galvanometer-
controlled or piezo-controlled scanning mirror, as well as single-axis
translational scanning employing, for
example, translational scanner or tilted glass plate scanner (US Pat.
7,443,554). Alternatively multichannel
imaging may be achieved, for example, by piezo-scanning the sample on a XYZ
nanopositioning scanning
microscope stage.
Said relay optics, schematically presented in the form of two lenses 53, 54
may be preferably implemented in the
form of relay optics of 4F-configuration. Said relay optics 53, 54 and said
scanning mirror 52 are arranged so that
the lens 53 is disposed at a distance from said illumination aperture IP2
equal to the lens focal distance F53, so
that said relay optics 53, 54 provide imaging of said illumination aperture
IP2 of said phase modulation and light
beam segmentation unit 3 onto conjugate image plane IP1 of the optical
microscope 4 via said scanning mirror
52, and via image rotator 55. Thus projection of said plurality of astigmatic
probe beamlets onto the microscope 4
image plane IPI is provided resulting in a corresponding plurality of regular
nearly diffraction limited focal lines of
excitation light overlaid with co-axial double-line shaped projections of
depletion light in said sample plane SP
conjugate to the image plane IP1.
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Said image rotator, schematically presented in the form of Dove prism 55 is
meant to provide the sample
scanning in at least two different directions, or preferably along three,
five, or more scanning axes. It may be
implemented in variety of forms, for example, in the form of Schmidt, Abbe,
Vee, Roentsch, Schmidt-Pechan
prisms, cylindrical lens rotator, or any other optical image rotator, as it
would be apparent to someone skilled in
the art. Said image rotator may be installed in any place between the
galvanometer-controlled scanning mirror 52
and the micro objective lens 41, for example between the lens 54 and the image
plane IPI , or preferably between
the scanning mirror 52 and said lens 54, as it is presented in FIG. 1.
Alternatively the sample scanning in a
number of different directions may be realized without said image rotator 55,
by a rotation of said sample 44 itself,
which must be placed onto an optional rotary stage (not shown), and deflecting
scanning beams in a direction
fixed relative the whole microscopy system.
Said light detection and imaging module 6 comprising said 2-dimensional
imaging detector 61 is meant to detect a
multitude of optical signals directed to it by said beam sampling optical
element 62 and provided by fluorescence
light spontaneously emitted by the fluorophore in said plurality of
diffraction limited focal strips of excitation light
constrained by overlaying co-axial double-line shaped strips of depletion
light in the sample plane SP. and to
provide row data for building digital STED images of the sample ROI.
Said light detection and imaging module 6 preferably comprises a 2-dimensional
imaging detector 61 with an
image sensor 611, high resolution imaging optics schematically presented in
the form of a lens 63, which, in
combination with said lens 54, forms s relay optic setup providing imaging of
said image plane IPI and
consequently said sample plane SP of the optical microscope 4 onto conjugate
image plane IP3 coinciding with a
face of said image sensor 611 with a desired magnification, and one or more
blocking filters 64 for rejection of
scattered, reflected by a specimen, and stray light of excitation and
depletion wavelengths. Said light detection
and imaging module 6 may be coupled to a remaining part of the STED microscopy
system with said beam
sampling optical element 62, to detect a corresponding multitude of optical
signals.
Said light detection and imaging module 6 may optionally comprise additional
optical and opto-mechanical
components (not shown), such as a confocal slit array with a number of slits
equal to the number of individual
STED probe beamlets, positioned in the image plane so that every slit aperture
transmits fluorescence light to a
corresponding row of image sensor pixels and rejects stray and ambient light,
second microlens array, image
scanner, and high resolution relay optics.
Said beam sampling optical element 62, transmitting light of said excitation
an depletion wavelengths A1, A2, and
reflecting fluorescence light, may be implemented in the form of appropriate
dichroic mirror, or preferable in the
form of a mirror slit placed into a focal plane of said lens 53 with the slit
orientation along Y¨axis, orthogonal to the
axes of cylindrical lenslets, and the slit width in X-direction superior to a
width of a focal light strip provided by said
lenticular lens 33 and said lens 53, when it focuses said collimated in X-
direction astigmatic probe beamlets.
