I AGING LARGE AREAS WITH MICROSCOPIC ^' RESOLUTION

CROSS-REFERENCE TO RELATED APPLICATIONS (000 J] This application relates to and claims priority from U.S. Patent Application No. 61/620,515, filed on April 5, 2012, and U.S. Patent Application No. 61,620,2] 5, filed on

April 4, 2012, the entire disclosures of which are incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

(0002] This disclosure was made with government support under Grant Nos. 08748 and 30CA08748 awarded by the National Cancer Institute, and Grant No. RO 1 EB012466 awarded by the National Institute of Health. The government may have certain rights in the present disclosure. This statement is included solely to comply with 37 C.P.R. §4 1.14(f)(4) and should not be taken as an assertion, or admission thai the application discioses and/or claims only a particular invention.

FIELD OF THE DISCLOS URE (0003] The present disclosure relates generally to imaging large areas, and more specifically to exemplary embodiments of systems, devices, methods and computer accessible mediums for imaging large areas with microscopic resolution.

BACKGROUND INFORMATION (0004] An accurate and complete removal of a tumor, with minimal collateral damage to ihe surrounding tissue, can be guided by the examination of pathology. However, the pathology prepared, either during surgery or after, can be time consuming, in the setting of Mohs surgery of non-melanoma skin cancers, frozen pathology that can be prepared dining the procedure can take 20-45 minutes per excision., and two or more excisions can be performed, which can .make the total preparation time several hoars (See e.g.. Reference 1). in other settings, such as head-and-neck and breast cancer, surgery fixed, pathology can be prepared following the surgery. Preparation of fixed sections can take at least 1 -2 days. Such time delays ca result in a inability to sample large amounts of tissue, and detection -of residual tumor margins, in real time. Consequently, insufficient sampling of tissue, incomplete tumor removal and positive margins are reported to be between 20 to 70% of patients (See e.g.. References 2 and 3). A large number of such patients subsequently undergo additional surgery, radiotherapy and/or chemotherapy. The optical imaging methods that can display nuclear morphology can offer real-time detection of tumors in large areas of freshly excised or biopsied tissue without the need for the processing that can he used in pathology. One well-know approach, is based on confocal microscopy (See e.g.. References 4 and 7} and another, more recent approach, is based on full-field optical coherence tomography (See e.g., Reference 8).

[00051 it can be possible to utilke confocal raosaieing microscopy procedures for imaging tumor margins in fresh tissue from surgery (See e.g., References 9 and .12). in one such exemplary embodiment ii can be possible to provide access to high-resolution images of large areas of tissue within a short time period (e.g., a few minutes).

jiMMMj For example, square confocal images can be collected and stitched together with custom software into a mosaic that displays a large field of view. The mosaicing of about 36 x 36 images (e.g., to display up to 12 x 1.2 mm" of excised tissue from Mohs surgery) can be provided in a short time frame, for example, in about 9 minutes (See e.g., References 4, 9, and 10). in a blind examination of 45 fluorescence mosaics by two Mohs surgeons, basal cell carcinoma margins were detected with an overall sensitivity of 96.6%, and a specificity of 89.2% (See e.g.. References 13 and 14).

|ΘΘ07] Indeed, obtaining the results in such time frame can certainly be faster than the hours or days generally required for preparing pathology; however, routine implementation in Molis surgical settings can benefit from even faster times. In other surgical settings, excisions can be larger, and thus mosaicing by such exemplary procedure can take longer. Therefore, the adaptation of such technology to be used during surgery may not be as effective. For practical and routine utility, the exemplary mosaicing should meet the surgeons ^* need to examine tumor margins in large areas (e.g., - cm*) within fairly short times (e.g., - one minute).

(0008] Thus, it may be beneficial to provide ^' exemplary systems, methods and computer-accessible mediums that can facilitate a shorter imaging time, and/or solve at least some of the deficiencies described herein abo v e.

