CA3035876A1 - Line scan photon reassignment microscopy systems - Google Patents

Abstract

Multiplexed confocal line scan microscopy systems realize the methods of computational and optical photon reassignment (PRA) microscopy for imaging fluorophore-labeled samples and comprise multiplexed line scan confocal image acquisition frameworks using focal line arrays instead of focal point arrays. Proposed embodiments are capable of resolving details fare below the Abbe diffraction limit. The multiplexed line scan photon reassignment microscopy is a technique whereby observations of fluorophore-labeled samples is realized by scanning ROI of the sample with a plurality of diffraction limited focal lines of excitation light. In some embodiments, images of fluorescence, provided by spontaneously emitting fluorophore molecules excited by the radiation of the excitation pattern, are acquired with an imaging device and are used as row data for building computational photon reassignment (CPRA) image of the sample ROI. In alternative embodiments imaging beamlets are optically reassigned prior their detection with an imaging device, thus providing optical photon reassignment (OPRA) images. The combination of the focal line scan and PRA
techniques merges the ability of a focal line scanning system to physically suppress out-of-focus light with the resolution enhancement by PRA. The multiplexed line scan PRA microscopes are seen as simple, cost effective instruments that provide with high acquisition rates supper-resolution specimen images with their resolution outperforming the diffraction limit by more than factor two.

Description

LINE SCAN PHOTON REASSIGNMENT MICROSCOPY SYSTEMS
FIELD OF THE INVENTION
The present invention relates to a confocal microscope for super-resolution imaging fluorophore-labeled samples, and more particularly, to a multiplexed line scan confocal microscope, configured to realize a super resolution by photon reassignment technique, and capable of resolving details fare below the diffraction limit.
BACKGROUND OF THE INVENTION
Confocal laser scanning microscopy (CLSM) is an established tool in fluorescence microscopy and well-known for its optical sectioning ability and high contrast. These characteristics are achieved by using detectors with a high dynamic range and collecting the emitted light through a pinhole, which is usually aligned to the position of the excitation focus. The resulting image is constructed by assigning the detected intensity to the corresponding excitation scan position. The imaging acquisition may be sped up by using a multifocal excitation scheme, wherein instead of a single pinhole, a pinhole array is used in cooperation with 2D imaging detector, e.g. swept field or spinning disk confocal microscopy. Line-scanning CLSM systems are fluorescence microscopes that use line-based illumination, i.e., the excitation light is focused into a thin line (e.g. US Pat. 5153428) or a line array (e.g. US Pat. US6288382) and swept across a sample while capturing the fluorescence on an area detector. The out-of-focus light is discarded by confocal slits. Since acquisition is massively parallelized compared to point-scanning techniques, line-scanning techniques offer much higher imaging speed.
Fluorescence CLSM is widely applicable for investigating structural organization and dynamical processes on the cellular level because of its noninvasiveness, sensitivity, and specificity. Its popularity has steadily grown despite the fact that it notoriously fails to image structures smaller than about half the wavelength of light (-200 nm), i.e. is limited by the so called diffraction barrier or Abbe limit.
In 1982 it was shown [1] that it is possible to achieve enhanced resolution by applying an off-axis pinhole. In a theoretical study in 1988, Sheppard [2] pointed out that confocal microscopy can surpass the diffraction limit without sacrificing signal-to-noise ratio. To enhance the resolution of a CLSM, the additional information of a pinhole plane image, taken at every excitation scan position, can be used and it is possible to double the resolution of a scanning confocal microscope by employing photon (or pixel) reassignment (PRA) technique. One can obtain after appropriate deconvolution of the recorded images, an image with doubled resolution. PRA
microscopy [2, 3] is based on the insight that the most probable origin of the detected photons is at maximum of the joint point spread function (PSF), i.e. the product of the individual excitation and (off-centre) detection PSFs.
This is illustrated in FIG. 1. The origin is located at intermediate point between the nominal focus of the excitation laser and the position corresponding to the point-like detector. Therefore, by reassigning the detected photons to this most probable place, an image with enhanced detection efficiency and resolution is obtained. This is contrary to a conventional CLSM where all detected photons are assigned to the nominal excitation position. Note that for identical excitation and emission PSFs this most probable position of an emitter in the sample appears at half way between the nominal excitation and detection centers. More importantly, the approach allows better imaging SNR
being compared with the standard confocal microscopy. In this technique, referred to as image scanning microscopy (ISM) by Muller and Enderlein [3], the point detector with a confocal pinhole is replaced by an imaging detector and each image from the detector is recorded as the illumination laser spot scans over the entire field.
The major drawback of this implementation was the slowness of the imaging. In 2012, York et al. [4]
demonstrated that this limitation may be overcome by using a multifocal excitation scheme. US Pat. 9696534 discloses a multi-focal selective illumination microscopy (SIM) system for generating multi-focal patterns onto a sample. The multi-focal SIM system performs a focusing, scaling and summing operation on each multi-focal pattern in a sequence of multi-focal patterns that completely scan the sample to produce a high resolution composite image. In 2013, Schulz [5], presented an alternative that achieves the same resolution enhancement by using a standard confocal spinning-disk microscope with minimal modifications, disclosed in EP Pat. 2871511.
This method, in principle, allows one to double the resolution of any existing microscope.
Roth et al. [6] presented optical photon reassignment microscopy (OPRA) which realizes this concept in an all-optical way obviating the need for time-consuming image-processing. In OPRA a similar reassignment to the optimal emission location is achieved optically, with the help of an additional intermediate optical beam expansion between descanning and a further rescanning of the detected light. Rescan confocal microscopy (RCM) is another realization of the OPRA technique, proposed by Luca et al. in 2013 [8]. RCM is based on standard confocal microscopy extended with an optical (re-scanning) unit comprising an imaging lens that projects light passing the pinhole onto a CCD camera. The optical photon reassignment is realized by twofold local contraction of the fluorescent focus spot with the imaging lens. Alternatively, the final fluorescence image is twofold magnified while maintaining the original fluorescence foci size. The last approach was used in US Pat. 20170254997 that discloses a rescan PRA technique for a single beam line scanning CLSM
generating two orthogonal consequent line scanning patterns onto a sample. Major drawbacks of the technique are a limited set of scan directions (two only), and indispensable computational rescaling anamorphic images of orthogonally scanned sample. OPRA
attains the same characteristics as computational reassignment without the need for high-speed pinhole cameras and without the increased read-noise of multiple fast readouts. OPRA
demonstrates much faster acquisition of super-resolution imaging. However a loss in confocal sectioning performance may be caused by working with relatively open pinholes.
The concept of enhancing resolution through the photon (or pixel) reassignment has been generalized to include techniques termed Image Scanning Microscopy (ISM) [3], Multifocal Structured Illumination Microscopy (MSIM) [4], Instant Structured Illumination Microscopy (ISIM) [7], Selective Illumination Microscopy (SIM) ¨ US Pat.
9696534, Re-scan Confocal Microscopy (RCM) [8], Optical Photon ReAssignment (OPRA) microscopy [6], and pixel reassignment microscopy [9]

