MICROSCOPY SYSTEM

Th s application c a ms benefit of aftd priority Jo LT.S. Provisional Application No. 62/527.232, filed June 3 , 2017, and is entitled to that filing date for priority. The complete Speeiieaiiori:, drawings, appendices, a id disclosure of U.S. Provisional Application No. 62/527,232 are incorporated therein- in their entireties by specific reference for all purposes.

This invention was made with the support oHhe United States government under SF Contract No, i 353904. 1¾e Government has certain rights it), this invention,

FIELD OF INTENTION

This invention relates to an . illumination module and related methods for multi-focal light-sheet structured iUuniis Jon fluorescence microscop for integration in a commercial fluorescent microscope,

BACKGROUND OF THE ΪΝ ΈΝΤΪΟΝ

Several areas of biological and biomedical research critically depend on three- dimensional (3D) imaging for accurate analysis of subcellular structures within thick cellular samples. Three dimensiona imaging with enhanced spatial and temporal resolution is an essential tool for in vivo and in vitro studies of sub-eellnlar dynamics,

Unfortunately, conventional imaging systems cannot capture the 3D smscture of a specimen from one single 2D image. In order to retrieve its structure, a 3D image may be composed .c mputationally by recording a stack of 2D images of different transverse sections within the sample. Such a · tec n que ^' is employed I wide-field microscopy ( FM}. wherein images are obtained by scanning the sample volume axiaiiy. However, this mechanical movement presents important issues such as a slow acquisition speed, ^' which makes the detection of highly- dynamic biological processes impossible and introduces distortions during the acquisition process _* A teelmique called multifocal plane microscopy (MMU) provides a simple solution to avoid mechanical scanning, by imaging <ttJ½^ _; sections -of the _. s eeiptso usin several sensors. However, the imaging capability of MM LI Is limited by diffraction in the same way as in. WF .

Additional limitations that prevent High qualif 3D images of specimens hen usin WFM f MMU Include: (1.) limited spatial resolution imposed by diffraction, which is mainly determined by the numerical aperture (NA) of the objective leas used; and (2) n bilit to obtain high-resoiutloa optically-sectioned images which means thai the final. three-dimensional image is missing iotbnoation present in the underlying sample.

Thus, there Is a specific need for high accuracy approaches to live-cell microscopy that are not constrained by the thickness of the sample ami do ^' not requir long data-acquisition times. As discussed In more detail, below, different imaging tec niques have been proposed and commercialized to overcome these drawbacks during th last decades, However, the shortcomings of con ventional microscopy ha vs. yet to be addressed in a single technique.

Light-sheet flrsorescen.ee microscopy (LSFM) is an alternate technique that combines optical sectioning wit multiple-view imaging, to observe tissues an living organisms. However, the resolution is, again, limited by diffraction, in LSFM,. the sample is illnrninsted from the side in the focal plane of the detection objective. The Olurnhiation and the detection paths are distinct and. perpendicular to each other, requiring thai the sample be placed at the intersection of the illumination and the detection axes. The light-sheet excites the sample within a thin, volume ¾round the focal-plane and the emitted fluorescence is collected perpendicularly with a standard objective lens. The main limitation of this technique is that since two objective lenses are required, gh-numefiesi aperture (NA) objectives cannot be used and. as a result, the resolution of the LSFM system is usually low. To overcome this issue, LSF systems may he combined with two-photon excitation and super-resolution techniques. Moreover;, in some LSFM syst ms, the sample has been illuminated using two light-sheets to provide multi-foeal LSFM, However,, such a combination introduces additional expense and complexity. Two. Widely-used microscopic techniques, eoaibeai scanning microscopy (CSM.) and structured ill.ujt»s»atio*i icrospopy (SIM) cars surpass the resolution limit associated with other teehrhqueS; CSM is based on. point-wise 3D scanning of a specimen, using a small pinhole to reject the un n ed out-of-foctss light, and SIM is based on the urodifleatlon of the illumination system of a conventional WFM so that the specimen, is illuminated by a structured excitation pattern. The use of a structured illumination (SI) pattern enables the recovery of high-frequency infersnatioo, which is filtered, out by the frequency response of the WFM imaging system, and the reconstruction of iiigh-resolsition. 3D images can be achieved via computational methods.

Different optica! schemes have been proposed to create the needed. Si pattern. One configuration is based on the incoherent imaging of a periodic one-dimensional (I D) grid in th object space, Although this method ha been commercially Implemented, this system is not suitable for Increasing the transverse resolution because the contrast of the structured pattern Is reduced significantly at high, spatial frequencies ^' by the- optical transfer function (OTF) of the Incoherent illunhnaiion system Another solution creates the structured patters using the ^' interference of two plane waves, which are produced b the coherent illumination of a ID- grating. This approach allows for doubling of the reso!utiors limit sf WFM but cannot produce simultaneously super-resolved images with optical-sectioning capability.