SUMMARY OF EXEMPLARY EMBODIMENTS

(00 91 Such needs can be addressed wi th the exemplary embodiments of the system, device, method and computer accessible medium for imaging large areas with microscopic resolution according to the present disclosure.

[00 9] To address this need, an exemplary approach (which can be called "strip mosaicing" herein) - which can be a faster approach - can be provided. An exemplary instrumentation utilized for strip moasicing was recently described (See e.g.. Reference 15). For example, exemplary strip mosaicing procedures can be performed with a combination of optical and .mechanical scanning. The sample can be mechanically translated across an optical, line allowing high aspect .ratios, instead of the standard 1 : i aspect ratio of previous mosaicing procedure. [fl l 1] According to certain exemplary embodiments of the present disclosure, mechanical arrangemenis, electronics and software can be provided to image, for example, about 1 en human skin tissue in 90 seconds. An exemplary tissue translation stage can be provided which can. increase and improve speed, accurac and precision. The optical and mechanical scanning arrangement can be provided which can be synchronized to optimize alignment among strips, and image strips can. be stitched, during acquisition with eastern software.

[0032 | Previously, mosaicing on, for example, 12 x .! 2 mm ^" of excised tissue from

Mohs surgery, and detection of basal cell carcinoma margins, was demonstrated in 9 minutes, A faster approach called "strip mosaicing" can be utilized, according to certain exemplary embodiments of the present disclosure. Exemplary strip mosaicing on, for example, 10 x 10 mm ² of tissue was demonstrated in 3 minutes. Exemplary instrumentation, systems, methods and computer accessible medium according to the present disclosure can be provided which can facilitate mosaicing of about 10 x 10 mm ^"1 tissue in about 90 seconds. For example, rapid mosaicing of confoca! images on large areas of fresh tissue can offer a way to perform pathology at the bedside. Thus, exemplary strip mosaicing confoca! microscopy procedures can serve as an adjunct to pathology for imaging tumor margins to guide surgery.

[0013] Exemplary systems, method and non-transitory computer-accessible medium can be prov ided for generating an image of at least one tissue that can be subdi vided into a plurality of strips. Using such exemplary systems, method and computer-accessible medium, it is possible to scan., using an optical arrangement to generate first information, a first portion of the tissueis) along a first one of the strips while the tissue(s) can be mechanically moved i a first direction. Move the one tissueis), by a distance equal to or less than a width of the first portion, in a second direction approximately perpendicular to the first direction. Use the optica! arrangement to generate second information, a second portion of the tissueis) along a second one of the strips while the lissue(s) can be mechanically moved in a third direction, which can be approximately opposite to the first direction. Then the image can be generated based on the first information and the second information.

| _. ΘΘ14] According to further exemplar embodiments of the present disclosure, the moving procedure can be performed wit hoist scanning the at least one tissue. The .first portion can. be scanned from a proximal end of the first strip to a. distal end. thereof, and. the second portion can be scanned from a. distal end of the second strip to a proximal end thereof. The scanning of the first portion and the second, portion can be performed without stopping, and the scanning of the first portion and the second portion can be performed during a continuous and uninterrupted motion of the tissne(s).

|ΘΘ15] to additional exemplary embodiments of the present disclosure, the scanning of the first portion and the second portion can be synchronized with the moving of the iissiie(s) using a synchronization procedure. The hardware synchronization procedure can include scanning the first portion and the second portion based, on a predetermined clock signal, or initiating the scanning of the first portion and the second portion based on the predetermined clock signal. The tissue(s) can be moved from a first location such that the opiical arrangement can be away from an end of the first portion or the second portion to a second location such that the optical arrangement can be at the end of the first portion or the second portion, and the scanning of the first portion or the second portion can be started based on the predetermined, clock signal and the second, location of the at least one tissue.