2 Development of OPRA 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. Although the spatial resolution of the PRA technique is still lower compared with other super-resolution methods, such as STED and Localization-based techniques, e.g. STORM, PALM, it overcomes some of their shortcomings. This technique inherits all advantages of the standard confocal microscopy, including high-speed imaging rate, acceptable excitation intensity, optical sectioning capability, and a broad choice of fluorescent dyes and/or proteins, making it a readily accessible technology in a variety of biological investigations.
More than that, the peak intensity of the reassigned PSF significantly increases, as compared to that in a conventional confocal microscope, because all the light is detected and after reassignment concentrated into a smaller PSF
[10].
As such, there is a need in the art for a multiplexed confocal microscopy technique based on the photon reassignment and providing more than doubled resolution enhancement, as compared to a standard confocal microscope, that is providing resolution exceeding the same of the state-of-the-art PRA techniques without sacrificing their advantages.
SUMMARY OF THE INVENTION
In the PRA techniques, illustrated by FIG. 1, a reassignment to the optimal emission location is achieved computationally or optically. A pixel on a digital camera, if it is small enough, detects light from a diffraction-limited region of the sample. A pixel disposed at a distance s from the excitation focus 0 will detect light signals mostly originated from the location of the peak of the product of excitation PSFs E(r) and scaled detection (shifted) PSF
D(r-s). The peak occurs at a distance of s/m from the excitation focus. The measured spot on the camera is to be mutually displaced proportional to (1 ¨ 1/m) due to the reassignment operation. The resulting PSF is thus the convolution of the excitation PSF with the scaled detection PSF. The separation of two points also scales with factor m so the effective width of the detection PSF scales as //m and the effective width of the excitation PSF
scales as (m-1)/m. A measure for the spot width CR, such as standard deviation, is thus effectively given by [8, 9]:
-\
alt =
M jm (1) Here 0-E and OD are standard deviations for the corresponding excitation and detection functions. Clearly, in the limit m = 1 we find o-R = op, i.e. the microscope is equivalent to a widefield fluorescence microscope. In the opposite limit m co we find o-R = GE, i.e. now the microscope's resolution is equivalent to that of a scanning fluorescence microscope. The optimum (minimum) spot width is found for a scale factor:
m = (up /ffE)2 + 1 = p2 + 1 (2)

3 For which the optimum (minimum) spot width is equal to:
aTeri) a a CT T= + 0 (3) All PRA techniques, disclosed in prior art under alternative names, are generally developed for equal excitation and detection PSFs, aE = up. This boils down to m = 2 and a -µ12 reduction in spot width aR = a012 = a01l\l2. But the effect may also be fruitfully incorporated for any case where the excitation PSF differs from the detection PSF.
In the case the reassignment distance is scaled down by the factor m defined by the equation (2). This difference can be induced by the Stokes shift of the used fluorophores or can be a feature of microscopy techniques used.
Examples include two-photon fluorescence microscopy or the use of pupil spatial filters.
One approach to provide a higher optimal scale factor m for getting more than two-fold resolution enhancement via realizing substantially different widths of excitation and detection PSFs is using focal line scan of samples and slit detection apertures, rather than focal point scan and pinhole detection apertures to construct PRA confocal system. In line-scan confocal microscopy, illumination and detection PSFs are significantly different, especially in the cases when linearly polarized light with an electrical field polarization vector parallel to the scan focal line of excitation light (s-polarization) and a fluorophore with a large Stokes shift are used. This difference is illustrated by FIG. 2 that presents ratio of the excitation and detection PSF widths Po = at, /o-E, and corresponding optimal reassignment scale factor m as a function of an objective lens acceptance angle a = arcsin{NA/n} for line scan PRA sample imaging with s-polarized excitation radiation with no Stokes shift.
If a fluorophore with a substantial Stokes shift, for example eFluor 506, is used, detection to excitation wavelength ratio may be AD /AE 1.3 leading proportional increase of the ratio p = Po AD /AE and the factor m, according the eq. (2). Resolution in PRA
microscopy system is enhanced in a direction, normal to the scan lines only, therefore it is desirable to acquire a set of images with scan in a number of directions for providing the enhanced omnidirectional lateral resolution, Multiplexed line scan PRA microscopy systems offer several useful advantages over single- and multi-focal point scan PRA microscopes; for example, systems provide significantly higher lateral super-resolution, higher fluorescence imaging signals and SNR, and higher attainable frame rate.
Multiple illumination and scanning methods, developed for confocal microscopy, may be applied for implementation of a multiplexed line scanning PRA microscope to great effect.
The major objective of the present invention is a provision of a multiplexed line scan computational photon reassignment (CPRA) microscopy system capable to provide imaging biological specimens with super resolution m-folds (m > 2) better than diffraction limit. Another objective of the present invention is provision of a multiplexed line scan optical photon reassignment (OPRA) microscopy system, capable to provide high frame rate wide ROI
imaging of live micro-objects and/or other biological specimens with super resolution m-folds (m > 2) better than

4 diffraction limit. Yet another objective of the present invention is a provision of a multiplexed line scan PRA
microscopy systems, attractive and affordable for 2D and 3D super-resolution photon reassignment microscopy of biological specimens and particularly in-vivo super resolution imaging.
The present invention provides a number of exemplary embodiments of multiplexed confocal line scan microscopy systems, which realize the methods of computational and optical photon reassignment microscopy for imaging fluorophore-labeled samples, capable of resolving details fare below the diffraction limit. The combination of the focal line scan and PRA techniques, which is the focus of this application, merges the ability of a focal line scanning system to physically suppress out-of-focus light with the resolution enhancement by PRA. The multiplexed line scan PRA microscopes are seen as simple, cost effective instruments that provide with high acquisition rates supper-resolution specimen images with their resolution outperforming diffraction limit by more than factor two, that is significantly higher than resolutions demonstrated by the prior art PRA systems.
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.
SHORT DESCRIPTION OF THE DRAWINGS
Embodiments according to the present invention will now be described hereinafter with reference to the accompanying drawings, where similar reference numerals indicate similar or identical features throughout the drawings. The components in the drawings are not necessarily to scale, emphasis instead being placed upon clearly illustrating the principles of the present invention.
FIG. 1 is a one-dimensional schematic diagram illustrating the principles of the photon reassignment.
FIG. 2 presents ratio of the excitation and detection PSF widths and optimal reassignment factor in as functions of objective lens acceptance angle for the line scan PRA sample imaging with s-polarized excitation radiation.
FIG.3 depicts a schematic perspective view of a first exemplary embodiment of a multiplexed line scan computational photon reassignment (CPRA) microscopy system in accordance with the present invention.
FIG. 4 presents a simplified schematic diagram of an exemplary embodiment of the excitation light source.
FIG. 5 schematically depicts an alternative exemplary embodiment of a multiplexed line scan CPRA microscopy system in accordance with the present invention.
FIG. 6 presents a schematic perspective view of an exemplary embodiment of a multiplexed line scan optical photon reassignment (OPRA) microscopy system in accordance with the present invention.