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Said high resolution imaging optics 63 may be alternatively implemented in the
form of anamorphic imaging
optics, schematically presented in the FIG. 1 (insert) in the form of crossed
axes cylindrical lenses 631, 632,
which in combination with said lens 54, form anamorphic relay lens setup
providing imaging of said image plane
IP1 and consequently said sample plane SP of the optical microscope 4 onto
conjugate image plane IP3 with
changing its aspect ratio, wherein IP1 is reimaged onto IP3 with a
magnification mx along the width of imaging
beam lets (X-axis) equal to a ratio of focal lengths of the lenses 631 and 54,
and with a magnification my along the
height of imaging beam lets, i.e. in the scan direction (Y-axis), equal to a
ratio of focal lengths of the lenses 632
and 54:
M = F /F m =F IF
x 631 54 Y 632 54
Said imaging detector 61 is to be preferably mounted so that the face of said
image sensor 611 is disposed in or
close to the image plane labeled as IP3 that conjugate to IP1 and consequently
to the sample plane SP. The
imaging detector 61 may, be preferably built in the form of a digital camera,
such as EMCCD camera, or rather
sCMOS camera, having high sensitivity, low noise, high readout speed and frame
rate, and resolution adequate
to diffraction limited imaging STED scan lines (for example, iXon Ultra 888by
Andor Tchnology, or Prime 95B by
Photometrics), aligned relative to said phase modulation and light beam
segmentation unit 3 and microscope 4 so
that each fluorescence imaging astigmatic beamlet is projected onto a
corresponding defined set of image sensor
pixel rows or, alternatively, columns.
Said digital camera 61 is to be synchronized with said pulsed lasers light
sources1, 2, and to provide a separate
image frame for each pulse of the probe excitation and depletion light. Every
such image frame presents a digital
(noisy) image of the array of fluorescence imaging light stripes, having
spatially modulated light intensities along
strips and their equal widths defined by Rayleigh limit. A number of the
imaging light strips is equal to the number
of the astigmatic probe beamlets. After pre-processing said array of
fluorescence strip projections is transformed
into a similar array of strip images characterized by narrowed STED strip
widths with similar profiles and by
existing spatial modulation along strips. This procedure may be accomplished
in hardware by employing said
cylindrical imaging optics 631, 632, and by employing a sub-window scan mode
with rolling shutter, or
alternatively by operation said digital camera in so-called vertical binning
mode, when signals of multiple adjacent
rows are combined electronically before they are readout. Preliminary
calibration of the microscopy system
provides sub-pixel precision position coordinates of central lines of all the
fluorescence spatially modulated
imaging light stripe projections on the image sensor of digital camera 61 and
consequently precision positioning of
the corresponding probe light stripes on the sample.
Camera readout signals being pre-processed so in combination with the multi-
beam scanner data provides
intensities (photon counts) and coordinates of a multitude of points of the
partial STED frame. Said partial STED
frame has a number of the STED super-resolution lines, oriented across the
scan direction, equal to the number
of probe beannlets..
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Between two consequent compound laser pulses said galvanometer-controlled
scanning mirror 52 provides a
scanning deflection of array of the STED probe beamlets, projected into the
sample plane SP for a single scan
step, defined by the desired microscopy system resolution and the scan method
used. The scan step is followed
by a next compound laser light pulse providing a new array of probe beamlet
projections in the sample plane and
new partial STED frame, and so on. Co-processing of a plurality of said
partial STED frames, collected during a
full scan cycle, provides the intermediate single axis scan full frame STED
image of the sample FOV, which is
characterized by an elliptical PSF exhibiting a diffraction limited resolution
along the probe and imaging light strips
(along X - axes) and STED super-resolution across the probe and imaging light
strips, i.e. in the scan direction
along Y - axes.
Next step for providing a desired super-resolution STED image is a rotating
image rotator for a predetermined
angle and repeating the scan and imaging procedure. A resulting super-
resolution STED image of the sample
FOV may be reconstructed by combining and post-processing two or more single
axis scan intermediate STED
images taken along different scan directions.
The proposed multichannel line scan microscopy system may be successively used
for Z-stack scanning
providing multiple slice super-resolution STED images in order to obtain 3D
microstructure reconstructions. It may
be done by utilizing an additional one-dimensional (1D) Z-scanner, for example
a piezoelectric scanner. The focal
plane of the microscope is continuously adjusted and a series of 2D STED
slices are obtained with one of the
phase modulation masks. One can then reconstruct a stack of 2-D images from
the recorded intermediate single
axis scan STED images. The 3D model of the sample is then reconstructed by
combining and post-processing
the 2D slices by use of appropriate software.