[00 J 6] In certain exemplary embodiments of the present disclosure, the hardware synchronization procedure can include moving the tissue(s) ^' based, on a predetermined clock signal. The tissue(s) can be moved, based on the predetermined clock signal, from a first location such that the optical arrangement can be away from an. end. of the first, portion or the second portion to a second location such that the optical arrangement can be at the end of the first portion or the second portion, and the scanning of the first portion can be started based oo the second location of the at least one tissue.

[0017] I n some exemplary embodiments of the present, disclosure, the first strip and the second strip can be square shaped. For example, the first strip and the second strip can be rectangularly shaped, and the rectangularly shaped first strip and second strip each can have an aspect ratio of approximately 1 :25. The image can be generated using a. moasicing procedure, which can include stitching together the first information and the second, information.

|ΘΘ18| In a further exemplary embodiment, of the present disclosure, a system can be provided for generating an image of a tissueis). The exemplary system can include an optical scanning arrangement configured to scan a strip of the tissue(s), and a mechanical arrangement configured to move the tissue(s) when the strip of the tissue(s) can be scanned by the optical arrangement. An imaging arrangement can also be provided which can be configured, to generate the image based on information received from the optical scanning arrangement.

[00191 These and other objects, features and advantages of the exemplary

embodiments of the present disclosure will become apparent, upon reading the following detailed description of the exemplary embodiments of the present disclosure, when taken in conjunction with the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[ΘΘ20] Further objects, features and advantages of the present disclosure will become apparent from the following detailed description taken in conjunction with (he accompanying Figures showing illustrative embodiments of the present, disclosure, in which: [0021] Figure 1 A is an exempiarv schematic diagram illustrating exemplary mosaicing of square-shaped images in two dimensions according to an exemplary embodiment of the present disclosure;

10022] Figure 18 is an exemplar)? schematic diagram illustrating exemplary mosaicing of rectangular-shaped long strips in one dimension according to an exemplary embodiment of the present disclosure:

(0023} Figure 2 is an exemplary schematic diagra m of an exemplary strip-sc anning m echanism and/or procedure according to an exemplary embodiment of the present disclosure;

(0024] Figures 3A-3C are an exemplary signal diagrams illustrating exemplary synchronization procedures for strips according to an exemplary embodiment of the present disclosure;

(0025) Figure 4 is an exemplary signal diagram provided by an oscilloscope trace of the front end of a scan of a strip according to an exemplary embodiment of the present disclosure;

(0026| Figure 5 is an exemplary signal diagram provided by an oscilloscope trace of the back end of a scan of a strip according to an exemplary embodiment of the present disclosure;

Ϊ0Θ27) Figure 6 is an exemplary image illustrating a mosaic having a particular exemplary number of fluorescence image strips excised tissue from an exemplary Mohs surgery according to an exemplary embodiment of the present disclosure;

(0028] Figure 7 is an exemplary image of a frozen Hematoxylin and eosin ί 'Ή&Ε"}- stained pathology of excised tissue from an exemplary Mohs surgery according to an exemplary embodiment of the present disclosure; [0029] Figure is an exemplary flow diagram for imaging at least one tissue

according to an exemplary embodiment of the present disclosure; and

[0030] Figure 9 is an illustration of an exemplary bl ock diagram o f an exemplary system in accordance with ^' certain exemplary embodiments of the present disclosure.

[0031] Throughout the drawings, the same reference numerals and characters, unless otherwise stated, are used to denote like features, elements, components, or portions of the illustrated embodiments. Moreover, while the present disclosure will now he described in detail with reference to the figures, it is done so in connection with the illustrative

embodiments and is not limited by the particular embodiments illustrated in the figures, or the appended claims.