FIG. 7 depicts XZ and YZ projections of the imaging branch of the OPRA
microscopy system with OPRA unit employing a converging cylinder MLA, and a ray path in the system branch.
FIG. 8 presents XZ projection of the imaging branch of the OPRA microscopy system with alternatively designed confocal OPRA unit employing a diverging cylinder MLA.
FIG. 9 presents examples of focus mismatch correcting means for a confocal OPRA unit with a converging cylinder MLA.
FIG. 10 presents examples of focus mismatch correcting means for a confocal OPRA unit with a diverging cylinder MLA.
FIG. 11 depicts a simplified schematic diagram of an adjustable slit array comprising two identical close spaced mutually displaceable optical masks.
FIG. 12 depicts a simplified schematic diagram of an alternative exemplary embodiment of the multiplexed line scan OPRA microscopy system in accordance with the present invention.
DETAILED DESCRIPTION
Embodiments according to the present invention will now be described hereinafter with reference to the accompanying 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.
Terms "light" and "radiation" as well as "light source" and "radiation source"
may be used interchangeable and are related to the electromagnetic spectrum from infrared via visible to ultraviolet (IR-Vis-UV).
A "probe beamlet" and "imaging beamlet", as used herein, refers to any one of plurality of excitation light beams provided by a microlens array and corresponding fluorescence imaging beams. In other words, each probe beamlet with a corresponding imaging beamlet presents a single channel of the multiplexed PRA microscope.
Polarized light with its electric field normal to the plane of incidence is called s-polarized while light whose electric field is along the plane of incidence is thus denoted p-polarized. Therefore, electric field of s- and p-polarized light focused into a focal line, is oriented along the focal line and normal to it, respectively.
PSF ¨ "point spread function" describes the response of an imaging system to a point source or point object that may be presented mathematically as the Dirac delta-function.

OTF ¨ "optical transfer function" of an optical system specifies how different spatial frequencies are handled by the system. Formally, the OTF is defined as the Fourier transform of the PSF.
ROI ¨ "region of interest" is an observable part of an analyzed object to be captured on the system's imager.
SNR ¨ "signal to noise ratio" is a measure that compares the level of a desired signal to the level of background noise. SNR is defined in optics as the ratio of signal power (photon counts) to the noise power (photon counts) in a given bandwidth.
A "mirror contrast," as used herein, refers to differences of reflectivity specified by mirror characteristics. A "mirror contrast pattern" is a pattern consisted of highly reflective and highly transparent areas.
Referring now to FIG. 3, which is a schematic perspective view of the first exemplary embodiment of the multiplexed line scan computational photon reassignment (CPRA) microscopy system in accordance with the present invention. In addition to the major optical components and units there are shown in FIG. 3, chief rays of aggregated excitation and imaging light beams, illustrated by outlined and by bold arrows, respectively.
The entire assembly of the proposed embodiment is preferably comprising: an excitation light source 1 that provides excitation laser radiation; an optical multiplexing unit 2 presented in the form of an array of cylindrical focusing lenslets 21; an optical microscope 4 comprising a high magnification objective lens 41, a tube lens 42, a XYZ microscope translation stage 43, and a fluorophore-labeled microscopy sample 44 placed on it; a beam scanner module 3 comprising a galvo-scanner 31 with a scan mirror 32, first and second scan lenses 33, 34, and an image rotator 35 slewable about the optical axis with a rotary actuator 36;
and a light detection module 5 comprising an imaging device 54, a beam splitter 51, an imaging optics 52, and a blocking filter 53. All said components are arranged to provide an optical train with a set of principal conjugate focal planes, namely, a sample plane SP, microscope image plane IP1, illumination aperture plane AP, and an image sensor plane IP3.
The excitation light source 1 provides a collimated highly uniform ("tophat") light beam of laser radiation of an excitation wavelength AE for exciting, in single- or multi-photon mode, a fluorophore in the sample 44 to be imaged. The light source 1 may be built in a variety of forms, previously developed for confocal microscopy. One exemplary implementation of said light source 1 is schematically depicted in FIG. 4. Said light source 1 may comprise at least one laser 11 for providing a beam of excitation light of an excitation wavelength AE; light delivery optics, schematically presented in the form of a fiber coupling lens 12 and an optical fiber 13; beam conditioning means schematically presented in the form a collimation lens 14, a beam shaping means 15, and a projection lens 16. Said light source 1 may comprise additional components, for example, an acousto-optic or another ON/OFF light switch 17, polarization control means18, and other components, such as lenses, dichroic and turning mirrors, prisms, etc.