EXAMPLE 1: If it is desired to get STED images of a sample labeled by a
fluorophore Alexa Fluor 488 with ROI
80 pm x 80 pm and a desired resolution 60 nm (-4 folds better than
"diffraction limit") in the first proposed
multichannel line scan STED microscopy system of FIG. 1, providing parallel
scan by the array of 64 probe
beamlets, and comprising a 100x oil immersion objective lens with NA = 1.4 and
EMCCD camera with its sensor
active pixels 1024 x 256 providing frame rate 240 frames per second at sub-
window rolling shutter mode, pulsed
laser light source 1 providing 473 nm excitation laser pulses with the pulse
duration 8 ns, and pulsed laser light
source 2 providing 561 nm depletion laser pulses with the pulse duration 10
ns. Laser pulse power densities on a
sample required for getting desired STED resolution are 5200 kW/cm2 for the
excitation laser pulse, and - 30
MW/cne for the depletion laser pulse, corresponding total energies delivered
to the sample are: excitation pulse
energy 50.4 nJ per line, depletion pulse energy - 30 nJ per line. The
corresponding required total laser pulse
energies delivered to the sample are: excitation pulse energy 525 nJ,
depletion pulse energy - 2 pJ, i.e. average
laser powers at said frame rate 240 FPS are 512 pW and - 1 mW respectively, if
an estimated transmission of the
STED microscope optical tract is - 50%. It is required for getting a full
resolution image at least two scans over
42 steps that means capturing and processing 84 partial STED frames. So the
STED image of the ROI 80 pm x
80 pm may be gotten with a frame rate <3 FPS depending on a rotation time t55
of the image rotator 55, that
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means - 2.8 FPS for the two axis scan and t55 = 10 ms, and - 1.8 FPS for three-
axis scan. Application of a digital
camera with a higher readout frame rate may proportionally increase the
reachable imaging rate.
A simplified schematic diagram of the second exemplary embodiment of the
multichannel line scan STED
microscopy system in accordance with the present invention is presented in
FIG. 4, where like reference
numerals are applied to like parts. There are also shown in FIG. 4 chief rays
of aggregated excitation light beam,
illustrated by outlined arrows, aggregated depletion light beam illustrated by
bold arrows, and imaging light beam
illustrated by stripy arrows, sample plane marked SP, and conjugate image
planes marked as IP1, IP2, IP3, and
IP4'.
This second embodiment is similar in general to the first one shown in FIG. 1,
and comprises the same major
modules: a pulsed excitation laser light source 1, a pulsed depletion laser
light source 2; a phase modulation and
light beam segmentation unit 3 incorporating a phase modulation mask 31 beam
combining means 32, and light
beam segmentation means 33, an optical microscope 4 incorporating a high
magnification objective lens 41, a
tube lens 42, a XYZ scanning microscope stage 43, and a microscopy sample 44
placed on said microscope
stage 43, and multi-beam scanner module 5 incorporating a galvo-scanner 51
with a scanning mirror 52, relay
optics 53, 54, and an image rotator 55, but has an alternatively implemented
light detection and imaging module
6.
The light detection and imaging module 6 of the second exemplary embodiment,
unlike the first embodiment,
preferably comprises a high resolution imaging device 65 with one or more
blocking filter 64, a beam sampling
optical element 62, a second cylinder lenslet array 66, an optional slit array
67 disposed close to the focal plane of
said second lenticular lens 66, a single axes image scanning head,
schematically presented an the form of an
image scanning mirror 58 controlled by a galvo-scanner 57 both identical to
said scanning mirror 52 and said
galvo-scanner 51, and a second relay optics, schematically presented in the
form of two achromatic doublets 68,
69.
Said light detection and imaging module 6 is meant to detect a multitude of
optical signals directed to it by said
beam sampling optical element 62 and provided by fluorescence light
spontaneously emitted by the fluorophore in
said plurality of diffraction limited focal strips of excitation light
confined by overlaying co-axial double-line shaped
projections of depletion light in the sample plane SP, and to provide row data
for building digital STED images of
the sample ROI.