DETAILED DESCRIPTION OF EXEM PLARY EMBODIMENTS

[0032) Exemplary system, method and computer-accessible medium, according to exemplary embodiments of the present disclosure, can be provided so as to utilize an exemplary mosaicing procedure (see, e.g., Figure 8 - Procedure 825) by stitching square* shaped images, for example, with a standard 1:1 aspect ratio, in two dimensions, as shown, in. Figure I A. According to another exemplary embodiment of the present disclosure, it can be possible to utilize a further exemplary mosaicing procedure which can acquire rectangular- shaped long strips of images, with, for example, a 1:25 aspect ratio for approximately 10 mm long strip, and stitches along the length of the strip, as shown i Figure IB. For example, art elimination of a dimension can reduce the acquisition time, stitching time and the artifacts due to illumination fall-off, for example, by about 50%.

[0033] A combined optical and mechanical scan strip mosaicing procedure apparatus, according io exemplary embodiments of the present disclosure, can be seen in the exemplary illustration of Figure 2, which can show a fast optical scan procedure that can image a line 205 (e.g., in x -directi on) along the width of a strip (e.g., x-axis) (see, e.g., Figure 8 - Procedure 800). This exemplary tine am be scanned along the length of the strip 210 (e.g., y- axis) by translating the stage 250 in the direction orthogonal to the optical line (see, e.g., Figure 8 ~ Procedure .805). When the acquisition of a strip image 215 is completed (e.g., "forward scan" in y-direction), the stage 250 can step through a distance equal to or less than the width of the strip is the x-direction (see, e.g.. Figure 8 - Procedure 81 ). Th en, the stage 250 can translate along the length, e.g., in the direction opposite (e.g., "return scan'"} to the previous scan, and another strip image can be acquired (see, e.g.. Figure 8 - Procedure 815). This process of acquiring strip images, cyclically in. opposite y-directions (e.g., forward scan followed by return scan), can be repeated until th entire tissue 220 can be imaged (see, e.g., Figure 8 - Procedure 820).

Exemplary Instrumentation

Exemplary Confocal Microscope

[0034) According to an exemplary embodiment of th present disclosure, it can be possible to provide exemplary system, method, and computer-accessible medium which, can utilize a modif ed breadboard versio of a commercially-available confocal microscope 225 (e.g., Vivascope 2000, Lucid Inc., Rochester, NY). The exemplary microscope can be modified to image in tw modes, reflectance and fluorescence, and can have two or more detector channels. Exemplary control commands can be transmitted to the microscope's control system, for example, via a RS-232 port, or other suitable communication mechanism. The exemplars' microscope can be provided in an inverted configuration that can. be designed to image excised or biopsied tissue ex vivo. The illumination, can be performed with, for example, a 488 nra laser wi h power of- 5 m.W on the tissue. The exemplary 24-sided polygonal mirror can be used to scan the laser in the x-direction sweeping a line at - 8.9 kHz through a 30x, 0.9 numerical aperture ("NA") water immersion, objective lens (e.g.,

Stabieview, Lucid inc.). The length of the line can be 485 .urn. The lateral resolution, per Rayleigh criterion, can be about 0.33 p.m (Airy radius) and the optical sectioning can be about 1 .61 pr The exemplary objective lens can be custom-designed to image through a S ~ ni-thick glass slide. In normal operation, this line can be scanned in the orthogonal direction (e.g., y-direction) by a galvanometricaHy-d.ri.ven mirror to produce a square-shaped image. To acquire a long rectangular-shaped strip image stop the ga!vanometric scan can be stopped and "lock" the position of the polygon-scanned line on the optical, axis. Exemplary Tissue preparation and Mounting

0035j Exemplary tissues used from Mohs surgery and staining methods have been extensively described (See e.g.. References 9 and 12). The staining of nuclear morphology can be accomplished by soaking the tissue in, for example, 0.6 mM acridine orange for about 30 seconds followed by rinsing the excess with isotonic phosphate buffered saline solution, [ΘΘ36] Fresh tissue 220 from surgery can present irregular shapes and topography, and variable sizes. Imaging of large areas of such tissue to create mosaic can benefit from accurate and repeatable mounting. A tissue fixture can be engineered for mounting Mohs surgical excisions and to control the position, flatness and orientation of the surface to be imaged. The fix ture can be mounted on to a translation stage 250, and the stage can be fitted to the microscope breadboard with a custom-made holder.

