CA2973361A1 - Multichannel line scan sted microscopy, method and device - Google Patents

Abstract

The invention discloses a multichannel line scan STED microscopy method and devices providing high imaging frame rates and wide field of view super-resolution STED microscopy capable of resolving details below the Abbe diffraction limit. The multichannel line scan STED microscopy is a technique whereby observations of fluorophore-labeled samples is realized by scanning ROI of said sample with a plurality of diffraction limited excitation light focal lines spatio-temporally overlayed with a plurality of co-axial double-line shaped projections of red shifted depletion light having zero intensity central lines. The depletion beamlets suppress the fluorescence everywhere within the focal region, except at the zero intensity lines and their proximity, thereby confining the width of line of effective molecular excitation and hence of fluorescence. The fluorescence from these narrowed lines is registered after each scan step followed by at least one excitation and depletion light pulse, thus providing after finishing one direction scan cycle an intermediate image exhibiting a STED super-resolution in the direction across the probe light lines and diffraction limited resolution along said probe light lines. Post-processing a multitude of such intermediate images taken along two or more different scan directions provides a full super-resolution STED image of the sample ROI.

Description

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) 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.

2 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.

3 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

4 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.

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.

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 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.

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) 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:

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.

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:

= 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 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.

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.

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..

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 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 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 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.

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 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

Claims (12)

1. A method of multichannel line scan stimulated emission depletion (STED) microscopy imaging a structure of interest in a sample, the method comprising the steps of:
- providing a sample, comprising structures of interest labeled with a fluorescent dye;
- providing one or more astigmatic probe beamlets each of which is focused in a first plane and collimated in an orthogonal second plane and comprises light pulses having a first wavelength .lambda.1 for exciting, in single- or multi-photon excitation mode, a fluorophore labels in a sample to be imaged and synchronized and spatio-temporally overlaying .pi.-step phase-modulated light pulses having a second wavelength .lambda.2, for de-excitation of excited states of fluorophore molecules;
- directing and focusing said probe beamlets onto said sample, providing projection of said one or more astigmatic probe beamlets into corresponding one or more probe areas in the form of nearly diffraction limited focal lines of the first wavelength light providing label excitation overlaid with co-axial double-line shaped projections of the second wavelength light having zero intensity central lines, and depleting the fluorescence everywhere within the focal regions, except at the zero intensity lines and their proximities, thereby confining the width of line of effective molecular excitation and hence of fluorescence, - repeatedly scanning the sample with said one or more astigmatic probe beamlets in the predetermined scanning range in at least two different directions;
- collecting and imaging detection of fluorescence light spontaneously emitted by the fluorophore in said one or more STED narrowed fluorescent areas after each scan step followed by at least one pulse of the excitation light overlaid with the de-excitation light, providing after finishing one direction scan cycle, in combination with the beamlet scanning data, an intermediate image exhibiting a STED super-resolution in the direction across the probe light lines and diffraction limited resolution along the probe light lines; and - post-processing of said intermediate images taken along said at least two different scan directions providing a full super-resolution STED image of the sample ROI.

2. A method of Claim 1, wherein a duration time T1 of said pulse of the light of the first wavelength .lambda.1 is equal or more than 0.5 ns, and a duration time T2 of said temporally overlaying pulse of the light of the second wavelength .lambda.2 exceeds the duration of said first wavelength light pulse.

3. A method of Claim 1, wherein .pi.-step phase modulation of the light of the second wavelength A2 is provided to semi-bandwidth of each of said one or more probe beamlets relative to its center line.

4. A method of Claim 1, wherein .pi.-step phase modulation of the second pulsed light is provided to the central part along each of said one or more probe beamlets.

5. A method of Claim 1, wherein an angle .theta. between two or more scan directions is defined by a number m of different scan directions, and may be calculated according the formula:
.theta. = 180°/m.

6. A STED microscopy system comprising:
- a first laser light source that provides a collimated millimeter-scaled light beam of pulsed laser radiation of a first wavelength .lambda.1 for exciting, in single- or multi-photon excitation mode, a fluorophore in a sample to be imaged;
- a second laser light source that provides a collimated millimeter-scaled light beam of pulsed laser radiation having a second wavelength .lambda.2 for suppressing spontaneous emission of fluorescence light by the fluorophore in the sample;
- a phase modulation mask in the form of square wave phase modulator comprising one or more .pi.-step retarding bars;
- a beam combining means merging said light beam of pulsed laser radiation of the first wavelength .lambda.1 and said light beam of pulsed laser radiation of the second wavelength .lambda.2;
- light beam segmentation means in the form of a first lenticular comprising one or more cylindrical or acylinder lenslets of equal focal distances, and providing one or more probe beamlets each of which is focused in a first direction orthogonal to the lenslet axes and collimated in a second direction along lenslet axes, and comprises light pulses having a first wavelength .lambda.1 and spatially and temporally overlaying .pi.-step phase-modulated light pulses having a second wavelength .lambda.2;
- a projection lens;
- an optical microscope comprising a high magnification objective lens, a tube lens, a microscope stage, and a labeled with a fluorescent dye microscopy sample placed on said microscope stage;
- a multi-beam scanner module comprising a first scan means with a scanning mirror, relay optics, and an image rotator for scanning the probe light beamlets, delivered from the light beam segmentation means, across the sample in two or more different directions and for de-scanning the imaging fluorescence light beamlets from the sample;

- a beam sampling optical element in the form of a mirror slit placed into a focal plane of said projection lens with the slit oriented orthogonally to the axes of cylindrical lenslets, or in the form of dichroic mirror transmitting the light of the first and of the second wavelengths and reflecting the fluorescence light; and - light detection and imaging module.

7. The STED microscopy system according to Claim 6, further comprising an operation unit for analyzing and post-processing of preliminary image data and providing a super-resolution STED image of said sample.

8. The STED microscopy system according to Claim 6, wherein said phase modulation mask, said dichroic mirror, and said lenticular are arranged so that said phase modulation mask is disposed close to the back focal plane of said lenticular with their bars parallel to the axes of said cylinder lenslets and providing .pi.-step retardation of said light beam of the second wavelength in parallel lines, projected equally into apertures of said cylinder lenslets, and said lenticular lenslets focus said astigmatic probe beamlets in a common focal plane conjugate to an image plane and to a sample plane of said microscope.

9. The STED microscopy system of Claim 6, wherein said light detection and imaging module comprises:
- a 2-dimensional imaging sensor in the form of a digital camera, - an imaging lens, - at least one blocking filter, all the things are arranged so that image sensor face of said digital camera is disposed in a plane conjugate to the microscope sample plane, and providing a single frame for every scan step.

10. The STED microscopy system of Claim 6, wherein said light detection and imaging module comprises:
- a high resolution imaging device in the form of a high resolution digital camera;
- at least one blocking filter;
- a second lenticular comprising the same number of similarly arranged cylindrical lenslets as the first lenticular, but having focal distances 2 to 10 folds shorter and correspondingly numerical apertures 2 to 10 folds more than those of cylindrical lenslets of the first lenticular;
- a second scanning means with an image scanning mirror both identical to said first scanning means;
and - second relay optics.

11. The STED microscopy systems of Claim 6 and Claim 10, wherein said scanning means is selected from the group consisting of a galvanometer-controlled mirror, a pieso-controlled mirror, an acousto-optical deflector, a polygonal scanner, a diffraction grating, and a microelectromechanical system.

12. The STED microscopy systems of Claim 10, wherein said first and said second scanning means are implemented in the form of a single scanning means with a bilateral scanning mirror, which 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.