Said beam conditioning means 14, 15 transform the diverging light beam outgoing from the distal end of said fiber 13 into collimated tophat light beam of the laser radiation having desired polarization state and cross section, and exhibiting high evenness of the transverse intensity distribution. Said beam shaping means 15 may be built in a variety of forms, for example, those based on Gauss-to-tophat diffractive optical elements, refracting optical components, such as Powell lenses, or filter means.
Said optical multiplexing unit 2 may be a converging cylinder microlens array (MLA) comprising one or more identical positive cylindrical lenslets 21 elongated along their cylinder axes oriented in Y-direction. A focal plane of said cylinder MLA 2 lays in an illumination aperture plane AP of the proposed system, conjugate to the image plane IP1 and to the microscope sample plane SP. Alternatively, said optical multiplexing unit 2 may be a diverging cylinder MLA comprising one or more identical negative cylindrical lenslets elongated along their cylinder axes oriented in Y-direction. Said diverging cylinder MLA provides imaginary focal lines in the illumination aperture plane AP upstream disposed.
Said beam scanner module 3 comprising a single axis galvo-scanner 31 with scan mirror 32; scan lenses 33, 34 disposed on the optical axis on either side of the scan mirror 32 in a conventional 4F configuration; and an image rotator 35 with a rotary actuator 36. A rotation axis of said scan mirror 32 is oriented along Y-axis, i.e. parallel to the axes of said cylindrical focusing lenslets 21. Said image rotator 35 provides capabilities of sample scanning in at least two different directions and may be implemented in variety of forms including but not limited to image rotation prisms (by Dove, Schmidt, Abbe, Vee, Schmidt-Pechan, etc.), mirror structures, or cylindrical lens rotator.
Said image rotator may reside at any place between the galvo-driven scan mirror 32 and the micro objective lens 41, for example between the scan lens 34 and the image plane IP1, as it is presented in FIG. 3; its slew axis is oriented along optical Z-axis. Alternatively the sample scanning in a number of different directions may be realized by a rotation of said sample 44 itself with an optional rotary stage (not shown), while scanning beams are deflected in a direction fixed relative the whole microscopy system.
Said light detection module 5 comprises an imaging device 54 with a 2D image sensor 55, imaging optics 52 arranged with said scan lens 34 in 4F configuration, and at least one blocking filter 53 for rejection of stray and ambient light. The imaging device 54 may be built in the form of a high speed digital camera, such as EMCCD or sCMOS camera, Said imaging device 54 may be mounted so that a face of the image sensor 55 lays in the plane IP3 conjugate to the image plane IP1 and to the sample plane SP. Said beam splitter may be a spatial beam splitter comprising a mirror-contrast (mirrored vs. transparent) line shaped item oriented along the first (X) direction and disposed between said first scan lens and first scan mirror, close to a pupil plane of said 4F
configured first and second scan lenses 33, 34. A width of the mirror-contrast item may superior to a width of a focal line, provided by said lens 33 focusing the collimated in Y-direction probe beamlets. It may be either a beam splitter 51 consisting of a line shaped highly transparent area allowing the excitation light to pass through to the sample 44, but the remaining part of the beam splitter is mirrored for reflecting the fluorescence path to the light detection module 5, as depicted in FIG. 3, or a spatial beam splitter 56 consisting of a line shaped reflective area on a highly transparent substrate to reflect the illumination beam path and to pass through the fluorescence.
Alternatively, said light detection module may be coupled to the rest system with an appropriate dichroic mirror 57, disposed at any point of the optical axis between said cylinder MLA 2 and objective lens 41, but it may be required additional imaging optics. The dichroic mirror 57 may be disposed, for example, between the illumination aperture plane (AP) and the scan lens 33, or between the tube lens 42 and the image plane IP1, as depicted in FIG. 5. In the case, the light detection module may not comprise the imaging optics 52, functions of which would perform said scan lens 33 or tube lens 42 respectively. Placement of the dichroic mirror 57 somewhere between the image rotator 35 and the objective lens 41, e.g., as depicted in FIG. 5, leads more effective use of said (usually) rectangular 2D image sensor 55 in the course of sample scanning in at least two different directions and enables getting a full frame rectangular ROI of the microscopy system, rather than inscribed polygonal one.
The first exemplary embodiment of FIG. 3 may optionally comprise additional optical components including but not limited to mirrors, lenses, prisms, microlens array. For example, it may comprise a matched confocal slit array disposed in an intermediate image plane in order to reject stray and ambient light, and additional optics to relay the microscopy image of a sample to the sensor plane IP3.
In operation, the optical multiplexing unit 2 splits the collimated tophat light beam of excitation radiation, provided by the light source 1, into one or more probe beamlets forming an aggregate excitation light beam traveling along Z-axis. Each of the beamlets stills collimated in Y-direction parallel to the lenslet cylinder axes and is focused in a normal X-direction, thus providing in the illumination aperture plane AP an excitation pattern comprised of one or more equidistant focal lines of excitation light. Said excitation pattern is relayed with said lenses 33, 34 onto the conjugate image plane IP1 via said beam splitter 51, scan mirror 32 and image rotator 35. Said optical microscope 4, provides projection of said one or more probe beamlets onto the sample plane SP to form there a scaled down array of diffraction limited lines of the excitation light pattern, collects fluorescence provided by spontaneously emitting fluorophore molecules excited by the radiation of said excitation pattern, and provides a magnified image of thus built up fluorescence emission pattern in said image plane IP1. Said emission pattern is relayed with lenses 34, 52 onto conjugate image sensor plane IP3 via said image rotator 35, scan mirror 32, beam splitter 51, and blocking filter 53 to be detected by said imaging device 54.
Said beam scanner 3 provides the multiplexed line-scanning technique, that is directs via the image plane IP1 said one or more probe beamlets focused to provide said excitation pattern in the sample plane SP, provides parallel sweep scan of ROI of said sample 44 by said array of diffraction limited focal lines of excitation light, and descan corresponding imaging beamlets of fluorescence from the emission pattern in the ROI of the sample plane SP for being further projected onto conjugate image sensor plane IP3. Each fluorescence imaging beamlet is projected onto a corresponding set of pixels of the image sensor 55. Said imaging device 54 detects a multitude of optical signals. Images of said emission patterns, acquired with said imaging device 54 are used as row data for building CPRA image of the sample ROI. It should be noted that the multiplexed line-scan microscopy imaging may be alternatively realized by single-axis translational scanning employing, for example, a piezo-scanning microscope translation stage, as would be apparent to those skilled in the art.
Computational photon reassignment data acquisition and processing may be conceptually divided into multiple steps: (i) multifocal sparse line illumination of the sample; (ii) recording the resulting fluorescence image with a camera; (iii) digital slit-cutting to reject out-of-focus emission; (iv) digital m-folds contraction of each slit-cut emission stripe (without changing overall image dimensions); (v) digital emission strip shift for a distance proportional to the scan angle; (vi) repetition of steps (i¨v) at different positions of the excitation pattern until the entire field has been scanned; (vii) digital summation of the resulting images to generate a super-resolution image with \Im improved resolution in the scan direction; (viii) single axis deconvolution to recover the full m-folds resolution improvement in the scan direction; (ix) repetition of steps (i¨viii) for a number of different scanning directions; (x) joint digital postprocessing the resulting images of step (ix) (including sub-step (xi) of digital image de-rotation), to generate an image with m-folds enhanced omnidirectional 2D
super-resolution.
Said digital camera 54 is to be synchronized with said scan mirror 32 that may be driven by galvanometer 31 in a stepwise mode and with the optional ON/OFF light switch 17 to acquire a separate image frame for each scan step. Camera readout signals being pre-processed in combination with the beam scanner 3 data, provides intensities (photon counts) and coordinates of a multitude of pixels of the partial CPRA frame. Every such partial CPRA frame presents a digital image of said magnified emission pattern.
Between two consequent exposures of said digital camera 54 said galvo-driven scan mirror 32 provides a scanning deflection of array of the probe beamlets, projected into the sample plane SP for a single scan step, defined by the desired microscopy system resolution. The scan step is followed by a camera 54 exposure capturing next magnified emision pattern and providing a new partial CPRA frame, and so on. Alternatively, the laser of light source 1 may be operated in a short pulse mode, while said galvo-scanner 51 may be operated in a continuous scan mode, to provide a separate image frame for at least one excitation laser pulse.
Pre-processing of a plurality of said partial CPRA frames, collected in the course of a full scan cycle (steps ii-v) with following their co-processing (steps vi-viii), provides the intermediate single axis scan full frame CPRA image of the sample ROI, which is characterized by an elliptical PSF exhibiting a diffraction limited resolution along the excitation focal lines (along Y ¨ axes) and CPRA super-resolution across the excitation focal lines, i.e. in the scan direction along X ¨ axes. Next step is the image rotator 35 slewing by a predetermined angle and repeating the scan, imaging, and image processing procedures (steps / - viii). A resulting super-resolution CPRA image of the sample ROI may be reconstructed by combining and post-processing two or more single axis scan intermediate CPRA images taken at different scan directions (step x). Data acquisition and processing procedures of the multiplexed line scan CPRA system of FIG. 5 does not include the step (v) of digital emission strip shift and sub-step (xi) of digital de-rotation of single axis scan images.