[0037] The tissue 220 can be placed on the glass window 230 of the fixture and imaged through it. An acquisition of a large number of images over a large area at a constant depth can use imaging and scanning planes parallel to each other. This parallelism can be obtained by adjusting the tip-tilt adjustments of the tissue fixture in an iterative manner, and observing images of the glass window at four comers. When the images at the four comers appear uniform and idetuical to each other,, the image plane can be parallei to the x-y scan plane.

[0038] The exemplary mechanical specifications of the translation stage such as straighmess, flatness, repeatability and accuracy can be exemplary factors for long strip scans. It can be possible to use a high-quality dual-axis stage (e.g., BioPrecisioiil, Ludl Electronics, Hawthorne, NY) to .mechanically scan (e.g., translate) the tissue with respect to the objective lens. The straighmess and flatness of this stage can be about Ι ηι /25mm, Therefore, for a scan (e.g., translation) of 10 mm travel, for example, the .maximum deviation in straighmess can be 0.8 μιη between any two strips. This can be well within the overlap between any two strips. The maximum deviation in flatness can also be within the optical sectioning of about 1.61 μ ^η ν

0039] The exemplary translation stage 250 can be equipped with a linear encoder.

The exemplary encoder can be read via its controller (e.g., MAC5000, Ludl). The encoder outputs can be sent to a fast data acquisition ^* DAQ") card 260 (e.g., via synchronization signals 280) for synchronization in real time. The commands 285 to initiate and control the movement of the stage 250 can. be transmitted to a controller 290 through a USB bus, or other suitable communication mechanism.

Exemplary: Hardware Synchronization

[0040] li can be beneficial to have the a plurality of scans, (e.g., the fast optical scan and the slow mechanical scan) to ^' be synchronous to avoid mismatch between any two adjacent strips, as such mismatches can introduce artifacts in the final image. Since the scanning mechanisms can be independent of one another (e.g., not coupled to one another) they can be asynchronous. Furthermore, the speeds of the two scans can he are vastly different from one another (e.g., milliseconds v. seconds). The optical scan system can be faster (e.g.. an angular scan of approximately 10 milliseconds per revolution) and the mechanical stage scan can be slower (e.g., a liner scan of approximately 2.5 millimeters per second), i order to overcome such varying scan times, various techniques can be used to synchronize the optical scan and the mechanical scan.

First Exemplary Hardware Synchronteation Procedure

{604 ij In the first, exemplary procedure, the relative time stability of each system can be utilized and synchronized only at discrete points in the image acquisition procedure (e.g., at the beginning of each image strip and at the end of each image strip).

f0042| Referring to the exemplary diagram of Figure 3B. the optica! scanner 325 can run tree (e.g., independently), and generate a clock signal HSYNC 330, using HSYNC generator 340. The mechanical stage (e.g., motorized stage, etc.) can initiate the scan of the first strip, e.g., the Forward Scan 315, at P0. When the motorized stage reaches the position PI the image acquisition can be triggered at the first available HSYNC 330 (e.g., HSYNC If)- When the stage reaches the position P2, the image acquisition for the Forward Scan 3 5 can stop. The mechanical stage can slow down .and stop at position P3 while the optical scanner 325 can continue to generate HSYNC pulses 330. Then, the mechanical stage can move a distance equal or less than the width of the most recently scanned strip in the direction orthogonal to the Forward Scan 31 5, and begin the Reverse Scan 320, of the adjacent strip, at the position P3. When the stage reaches the position P2, the image acquisition can he triggered at the first available HSYNC (e.g., HSYNC fr). When the stage reaches the position Pi, the image acquisition, can stop, .finishing the image strip.