Super-resolved optical-sectioned images have been achieved by producing a 3D S| pattern, which modulates excitation light in both the lateral and axial, direction. This structured pattern was obtained by the coherent Interference of three plane waves proceeding from a special IB diffraction grating. However, these illumination systems use coherent illumination that results In coherent noise in. the recorded images. _· . Furthermore^ these systems also, fail to provide the ability to change the frequency of the fringe illuminating pattern because the pattern depends ^' on the grating.

A spatial light modulator (SLM) or other electro-optical device can he used to create rapid frequency-tunable structured fringes In a .controlled and accurate mode. However, these technolo ies resen t eir own drawbacks, inektdmg, but not. limited to, the following: (1) they ar expe sive (due to die cost of the SLM or other type of eiectp optical device); (2) the enerated structured patterns■ ^■ usually are distorted due to the oblique illumination beeatise mode of them operate in reflection mode; (3} there are problems displaying high spatial-frequencies su¾ctt«*4 patterns due to their finite pixel $ ^' m an crosstalk effects:, strut (4) experimental Implementation of the SLM Ill umination system is extremely complex .

Alternatively;,, a Fresno! biprism illuminasd fey a set of in.eofeerea ly-ill iPtuated silts may be used to generate a suitable SI pattern. Using this system, lateral modulation of light depends the distance between die biprism and the sourc plane, while axial modulation of light can be governed by the number of slits and die separation between them. Although this s stem provides a tunable structured pattern that can -modulate light laterally and axialiy and lacks coherence noise, use of the Fresael biprism constrains, the available field of view and results in sub-optimal contrast.

Accordingly, what is needed is a system and approach that addresses the above deficiencies in the prior art,

SU ARY OF INVENTION

In various exemplary embodiments, the present inventio comprises a multifocal light- shee structured illuminatio module that can be implemented in any commercial fluorescence microscope. The present invention provides S8»«!taneons capture of ID images from multiple planes within a 3D volume, which are resolved laterally and axialiy to provide improved .resolution along the three dimensions (χ,ν,χ). The illumination method of this ^' invention employs a Wolfaston prism, thereby allowing several xislly-locallised high-contrast ^structured illnm snations pattern .

hi several embodiments, the illumination system provides light-sheet structured patterns ont the sample s ace of tunable spatial period ia order to obtain high resolution images of fluorescent specimens under research. These light-sheet structured patterns are generated by illuminating the Wollaston prism through the emerging spherical waveffent ftom set of equidistant and parallel, mcohereniiy^ilommated slits. The slits can he illuminated by a spatially- Incoherent light. The light source employed may -comprise light fitting diode (LTD; or a laser, wherein the spatial coherence of the light emission is broken usin , for example, a rotating difihser. Alternatively, the light source may fee. a white lam with a narrow bandwidth: filter. Ad it onal light sources may be appropriate or nse with the present illumination system in various embodiments,

BRIEF DESCRIPTION OF T E DRAWI GS

Figure ! A shows a view of the optical configuration of a conventional fluorescence microscope ^' mplemented in an upright scheme.

Figure IB shows a view of the optical c nfi uration of a m»hi-focal light sheet structured illumination microscope implemented in an upright scheme in accordance with an embodiment of the present Invention.

Figaro 2 A shows a view .of the optical configuration of a conventional fluorescence ^■ microscope implemented in an inverted scheme,

Figure IB shows a view of the optical configuration of a multi-focal light sheet structured illumination mic oscope implemented i .an inverted scheme n accordance with an embodiment of the present invention.

Figure 3 shows a schematic description: of the steps required to retrieve restored super- resolved (SIM) images from the recorded srrisctus-ed-illuminatiori micmscopy images through the acquisition of 3 images with, different phases.

ETAiLED DESCRIPTIO OF E5 E FLARY EMBODIMENTS in various exemplary embodiments, the present system comprises a multi-focal light- sheet structured illumination module that can. be implemented in any commercial fluorescence microscope. The present invention provides simultaneous capture o 2D images from multiple planes withia a 3D v lume, which are resolved laterally and axially to provide improved resolution along the three dimensions (x,y,2>, The illumination method of this invention employs a Woilaston prism, thereby allowin several axially-loea!ixed high-contrast structured illuminations patterns.