Said image pre-processing and co-processing can be described by equations, as follows. If we assume an imaging system with unity magnification, for simplicity, a position on the sample plane, at which the detection (fluorescent) light is generated, denote as r, a light receiving amount U(r) at a pixel position s on the image plane is described by the photon reassignment PSF expressed as follows [5, 8]:
m2 7/1 U (r) = f D(m = s)E(¨ (r ¨ s))ds (4) m-1 m-1 where s is the pixel position on the image sensor 55, D(r) is the detection PSF of a wide field microscope, and E(r) is the excitation or illumination PSF. In the line-scan confocal microscopy, the excitation and detection PSFs may be significantly different (see, e.g., [11, 12]). The difference is a feature of the line scan confocal microscopy technique itself, additional difference can be induced by the Stokes shift of the used fluorophores. Nevertheless, approach developed by [3] for a point scan PRA technique with identical excitation and detection PSFs may be adopted with a great effect for the line scan technique as well. This may be demonstrated by a simplified discussion of the imaging properties of PRA technique for objects within the focal plane of the microscope. Thus substituting for the sample plane r = (x, y), s = (s, t) and taking into account that the illumination PSF is a function of a single X-coordinate (does not depend on Y-coordinate) simplifies the reassignment PSF (4) to m2 U(x, y) = f D(m = s,y)E(-- (x ¨ s))ds (5) m-1 m-1 Equation (5) shows that the reassignment PSF in the X-direction is equal to the detection PSF rescaled by factor 1/m convolved with the illumination PSF rescaled to (m-1)/m of size, whereas it is equal to the wide field detection PSF in the Y-direction. In other words, the response is significantly improved in the X-direction, but the same as in a conventional wide field microscope in the Y-direction. In Fourier space, this reads:
U(k) = (1E, 1) ( k 1) S(1) (6) Here k and / are x- and y-components of the wave vector k, 5(1) is the Dirac delta function. Thus the reassignment image is presented in the focal plane as (x, y) = C(x, y) 0 U(x, y), (7) where gx,y) is the spatial density of fluorescent emitters in the focal plane and 0 is the convolution operator. It contains information of spatial details in the X-direction m times as small as those seen by a conventional wide-field microscope with an OTF (k), while keeping the microscope resolution in the Y-direction unchanged. If we assume for (6) a Gaussian shaped excitation and emission PSFs f(x) = exp{-x2/(2a2)} (8) (standard deviation o-E for the excitation and ap for the detection functions), the integral can be solved analytically. The final PSF is a product of two Gaussians, one for X-direction another for Y-direction, and is found to have the standard deviation in the X-direction presenting the reassignment outcome (1) with the minimal total extent along X-axis presented by (2), (3). Therefore, one can obtain a lateral resolution enhancement by a factor of -Nim over what would be expected for conventional wide field microscope.
This shows that reassignment microscopy realizes super resolution at the theoretical overall detection efficiency of a wide field microscope and of a conventional slit scan confocal microscope.
A full m-fold enhancement of the X-resolution of PRA may be achieved in the following way: (i) transforming the image U(r) into Fourier space, thus obtaining U(k), (ii) applying an appropriate weight function W(k), and (iii) back-transforming the result to real space. However, the reassignment OTF U(k) overweighs components with large amplitude (small values of Ikl) and underweights components with small amplitude (large values of Ik1). This can lead to an unwanted amplification of image noise at spatial frequencies close to those divergences. To avoid such effects, a better choice for the weight function 171(k) is, for example, 171/(k, = [E. (k -m-1,1) + Er (9) The parameter should be by at least one order of value smaller than the maximum amplitude of IE(k)I.
The scale factor m is a function of the different X-axis widths of the excitation and emission PSFs. FIG. 2 presents ratio of the excitation and detection PSF widths po = o-o /ak, and corresponding optimal reassignment scale factor m as functions of objective lens acceptance angle a = arcsin{NA/n} for line scan PRA sample imaging with s-polarized excitation radiation with no Stokes shift. For example, employing an oil immersion objective with, NA =
1.46, i.e., sin a = 0.964 (Plan Apo 100x1.46 by Leica), results in Po = 1.40 and PRA scale factor in = 2.96. More prominent results may be obtained, if a fluorophore with a substantial Stokes shift, for example eFluor 506, is employed. In the case, a detection to excitation wavelength ratio AD/AE ?.
1.28 and p = PO (AD IAE) 1.79 for s-polarized excitation light. It says that the PRA resolution enhancement of the slit scan confocal microscope is as high as Alm = \l(p2 + 1) ?. 2 and can be further improved by the deconvolution algorithm up to a factor m 4.2.
Steps (ii¨vii), discussed hereinabove with reference of CPRA microscopy system of FIGS. 3, 5 may be performed entirely in hardware in an optical photon reassignment (OPRA) system. A
perspective view of an exemplary embodiment of a multiplexed line scan OPRA microscopy system in accordance with the present invention is presented in FIG. 6, where like reference numerals are applied to like parts, and like notations are applied to like events. This microscopy system is similar, in a certain sense, to the embodiments of FIGS. 3, 5 and comprises identical or similar major components: the excitation light source 1, the optical multiplexing unit 2 that may be an array of cylindrical focusing lenslets 21; the optical microscope 4 comprising the objective lens 41, the tube lens 42, and the microscope translation stage 43 with a microscopy sample 44, fixed on it; the beam scanner module 3 comprising the galvo-scanner 31 with the scan mirror 32, the scan lenses 33, 34, and the image rotator 35 with the rotary actuator 36; and a light detection module 5 comprising the imaging device 54, the beam splitter 56, and the blocking filter 53, but unlike the former embodiments it comprises some additional units, namely a rescan module 6, comprised of a scan mirror 62 controlled by a galvo-scanner 61, two scan lenses 63, 64, and, optional, an image de-rotator 65 with a rotary actuator 66; a confocal OPRA
unit 7 comprised of a slit array 71 and a refocusing element 73, and imaging optics 8.
FIG. 7 depicts the imaging branch of the OPRA microscopy system downstream to the scan mirror 32 and XZ and YZ projections of a fluorescence imaging beam; one of imaging beamlets is highlighted. Said confocal OPRA unit 7 is matched to the optical multiplexing unit 2 and comprises the refocusing element 73, which is a converging cylinder MLA comprised of one or more positive cylindrical lenslets 74, and the confocal slit array 71, comprising fit slits 72. Said imaging optics 8 may be a bi-cylindrical lens assembly comprising two cylinder lenses 81, 82 with their axes mutually at right angle. The lens 82 performs X-meridian focusing imaging beamlets and provides a set of focal lines in image plane IP2. The fluorescence imaging beamlets travel through corresponding lenslets 74, that additionally focus them to provide an array of m-fold contracted lines of the emission pattern in the refocus plane RP, which is a confocal plane of the system, wherein the slit array 71 resides. Each imaging beamlet travels through a matched slit 72. Because the refocus plane RP does not coincide with the image plane IP2, said cylinder lens 81 is disposed at a distance from the refocus plane RP, equal to its focal length, to provide Y-meridian focal plane coinciding with the refocus plane RP. Thus said bi-cylindrical lens assembly 8 corrects the mismatch of the Y-meridian focal plane with the X-meridian focal plane. In some embodiments the imaging optics 8 may be replaced with a spherical imaging lens, while the mismatch of the X-meridian and Y-meridian focal planes may be corrected by the bi-cylindrical lens assembly 81, 82 replacing the scan lens 63.
An alternatively implemented imaging branch of the OPRA microscopy system is depicted in FIG. 8, wherein XZ
projection of a fluorescence imaging beam path and principal optical components are presented; one of imaging beamlets is highlighted. Said OPRA system may comprise an imaging lens 52, matched confocal slit array 71, a matched diverging cylinder MLA 75, comprised of negative cylindrical lenslets 76, and imaging optics 8' that is a bi-cylindrical lens assembly 81, 82 with lens axes mutually at right angles.
The imaging lens 52 provides a second image plane IP2, which is a confocal plane of the microscopy system wherein the matched slit array 71 is disposed. The diverging cylinder MLA 75 provides a reassigned emission pattern in an imaginary refocus plane RPi. The imaging optics 8' is disposed downstream to the MLA 75 so that the crossed cylinder lenses 81, 82 reside at distances equal to their focal lengths from the image plane IP2 and the refocus plane RPi respectively.
Some alternative embodiments of the OPRA microscopy system may comprise both spherical lenses 52, 63 and appropriate focus mismatch correcting means 9 that may be implemented in a variety forms including but not limited to examples depicted in FIGS. 9, 10 and may comprise a number of cylindrical and spherical lenses and/or mirrors, prisms and plates to accomplish the goal of correcting the mismatch of the Y-meridian and X-meridian focal planes. FIG. 9 presents examples of correcting means 9 for a confocal OPRA unit 7 with a converging cylinder MLA 73. It may be implemented in the form of a positive cylinder lens 91 with its axis orthogonal to ones of microlenses 74, disposed on the optical axis upstream (a) or downstream (b) to the confocal unit 7, may be composed of two cylinder lenses 92 disposed on either side of the unit 7 (c), or it may be a negative cylindrical lens 93 (d), disposed downstream to the confocal OPRA unit 7 with its axis parallel to ones of MLA 73. FIG. 10 presents examples of correcting means 9 for the OPRA unit 7 with a diverging cylinder MLA 75. The means 9 may be built of properly oriented and disposed cylindrical elements, such as one negative lens 94 (a, b), two negative lenses 95 (c), or positive lens 96 (d).
By virtue of the fact that the optimal scale factor m is a function of excitation and detection wavelengths AE, AD, and of the excitation light beam polarization state, said cylinder MLA, converging 73 or diverging 75, and cylinder lens 82 may be mounted linearly adjustable along the optical axis to control the scale factor of the OPRA
microscopy system. The scale factor control in embodiments comprising both lenses 52, 63 and the focus mismatch correcting means 9 may be done by axial linear adjustments of the MLA
73 or 75, at least one lens in the correcting means 9, and one of lenses 52 or 63. Opening widths of said confocal slits 72 may also be adjusted for OPRA imaging with a maximal SNR.
Said slit array 71 may be a one piece fixed slit array or an adjustable device, such as, e.g., depicted in FIG. 11 The adjustable slit array is comprised of two identical periodic optical masks 77 comprising constant-interval bars 78 and spaces 79 deposited onto optical substrates. Said optical masks 77 are to be face-to-face close spaced and aligned to mutually parallelize their bars. Width of said slits 72 is adjustable by mutual displacement of the optical masks 77 in X-direction, illustrated by arrows. Alternatively, said adjustable slit array 71 may be a spatial light modulator, such as, e.g., a liquid crystal modulator or a digital micromirror device.
Said rescan module 6 may be similar to said scan module 3 and comprises a single axis scan mirror 62 controlled by a galvo-scanner 61; two scan lenses 63. 64, disposed on the optical axis on either side of the image scan mirror 62 in a conventional 4F configuration; and, optional, the image de-rotator 65, driven either with the same rotary actuator 36, or with an own actuator 66 synchronized with the former one. Said image de-rotator 65 may be disposed on the optical axis downstream to the scan mirror 62, e.g., so as presented in FIG. 6.
Said imaging device 54 of the light detection module 5 of this embodiment may be implemented in the form of a high resolution digital camera, such as EMCCD or sCMOS camera. Said beam splitter, presented as the spatial beam splitter 56 reflecting excitation light and transmitting fluorescence, may be the properly arranged spatial beam splitter 51 that, contrary, transmits excitation light and reflects fluorescence, a polarizing bean splitter, or an appropriate dichroic mirror 57.