Said high resolution imaging device 65 may be preferably implemented in the
form of a high resolution, high
sensitivity and low noise digital camera, such as EMCCD camera, or rather
sCMOS camera (for example, Neo
5.5 by Andor or ORCA-Flash4.0 v2 by Hamamatsu).
Said beam sampling optical element 62 may be implemented, for example, in the
form of a dichroic mirror
reflecting excitation and depletion light and transmitting fluorescence light.
Said sampling optical element 62 may
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CA 2973361 2017-07-14
be alternatively implemented in the form of a mirror slit with an additional
lens 63 in the manner similar to that of
the first and the third exemplary embodiments depicted in FIGS.1, 5, as it
would be apparent to someone skilled
in the art.
Said relay optics, schematically presented in the form of two achromatic
doublets 68, 69 may be implemented in
the form of a high resolution and low aberration relay lens meant to relay
with a desired scale the image plane
IP4' onto the image plane IP3 where face of sensor 651 of said digital camera
65 is disposed.
Said lenses 54, 53 form a relay optics providing a transfer of the microscope
image plane IP1 onto a conjugated
image plane IP4, close to which said second cylinder lenslet array 66 is
placed orthogonally to the optical axis.
Said second cylinder lenslet array 66 is similar but not identical to said
lenticular lens 33, i.e. comprises an equal
number of similarly arranged cylindrical lenslets 661, having unlike to
lenslets 331 focal distances 2 to 10 folds
shorter and correspondingly numerical apertures (NA) 2 to 10 folds more than
those of cylindrical lenslets 331.
Said second cylinder lenslet array 66 is designed to provide projection of the
sharp imaging fluorescence light
strips, whose widths dl are dennagnified by said cylindrical lenslets to be
equal to or less than the STED super-
resolution line width d3, that is
d3 / K ,
where dl is a width of a diffraction limited line image:
d1=
2 F42.3
NA F =F
41 ,41 54
F41, F42, F64 and F63 are focal distances of corresponding lenses 41, 42, 54,
and 63; K is the STED super-
resolution factor:
IC r-t,' 1 + -D- . (6)
/s
Lateral dimensions of individual cylindrical lenslets and of whole lenslet
array 66 are proportional and may be
equal to those of lenslet array 33. Said second cylindrical lenslet array 66
is preferably placed close to the image
plane IP4, so that every cylindrical microlens is disposed co-axial with a
corresponding imaging beam let and
focus individual imaging beams into the modified image plane IP4', thus
providing a corresponding multitude of
sharp focal lines of the detected fluorescence light. Said modified image
plane IP4' may not coincide with the
original image plane IP4. Said relay optics 68, 69, said scanning mirror 58,
and said high resolution digital camera
65 are arranged to project via said scanning mirror 58 and said blocking
filter 64 the multitude of sharp focal light
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strips provided by said second lenslet array 66 in the modified image plane
IP4' onto the image sensor 651 face
that disposed in the conjugate image plane IP3.
Said optional slit array 67 may be disposed in the modified image plane IP4',
so that every slit aperture is co-axial
with a corresponding cylindrical light focusing element of said second
lenticular lens 66, which projects into a
corresponding slit aperture a focal line of fluorescence light that is to be
imaged. The reasons for using a slit array
are the rejection of stray and ambient light and the convenient confocal axial
sectioning provided by this scheme.
It should be noted that confocality is not an ingredient of the concept of the
line STED microscopy, because the
STED microscopy resolution dominating factor is the depletion function q
(x,y,z), not the detection function of the
confocal slit or pinhole.
Said two pulsed laser light sources 1, 2 providing excitation and depletion
laser radiation are synchronized with
said scanner heads 51, 57 and said high resolution digital camera 65, whose
exposure time continues a number
of the laser pulse periods to get a required resolution of STED images in scan
direction (across the wide probe
beamlets). When a shutter of said digital camera is open, every pulse of
excitation and depletion light provided by
said pulsed laser light sources 1 and 2 generates on the image sensor of said
digital camera 65 a number of
sharp super-resolution imaging lines, equal to the number of astigmatic probe
beamlets, and having equal widths
and spatially modulated light intensities along strips. Between two consequent
probe laser pulses said
galvanometer-controlled scanning mirror 52 provides a scanning deflection of
array of the STED probe beamlets,
projected into the sample plane SP, for a single scan step, defined by the
desired microscopy system resolution
and the scanning method used, and said second galvanometer-controlled image
scanning mirror 58 provides a
synchronous and proportional single step deflection of said narrowed
projections of fluorescence imaging
beamlets across the digital camera 65 image sensor face, disposed in said
image plane IP3.