Figures IA and 2A show conventional fluorescence microscopes that are..sot equipped with the illumination system presently disclosed. Figures I B and 28 show a schematic view of a multi-focal light-sheet structured illumination system In accordanc with an embodusfent of the present ^■ invention. As shown in Figures. IB and 2B, the conventional microscopes of Figures IA _. and 2B can he readily adapted t include- the- Illumination system in accordance ife embodiments of the present inven tion.

In the upright configuration of f igure IB, incoherent light from an incoherent light source U} is passed through a first polarizer 1 a and a plurality of equidistant slit 12 that are parallel to one another (which may he in the font) of a binary mask). Subsequently, the light is. split by a Woilaston prism IS, The Woilaston prism 13 may he comprised of two blreffiugent wedges that are joined together along their respecti ve hypotenuses. In general, a Woilaston prism comprises two orthogonal prisms jointed- together along their base to. ibrrn two right triangle prisms with perpendicular optical axes, so that outgoing light beam diverge with the angle of divergence determined by the prisms' wedge angle and the wavelength of the light. Upon exiting the Woilaston prism 13, the light beam is divided into two spherical waves ith orthogonal polarization for each of the plurality of the slits 12, The light is sequentially transm itted through a first converging lens. 2f _t a quarter* ave piste (}J4) 14, and a second polarizer life. The second polarizer 11 b, may be configured to rot te aboot an axis, in this way, rotation o the second linear polarizer 1 lb permits a controlled shifting of a structured pattern.

The _. ligh beam next Is reflected via a .folded mirror ?b and transmitted through a second- converging lens 2b to be- reflected down ward via a diehroie mirror 3 and deflected off of a sample being held in a sample holder 5. The light beam cat) be focused on. th sample-b manipulation of a focusing scre 6 to move the sample holder relative to objective lens 2, The fluorescent light emitted by the sample is collected by an objective lens 4 and up through the diehroic mirror 3, A third converging tens 2e transmits ihe ^: light through the eyepiece 9 imaging path, or through the sensor 8 imag ng path using, a second folded mirror 7a. If the Sight is not reflected by the folded mirror 7a, then it is transmitted through a fourth converging lens M and fifth converging lens 2e _> and, ultimately, the image is projected onto a sensor 8 for eoHectio of the image data,

in the inverted conilgnration of Figure 2B, light from an incoherent light source 10 is passed through a first polarizer 11a and a plurality of equidistant slits 12.that are parallel to one another (which, as n ted above, may he in the form ^' of a binary mask). _. Subse u ntly, ^' the light is split by a oilastou prism (as described above) 13, The light is sequentially transmitted through a first converging lens 2f, a. quarter-wave plate (A/4) 14, and a second polarizer life. The second polarizer lib may be ^' configured to rotate about an axis, in this way, rotation of the second linear polarizer li permits a controlled shifting of a simc red pattern.

The light beam passes sequentially through a second converging lens 2g,. a first .diehroic mirror 3a, and a third eonvergmg lens 2b _* which directs light to the sample holder 4 with ^' a sample being held therein. The light can be focused on the sample by manipulation of a focusing screw 6. The fluorescent light emitted by the sample is collected by an objectiv lens 5, reflected by two mirrors 7a, 7b, and transmitted through two converging lenses 2e .2&. A folded -mirror ¾b projects the image through an eyepiece 9 or the sensor 8. Before- forming the image on the sensor for collection of the image data, the light passes through a sixth converging lens 2«.

As seen by comparing Figure. 1 with Figure IB, or f igure 2A with Figure 28, the above-described systems replace the . simple light source 1 and converging lens 21a used in the prior art.

It should be noted that the arrangement and number of lenses 3, and mirrors 3, 7 may he flexible, and that the arrangements shown in. Figures IB and 2B represent exemplary embodiments. Alternate arrangements that do not affect the optical resolution of the images obtained are envisioned. for a non-limiting, example, the Wollaston prism can be replaced by any polarixation- sensitive beam splitter. In another -alternative ^' the binary m sk could be generated using a programmable electro-optical device (e.g., a SL ), thereby providing control of a variable separation between the slits aitd the number of slits req ired,

As shown, the illumination system provides light-sheet, structured patterns onto the

^• sam le space 4 of tunable spatial period in order to obtain high resolution, images of fluorescent specimens trader research. These light-sheet structured patterns are generated by iilntthnatlag the Wollaston prism 1.3 through the emerging wayefrost from a set of equidistant and parallel slits 12, The slits 12 can be illuminated by a spatially-incoherent light HI, The light source W employed may comprise a quasi-monochromatic light emitting diode (LED) or a laser, wherein the spatial coherence o the light emission is broken using, for example, rotating diffuser. Alternatively, the- light: source SO may he a white-light lamp with a narrow bandwidth filter. As obvious to one of skill in the art, additional light sources may be appropriate for age with the present illumination system in various embodiments.