The optical multiplexing unit 2 splits the collimated tophat beam of excitation radiation, provided by the excitation light source 1, into an array of focused probe beamlets, which provides in the Illumination aperture plane AP a pattern comprised of one or more equidistant focal lines of excitation light.
Said excitation pattern is relayed with said lenses 33, 34 onto the conjugate image plane IP1 via said beam splitter 56, scan mirror 32 and image rotator 35. Said optical microscope 4 relays the excitation pattern onto the sample plane SP, to form there a scaled down array of diffraction limited lines of the excitation light pattern, collects fluorescence provided by spontaneously emitting fluorophore molecules excited by the radiation of said excitation pattern, and provides a magnified image of thus built up emission pattern in the image plane IP1. The lens 82 of the bi-cylindrical lens assembly 8 focus along X-axis the imaging beamlets passing via said image rotator 35, scan mirror 32, and beam splitter 56. MLA
73 additionally focuses the beamlets contracting locally the imaging focal lines, while the lens 81 focuses the beamlets along Y-axis. Thus a sharp resulting image of the array of m-fold contracted lines of the emission pattern is provided in a refocus plane RP, which is a confocal plane of the system. The matched confocal slit array 71, disposed in the plane RP, rejects out-of-focus emissions. The reassigned emission pattern is relayed by the scan lenses 63, 64 via said scan mirror 62 and optional image de-rotator 65 onto the conjugate sensor plane IP3 to be detected by said light detection module 3.
Said beam scanners 3, 6 provide a parallel multibeam line-scan imaging technique, that is scan mirror 32 provides parallel sweep scan of ROI of said sample 44 by said plurality of probe beamlets, and descan corresponding imaging beamlets of fluorescence from the emission pattern in the ROI of the sample plane SP, while the rescan mirror 62 provides synchronized proportional sweep scan of the plane IP3 of the image sensor 55 by said reassigned emission pattern. Said image rotators 35, 65 slewable about the optical axes provide capabilities for the sample 44 sweep scanning in at least two different directions and for de-rotating images of said reassigned emission pattern in the sensor plane IP3 to acquire resulting at least two images being not mutually rotary shifted.
Said digital camera 54 is synchronized with said galvo-driven scan mirrors 32, 62 and said optional ON/OFF light switch 17 of the light source 1. When a shutter of said digital camera 54 is open, fluorescence generates on its image sensor 55 the reassigned emission pattern. Exposure time of the digital camera 54 is long enough to acquire the fluorescence image while said scan mirror 32 sweeps said excitation pattern across the sample 44 plane SP for at least one pitch of the excitation pattern. Equally, said rescan mirror 62 sweeps in unison the reassigned emission pattern across the plane IP3 of the image sensor 55. After finishing one axis scan cycle the shutter of said digital camera 54 must be closed, the camera 54 readout signal provides a one axis scan full frame OPRA image of the sample ROI. PSF of the image exhibits super-resolution in the scan direction and a diffraction limited resolution in the normal direction. Next operation step is slewing said image rotators 35, 65 by a predetermined angle and repeating the scan and imaging procedures. A resulting super-resolution OPRA image of the sample ROI may be reconstructed by following combining and post-processing two or more OPRA images acquired in the course of sample scanning in different scan directions.