Every scan step is followed by a new laser light pulse, and so on. The scan
cycle may be finished, when a
distance between center lines of any two neighbor probe lines in the sample
plane becomes equal or less than a
half of the STED resolution. After finishing full scan cycle the shutter of
said digital camera 65 must be closed, the
camera 65 readout signal provides an intermediate single axis scan full frame
STED image of the sample FOV,
which is characterized by an elliptical PSF exhibiting a diffraction limited
resolution along the probe and imaging
light strips (along X ¨ axes) and STED super-resolution across the probe and
imaging light strips, i.e. in the scan
direction along Y ¨ axes. Next step for providing a desired super-resolution
STED image is a rotating said image
rotator 55 for a predetermined angle and repeating the scan and imaging
procedure. Post-processing of two or
more such intermediate STED images gotten in the course of sample scanning in
different scan directions
provides a full resolution STED image of the sample ROI.
The second exemplary embodiment of the multichannel line scan microscopy
system may be successively used
for Z-stack scanning providing multiple slice images in order to obtain 3D
model of microstructures by combining
and post-processing said Z-stack of 2D slices, as it is discussed hereinabove.
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EXAMPLE 2: The second exemplary multichannel line-scan STED microscopy system
providing parallel scan by
the array of 50 probe beamlets, comprises the pulsed laser light source 1
providing 473 nm excitation laser pulses
with the pulse duration 8 ns, the pulsed laser light source 2 providing 561 nm
depletion laser pulses with the pulse
duration 10 ns, a 100x oil immersion objective lens with NA = 1.4, and said
sCMOS camera ORCA-Flash4.0 v2 by
Hamamatsu, with the number of active pixels 2048 x 2048, and full resolution
frame rate up to 100 frames per
second. This multichannel line-scan STED microscopy system may be used for
getting STED images of a sample
labeled by a fluorophore Alexa Fluor 488 in the ROI 60 pm x 60 pm with a
resolution 60 nm (-4 folds better than
"diffraction limit"). It should be noted that the highest resolution of STED
images provided by the second
exemplary line scan multichannel STED microscopy system illustrated by FIG. 4,
is limited by the resolution of
said digital camera 65 and cannot be higher, than, for example 1024 lines in X
and Y directions, when it is used
said sCMOS camera ORCA-Flash4.0 v2 with its 2048 x 2048 sensor active pixels,
i.e. -60 nm for the desired ROI
60 pm x 60 pm. It is required 40 scan steps and 41 synchronized excitation and
depletion laser pulses for getting
a single axis scan intermediate STED image of the sample ROI. An optimal
repetition rate of excitation and
depletion lasers is 4.1 kHz being defined by the camera frame rate (100 FPS)
and by said number of excitation
and depletion laser pulses per a single frame (41 pulses). Laser pulse power
densities on a sample required for
getting desired STED resolution are 5200 kW/cm2 for the excitation laser
pulse, and - 30 MW/cm2 for the
depletion laser pulse. Corresponding energies delivered to the sample are:
excitation pulse energy 50.3 nJ per
line, depletion pulse energy - 20 nJ per line. The corresponding required
total excitation laser pulse energy
delivered to the sample is 515 nJ, total depletion pulse energy delivered to
the sample is - 1 pJ. The average
excitation and depletion laser powers at said pulse repetition rate 4.1 kHz
are 5120 pW and - 8 mW respectively,
if an estimated transmission of the proposed STED microscopy system optical
tract is - 50%. It is required for
getting the desired super-resolution STED image at least two scans over the
sample ROI. So that the STED
image of the ROI 60 pm x 60 pm may be gotten with a frame rate <50 FPS
depending on the laser pulse
repetition rate f and a rotation time t55 of the image rotator 55; that means -
40 FPS for the two axis scan, and
25 FPS for three-axis scan, if the laser pulse repetition rate f = 10 kHz and
the rotation time t55 = 10 ms.