The presentl disclosed illumination system provides the incoherent superposition of N high-contrast sinusoidal patterns. Each of these N structured- patterns are a iaily-extended and there is a lateral displacement among them which produces axially-ioeaiized structured hinges. Because of this lateral displacement, the visibility of the structured pattern changes periodically, defined by the following Equation 1:

where % ™ ~ - n ₀ ) i I td nW are the apex angles, of the birefringent wedges; and no are art extraordinar and an ordinary refractive, index: of the Wollaston material, respectively; η is an axial separation between the slits and the Wollaston prism; / is the focal length of th converging lens, inserted after the oi as&m prism; and. s is an axial distance etwee : the le s and the observation plane.

To provid a period-tunable- structured illumination system, the incohcrentiy-ilfenvinated slits 12 are set at the front focal lane of a converging lens 2f, whose focal length is and the W Hastoh prism 13 is inserted between them, m this orie:n¾ation _t axial dis lacement (j.e _s . •orthogonal, relative to the illuminating light beam) of the Woliaston .prism H, (tf produces ^■ continuous variation of the spatial-period, in the structured pattern, /? given by the following Equation. 2:

with terras -as defined above.

As previously mentioned, the structu e pattern created by he Wo!lastson prism 13 after the converging tens is Imaged ^' onto the sample plane by means of an afoeal teieeentric imaging system, (comprising lb and 4 of Figure IB, and 2g and 2b of Figure 2B>> Inside the 3D volume ^• of the specimen one can i¾d different axial planes with . high-contrast structured fringes. Thus, usuiii-focai light-sheet structured pattern, of period uuabi!ity can be obtained on the sample volume. While the lateral period of the fringes can be tuned by. axiaily displacing the Woilastou prism 13, see Eq, 2, the axial positions and the separation of the different structured planes i$ mainly determined by the separation between the slits t% u¾) and totally sndcpendent of the number of slits, as seen in the following Equation 3 :

where m Is a positive integer. From this equation, one can derive that the axial separation between two planes of maximum visibility is determined by the following Equation 4: in this illumination s ste , the higher the number of sUts ^' 12, the narrower the axial co fmement Of the fringes, :meamng ^: thatthe axial confinement {Az) of these patterns is inversely proportional- to the number of meoherenUy^ilnmiaated _. sifts 12 {N). the axial, extension, of the planes with m xima contrast is defined by the following Equation. 5:

77

',ν

^" !.¾8 illumination system may generate, a- l ight-sheet structured pattern in a sample plane of axial extension defined by the following Equation 6:

and a lateral period defined by the following .Equation 7;

where Mat is the lateral magnification between an ilium matioR plane a id the sample plane.

As shown In Figures IB and 28, the illumination system disclosed herein is uniquely designed to be implemented in any commerci l microscope as an external Illumination .module.

The l.lhnnmation system can. be fitted to commercial microscopes in a ^■ transmission or a re flection configuration and in upright and Inverted schemes. Since the disclosed multi-focal light-sheet

SiM system requires minimal implementation, the illumination system maintains the advantages of ^* any commercial microscope in terms of robustness, stability, and aser-friendjy operation while siimdtaneonsly offering ^' the additional advantages disclosed herein.

Advantages of the multifocal light sheet slructu ^' red-iliumination system include, but arc not limited, to. the following: ( 1 ) generation of high-contrast structured fringes with tunable spatial-frequency; (2) phase-shifting of the recorded structured image by rotating a polarizer wfee the light used to illuminate the oi!astou prism is: circularly-polarised^ (3) every 90-degree rotation of th anal ser providing a phase-shifting of 3t radians in the fringes, independent of the lateral modulation frequency of the structured pattern, and thns calibration is not needed; (4) creation of tat ti-focai light-sheet structured fr nges, inside the volume of the specimen whic permits recording of the 3D structure -of the sample without any movement of the sample, itself; (5) no coherence noise to distort the recorded images; (€) discrimination of different feateres of biological samples: based on their response to a specific direction of light polarization because the Wollaston prism Is a. polarization-sens ive device; (?) accomplishing optical-sectioning capability comparable to thai of LSFM without requiring two objectives lenses and Overcoming the resolution limit; and (8;) overcoming the resolution limit by eoo dkg three phase-shifted raw images instead of nine needed b odrer SIM systems (this redaction in data-acquisitlosi is m advautage tor live-cell imaging, which requires fast imaging and at lower light exposures).