The multiplexed line scan OPRA microscopy system of FIG. 6 attains the same characteristics as the computational PRA systems of FIGS. 3, 5 without the need for high-speed cameras and without increased noise of multiple readouts. This raises the acquisition speed as the whole single axis scan image is acquired in only one exposure frame. Said optional image de-rotator 65 precludes the sub-step of digital image de-rotation during post-processing two or more single axis scan OPRA images and may provide a full frame rectangular ROI of the microscopy system.
It should be noted that the rescan confocal microscopy technique with a single scanning focal line and a single confocal slit may be realized in the embodiment of FIG. 6, wherein the rescaling and astigmatism correcting means 7, 8 are not included. This process is accomplished by keeping a ratio of angular amplitudes of the scanners 6 and 3 equal to the scale factor m.
A perspective view of an alternative exemplary embodiment of the multiplexed line scan OPRA microscopy system in accordance with the present invention is presented in FIG. 12, wherein like reference numerals are applied to like parts, and like notations are applied to like events. This embodiment is similar, in general, to the embodiment of FIG. 6 and is comprised of similar major units: the excitation light source 1; the optical multiplexing unit 2 comprised of the cylindrical lenslets 21; the optical microscope 4 comprising the objective lens 41, the tube lens 42, and the translation stage 43 with a microscopy sample 44 fixed on it;
a beam splitter, e.g. the spatial beam splitter 51; a beam scanner module 3 comprising a bilateral scan mirror 37 controlled by the galvo-scanner 31, the scan lenses 33, 34, and the image rotator 35 with the rotary actuator 36; the imaging lens 52; the rescale OPRA unit 7 comprising, e.g., the slit array 71, matched to the optical multiplexing unit 2, and the matched diverging cylinder MLA 75, disposed downstream to the slit array 71 and providing said imaginary refocus plane RPi; rescan optics comprising the mismatch correcting and imaging bi-cylindrical lens assembly 8' comprised of the crossed cylinder lenses 81, 82, the rescan lens 64, at least two folding mirrors 58, and the optional image de-rotator 65 with the rotary actuator 66; and the light detection module 5 comprising the high resolution digital camera 54 with the 2D image sensor 55 in the image plane IP3, and the blocking filter 53..
This exemplary embodiment operates in the same manner, provides similar results as the embodiment of FIG. 6, and its similar components perform similar functions; the only difference is that the bilateral galvo-driven scan mirror 37 provides scanning deflection of probe beamlets, descanning fluorescence imaging beamlets, both by its front side, and synchronous rescanning deflection of imaging beamlets by its back side. As a result, it performs, in parallel, functions of two separate galvo-driven scan mirrors 32, 62 in the embodiment of FIG. 6.
The proposed multiplexed line scan CPRA and OPRA microscopy systems may be successively used for Z-stack scanning in order to provide 3D microstructure reconstructions. It may be done by rejection of out of focus light originating elsewhere than the focal plane, computationally or optically with narrow enough confocal slits 72 of said slit array 71, and by utilizing an additional one-dimensional Z-scanner, for example a Z-axis driven sample stage or nanofocus adjustable objective lens to provide a series of 2D slices.
One can then reconstruct 2D super-resolution images from the recorded intermediate single axis scan frames. The 3D model of the sample is then reconstructed by combining and post-processing a stack of the 2D slices by use of appropriate software.
It should be noted that the various lenses and other optical components, specifically the objective and tube lenses, the scan and imaging lenses, the scan and folding mirrors, and the cylinder lenses, the dichroic mirrors, and the blocking filters, are all described hereinabove and depicted in the drawings as single components. This may or may not be the case and is only used for brevity and clarity. Each "lens," "mirror," and "beam-splitter" as such, may comprise any number of lenses, mirrors and/or prisms to accomplish the functions desired. Thus, where appropriate, these structures are reference in terms of the functional result to be obtained. For example, several optical multiplexing units and refocusing elements are described hereinabove as a "cylinderl MLA" or "cylinder lenslet array". There are many different optical means that can be used to transform a light beam and to provide an array of real or imaginary focal lines in their focal or imaging planes. The optical means may be converging or diverging, cylinder or acylinder microlens and/or micromirror arrays, prism arrays, diffractive optical elements, or any other optical elements and assembles performing these functions. Similarly for sake of brevity and clarity only, in the discussion hereinabove, several scan mirrors are described as "galvo-driven." It may refer to any plurality of means to deflect a light path, such as acousto-optic deflectors (AOD), piezo-controlled scan mirrors, polygonal scanners, tilted glass plate image shifting scanners, and the like.
Finally, the specific exemplary embodiments has been particularly shown and described heretofore only for the purpose of explaining and illustrating the present invention. It will therefore be apparent to those skilled in the art that various changes, modifications or alterations to the invention as described herein, may be made without departing from the spirit and scope of the invention and essential characteristics thereof. Furthermore, although the preferred application field of the present invention, as set forth herein, is fluorescent confocal optical microscopy of biological specimens, other general applications are contemplated. For example, a similarly implemented system may be applied for high-resolution episcopic microscopy of non-fluorescent objects. All such embodiments and variations are believed to be within the sphere and scope of the invention as defined by the claims appended hereto.
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Claims (20)

LINE SCAN PHOTON REASSIGNMENT MICROSCOPY SYSTEMS

1. A multiplexed line scan photon reassignment (PRA) microscopy system for super-resolution imaging an observed area of a fluorescent dye labeled microscopy sample, comprising:
- an excitation light source for providing a beam of radiation of at least one excitation wavelength for exciting, in a single- or multi-photon excitation mode, fluorophore molecules in a sample to be imaged;
- an optical multiplexing unit for splitting the beam of excitation radiation into one or more probe beamlets being focused in a first direction (X) in a joint focal plane, but not in a second direction (Y), and providing an excitation pattern comprised of respective one or more real or imaginary focal lines in an illumination aperture plane (AP) of the microscopy system;
- an optical microscope comprising an objective lens and a tube lens for (i) projecting said one or more probe beamlets onto a sample disposed in a sample plane (SP) conjugate to the illumination aperture plane (AP), (ii) forming there a scaled down projection of said excitation pattern, (iii) collecting fluorescence provided by spontaneously emitting fluorophore molecules excited by the radiation of the excitation pattern, and (iv) magnified imaging thus built up fluorescence emission pattern in a first image plane (IP1) conjugate to the sample plane (SP);
- a beam scanner module comprising a scan mirror and first and second scan lenses, disposed on the optical axis on either side of said scan mirror, for parallel sweep scanning the sample in the sample plane (SP) by the focused probe beamlets and descanning corresponding imaging beamlets of fluorescence from the emission pattern;
- an image rotator slewable about the optical axis and providing capabilities for the sample sweep scanning in at least two different directions;
- a beam splitter for separating a path of the excitation light beam directed to the sample and a path of the fluorescence imaging beam to be detected; and - a light detection module comprising a digital camera with a 2D image sensor, disposed in a third image plane (IP3), conjugate to the sample plane (SP), for acquiring images of said fluorescence emission pattern, and at least one blocking filter for rejecting stray and ambient light.