A simplified schematic diagram of the third exemplary embodiment of the
multichannel line scan STED
microscopy system in accordance with the present invention is presented in
FIG. 4, where like reference
numerals are applied to like parts, chief rays of aggregated excitation light
beam are illustrated by outlined arrows,
aggregated depletion light beam is illustrated by bold arrows, and imaging
light beam is illustrated by stripy
arrows, a sample plane is marked as SP, and conjugate image planes are marked
as IP1, IP2, IP3, and IP4'.
This embodiment is similar in general to the second exemplary embodiment shown
in FIG. 4 and comprises the
same major modules: pulsed excitation and depletion laser light sources 1, 2;
the phase modulation and light
beam segmentation unit 3 incorporating a phase modulation mask 31, beam
combining means 32, and light beam
segmentation means 33, an optical microscope 4 incorporating the same
objective lens 41, a tube lens 42, a XYZ
scanning microscope stage 43 with a placed on it microscopy sample 44, and a
multi-beam scanner module 5,
but the last comprises indeed a bilateral galvanometer-controlled scanning
mirror 56, which in parallel performs
CA 2973361 2017-07-14
functions of the second galvanometer-controlled scanning mirror 58 of the
second embodiment depicted in FIG. 4,
and an alternatively implemented light detection and imaging module 6
preferably incorporating the same high
resolution imaging device 65 with the blocking filter 64, the beam sampling
optical element 62, the second
cylinder lenslet array 66, the optional slit array 67, the second relay optics
68, 69, an additional imaging lens 63
that forms in combination with said lens 54 a relay optic system, and folding
mirrors 70, 71, 72.
Said multi-beam scanner module 5, which is a part of the embodiment,
preferably comprises a beam scanning
head, schematically presented an the form of a bilateral scanning mirror 56
controlled by a galvanometer 51, relay
optics 53, 54 preferably of 4F-configuration, and an image rotator 55. Said
optical components 53 ¨55 are built
similarly to those of embodiment depicted in FIGS. 1, 4 and meant to perform
the same functions, while said
scanner head 51 with a bilateral scanning mirror 56 provides a scanning
deflection of probe beamlets and
descanning fluorescence imaging beamlets, both by a front side of the mirror,
and synchronous rescanning
deflection of imaging beamlets by the back side of the bilateral mirror 56.
Said image rotator 55, schematically
presented in the form of Dove prism, is meant to provide the sample scanning
in at least two different directions.
Said image rotator 55 is identical to one of embodiments of FIGS. 1, 4 and may
be installed in any place between
the scanning mirror 52 and the micro objective lens 41. Alternatively the
sample scanning in a number of different
directions may be realized by a rotation of said sample 44, as it is explained
hereinabove.
Said high resolution imaging device 65 may be preferably implemented in the
same form of high resolution, high
sensitivity and low noise digital camera, as one of the second embodiment, and
is meant to provide the same
function. Said beam sampling optical element 62, transmitting light of said
excitation an depletion wavelengths
and reflecting fluorescence light, is, in general, the same as one in the
first and in the second embodiments, and
may be implemented in the form of appropriate dichroic mirror, or in the form
of a mirror slit placed into a focal
plane of said lens 53. Said second cylinder lenslet array 66 is identical to
the lenticular 66 of the second
embodiment, and characterized by the shorter focal distance and the higher NA,
than ones of said lenticular lens
33.
Said lenses 54, 63 form a relay optics providing a transfer of the microscope
image plane IP1 onto a conjugated
image plane IP4, close to which said second cylinder lenslet array 66 is
placed orthogonally to the optical axis, so
that every cylindrical lenslet is disposed co-axial with a corresponding
imaging beamlet and focus individual
imaging beams into the modified image plane IP4', conjugate to said image
plane IP2 and said sample plane SP.
where may be disposed said optional slit array 67. Said relay optics 68, 69,
said bilateral scanning mirror 56,
folding mirrors 71, 72, and said high resolution digital camera 65 are
arranged to project said image plane IP4'
onto conjugate image plane IP3, where the sensor face of said high resolution
digital camera 65 is disposed.
The third exemplary embodiment of FIG. 5 operates in the same manner and
provides similar results as the
second exemplary embodiment of FIG. 4; the only difference is that the
bilateral galvanometer-controlled
scanning mirror 56 provides as a scanning deflection of probe beamlets and
descanning fluorescence imaging
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CA 2973361 2017-07-14