The images recorded by the camera should be computationally processed in order to obtain super-resolved images with optioal-secdoning capability; Figure 3 shows the acquisition and processing steps of this technique. This proces can be divided in four main steps, and with the proper code, the restored images cm be obtained almost in real-tirue. The first step is the acquisition phase, In the acquisition phase,. 30 forward images (p /> _¾ Bj) ar captured with three different hases{ ;, $) by axiaily scanning the speekner s vo!nme, with 3D irradiance distribution given by 0(F) , At each field of view, three images are acquired that correspond to three different phases of the structured pattern along the direction of the fringes, in the second step, die image are decomposed according to the following formulas, which includes, computation of the- fast 3D Fourie -transform of the.3D images: where H(r) stan s for the impgbe response _, of the imaging system, is the axt&l modulation of the visibility of the iUuininatkm _. pattern, and f _} . ₍ Is the lateral spatial frequency of this pattern. in the sample's volume. In this equation, X ~ F {X} represents the ^' 3D Fourier transform of the _. tunc, ion A'.

After ^■ decomposition, the deconvolntiou and shifting set occurs, wherein three new 3D components are calculated fey demodulating each SIM image using- a Wiener filter.- .During shifting, the positions of the n w c mponents are centered m the Fourier domain. Finally, in the fourth step, the- centered, deconvolved, 3D images are combined to form the restored images. This step is accomplished by wa of a linear mm _* calculating the inverse fast 3D Fourier transform of the combination. Alternatively, different processing methods applicable to any SIM approach, cm he used to process the data acquired from this multi-focal light sheet sectored illumination system

Note that, isotropic improvement in 3D resolution is achieved by rotating the structured pattern and sequi ing data, in 3 different orientation angles. This hinges ^* rotation can be produced by rotating jointly the first polarizer, the binary mask, the Woliaston prism and the quarter-wave plate. Commonly, one should acquire 3 phase-shifted SIM images per orientations (a. total no ber of 9 images per transverse section) and apply the reconstruction method described above to achieve the final 30 super-resolution image. However, takin advantage of Information, redundancy, th total number of recorded Images can he reduced to fear image (two taken in th first orientatio and one fo each of the remaining two orientations),

in some embodiments, the system further comprises polarisation rotator (which may be mechanically, electrically, or optically driven) _, disposed between the quarter-wave plate and the second linear polarizer. In additional embodiments, alternation of the orientation of the plurality -of light-sheet structured patterns is achieved by a tunable image rotator after the oiiaston prism, as (out not limited to) a mirror combination or a Dove prism. Embod ments of the illumination system disclosed her in provide a number .of novel aspects thai are not enrrently available in die rior art. The novel as ects include, but are not limited to: (1) continuous variation of the spatial-period of the structured pattern fringes; (2) axial localization of the structured fringes (light-sheet structured patterns); (3) simultaneous illumination of multiple axial plane within the specimen; (4) polarfeatioo-sensitrve illumination system; and (5) m exterftal illumination module adaptable to any commercial fluorescent microscope.

The illumination system disclosed herein also offers rsumeroas distinct advantages over current technology. Importantly, the: embodiments of the present invention permit tunabHi y of the structured fringes with the axial displacement of the Wollaston prism 13, This advantage circumvents the use of a spatial light modulator (S ^' L ). which is currently used by many- researchers- and manufacturers. Unlike the presently disclosed illumination system, SLMs are extremely expensive and are not designed to be easily implemented m a commercial microscope. In addition, embodiments of the present invention use incoherent light, which is a significant advantage because the coherent light used in commercially available microscopes produces coherent, noise that interfere with and distorts the ultimately recorded images. Further, in the present invention, there is no reduction of the contrast of the structured fringes for high spatial- frequency as in .commercially-available modules.

Aspects of th presently disclosed invention provide high optical-sectioning capability while simultaneously overcoming the resolution limit The ^' high- optical-sectioning is somewhat comparable to conventional light-sheet microscopy; however, unlike light-sheet microscopy, the present invention -accomplishes, such high-optical sectioning th o g he use of only a singl objective lens. The present illumination is also capable of simultaneous Hi uniuation of different transverse sections of a specimen. In. this wa , multi le focal ^' lanes can ^' be- detected through the use of multiple cameras , Thus, it should be understood t &i the embodiments and examples descri ed herein bave b en chosen and described in order to best illustrate the principles of the nvention and. its practical applications io thereby enable one of ordinary skill in the art to best aiiiize the invefttion m various .embodiments and with various modifications as are suited for particular uses contemplated. Even though specific embodiments of this invention have been described, t y are not to be takers as exhaustive. There are several variations that ill be apparettt to those skilled its