2. The microscopy system of claim 1, further comprising imaging optics for projecting image of said fluorescence emission pattern onto said image sensor in the third image plane (IP3).

3. The microscopy system of claim 1, further comprising an operation unit for control of the system and for (i) recording at least one frame for every scan step, (ii) digital slit-cutting recorded images to reject out-of-focus emission, (iii) digital photon reassigning the recorded images, (iii) digital summation and co-processing a plurality of resulting one direction scan images , and (iv) joint digital postprocessing the images generated for at least two different scan directions to provide a super-resolution PRA image of the sample.

4. A multiplexed line scan optical photon reassignment (OPRA) microscopy system for super-resolution imaging an observed area of a fluorescent dye labeled microscopy sample, comprising:
- an excitation light source for providing a beam of radiation of at least one excitation wavelength for exciting, in a single- or multi-photon excitation mode, fluorophore molecules in a sample to be imaged;
- an optical multiplexing unit for splitting the beam of excitation radiation into one or more probe beamlets being focused in a first direction (X) in a joint focal plane, but not in a second direction (Y), and providing an excitation pattern comprised of respective one or more real or imaginary focal lines in an Illumination aperture plane (AP) of the microscopy system;
- an optical microscope comprising an objective lens and a tube lens for (i) projecting said one or more probe beamlets onto a sample disposed in a sample plane (SP) conjugate to the illumination aperture plane (AP), (ii) forming there a scaled down projection of said excitation pattern, (iii) collecting fluorescence provided by spontaneously emitting fluorophore molecules excited by the radiation of the excitation pattern, and (iv) magnified imaging thus built up fluorescence emission pattern in a first image plane (IPI) conjugate to the sample plane (SP);
- a first beam scanner module comprising a first scan mirror and first and second scan lenses, disposed on the optical axis on either side of said scan mirror, for parallel sweep scanning the sample in the sample plane (SP) by the focused probe beamlets and descanning corresponding imaging beamlets from the fluorescence emission pattern;
- an image rotator slewable about the optical axis and providing capabilities for the sample sweep scanning in at least two different directions;
- a beam splitter for separating paths of the excitation light beam, directed to the sample, and a path of the fluorescence imaging beam to be detected;
- imaging optics for relaying, in conjunction with said second scan lens, image of said fluorescence emission pattern from the first image plane (IP1) onto a conjugate second image plane (IP2);

- a confocal OPRA unit comprising a refocusing element matched to said optical multiplexing unit for providing in a refocus plane (RP) reassigned confocal emission pattern, composed of a real or imaginary images of locally contracted emission stripes, and a confocal slit array comprising matched slits for transmitting one-axis focused imaging beamlets and rejecting out of focus light;
- a rescan module comprising a second scan mirror, synchronized with said first scan mirror, and third and fourth scan lenses, disposed on the optical axis on either side of said scan mirror, for parallel sweep scanning the third image plane (IP3) by the focused imaging beamlets;
and - a light detection module comprising a digital camera with a 2D image sensor, disposed in the third image plane (IP3), conjugate to the refocus plane (RP), for acquiring reassigned fluorescence emission images, and at least one blocking filter for rejection of stray and ambient light.

5. The microscopy systems of claims 1, 4, wherein said excitation light source comprise polarization control means for a control of polarization state of the provided beam of excitation radiation.

6. The microscopy systems of claims 1, 4. 5, wherein said excitation light source provides a beam of linearly s-polarized excitation radiation that is its electric field is oriented along the excitation pattern focal lines.

7. The microscopy systems of claims 1, 4, wherein said optical multiplexing unit is a converging cylinder microlens array (MLA), disposed upstream to said aperture plane (AP) and comprised of one or more equal power positive cylindrical lenslets.

8. The microscopy systems of claims 1, 4, wherein said optical multiplexing unit is a diverging cylinder MLA, disposed downstream to said aperture plane (IP2) and comprised of one or more equal power negative cylindrical lenslets.

9. The microscopy system of claim 4, wherein said refocusing element is a converging cylinder MLA, comprised of one or more equal power positive cylindrical lenslets and disposed upstream to said slit array and second image plane (IP2).

10. The microscopy system of claim 4, wherein said refocusing element is a diverging cylinder MLA, comprised of one or more equal power negative cylindrical lenslets and disposed downstream to said slit array and second image plane (IP2).

11. The microscopy system of claims 9, 10, wherein either said imaging optics or said third scan lens is a lens assembly comprising first and second cylinder lenses with their axes mutually at right angles for correction of a mismatch of the first (X) and second (Y) meridian focal planes, provided by said confocal OPRA unit.

12. The microscopy system of claims 9, 10 further comprising a focus mismatch correcting means that comprises at least one cylindrical optical component for correction of a mismatch of the first (X) and second (Y) meridian focal planes, provided by said confocal OPRA unit.

13. The microscopy system of claim 4 further comprising a de-rotator being a second image rotator, synchronized with the first one and disposed on the optical axis downstream to said second scan mirror, for de-rotating images of said reassigned emission pattern in the third image plane (IP3).

14. The microscopy system of claim 4, further comprising an operation unit for control of the system and joint digital post-processing of at least two single axis scan OPRA images of a sample, scanned along different directions.

15. The microscopy systems of claims 1, 4, wherein said beam splitter is a spatial beam splitter that comprises a mirror contrast line shaped item, oriented along the first (X) direction: said beam splitter is disposed close to a focal plane of said first scan lens.

16. The microscopy systems of claims 1, 4, wherein said beam splitter is a dichroic mirror.

17. The OPRA microscopy system of claim 11, wherein said refocusing element and one of said first and second cylinder lenses are linearly adjustable along the optical axis to control a scale factor of the OPRA microscopy system.

18. The OPRA microscopy system of claim 12, wherein said refocusing element, at least one cylindrical optical component of said focus mismatch correcting means, and either said imaging lens or third scan lens all are linearly adjustable along the optical axis to control a scale factor of the OPRA microscopy system.

19. The microscopy system of claim 4, wherein said first and second scan mirrors are implemented in the form of a single bilateral scan mirror for providing scanning deflections of the probe beamlets and descanning the fluorescence imaging beamlets, both by a front side of the mirror, and synchronous rescanning deflection of the reassigned imaging beamlets by the back side of the mirror.

20. The microscopy system of claims 1, 4, 19, further comprising Z-scanner means for providing a series of 2D
slice images of a sample to be used for reconstruction of a 3D model of the sample.