Second fae^¾ t ry Hardware S nc [0043] Referring to the exemplary diagram of Figure 3C, the optical scanner 325 can generate a start-of-scan ("SOS") signal with a circuit with SOS generator 335. Another circuit can be triggered arid/or gated to generate a clock using a HSYNC generator 340. The HSYNC signal 330 ca then be integrated, using an integrator 345, to make a voltage signal that, is proportional to the frequency of the SOS. This voltage can then be transmitted to a voltage-controlled oscillator ("VCO") 350 to generate a clock signal 360 having a particular frequency to drive the motors of the stage, using, e.g., a motor controller 355 , Motor pulses can be generated with a phase4ock.ed~l.oop ("PLL") circuit driven by the HSYNC signal 330. Ι0Θ44]

To conduct a scan of an image strip, the optical scanner 325 can run asynchronously, and the exemplary circuit can generate SOS pulses. When the first image strip is ready to be acquired, in the Forward Scan (e.g., in Figure 3B), a circuit can be triggered to generate the .first HSYNC 330 (e.g., HSYNCl f in Figure 3B). This can generate exemplary motor pulses for the motorized stage and the Forward Scan 15 can begin synchronously at position ΡΘ, When the stage reaches position PI the image acquisition can he initiated, and when the stage reaches P2 the image acquisition can be stopped and the stage can he brought to a halt at P3. Then, the exemplary stage can move a distance equal or less than the width of the most recently scanned strip in the direction which can be orthogonal to the Forward Scan 3.15, and the circuit can issue a trigger to generate HSYNC l.r (e.g., see Figure 3B) to initiate the Reverse Scan 320. The motor pulses for the motorized stage can then be generated

synchronously to HSYNC S , and the Reverse Scan 320 can begin at position P3. The image acquisition can be initiated at the position P2, and stopped at the position PI , completing the reverse scan image strip. Then, the stage can be brought to halt at the position P0. Exemplary Software Synchrooigation Procedure [0045] While hardware techniques can designed to minimize and errors between my two strips, errors can be ultimately dependent on the mechanical stability of each system. The mechanical instabilities can cause mismatch between any two adjacent image strips along the ■length of the image strip . Therefore, an. exemplary software technique can. be used to compensate for any errors by registering the image strips.

[0046] The registering of the _. strips can.be performed in parallel with th acquisition, for example, where each new strip can be stitched to the previoits strip while the next strip is acquired.. This exemplary procedure can help routine clinical care in two ways. First, such exemplary procedure can provide a smoother image without any mosaicing artifacts, (e.g., illumination fail off), with minimal additional time to the scanning process. Second, the exemplary procedure can facilitate the clinician to view the image for various pathologies as it is being acquired, without having to wait for ihe end of the scan, thus shortening the time to diagnosis. Exemplary Synchronization of strips in the Y-direciion

10047] The mechanical, scan by ihe translation stage (e.g., slow scan in Y-directiori) can be dri ven by a stepper-motor without a position encoder, it can be possible to accomplish the synchronization of the optical scan by the polygon (e.g., fast scan in. X- directton) and the mechanical scan by the translation stage (e.g. slow scan in Y-directioh) by counting the step-pulses from an exemplary stepper-motor. A mismatch of up to 75 l ines, between any two strips, can be observed, which can result from missing steps, and the lack of posi ion accuracy. This mismatch can be corrected daring stitching of the strips, although at the expense of a likely high demand for computer processing power and increased, time for creating mosaics. Therefore, position synchromjtation in. Y-direction can be important to reduce the work of the stitching procedure, and to reduce the time used for mosaicing. The exemplary synchronization mechanism, according to an exemplary embodiment of the present disclosure, can. facilitate thai the maximum expected mismatch between, any two strips to be, for example, one line in the Y-direction, and zero pixels between any two lines within a strip.

(0048) Figure 3 A shows an exemplary signal diagram provided by an exemplary synchronizing procedure for the strips, in accordance with an exemplary embodiment of the present disclosure. For example, a Y-translatien stage cycle can include two or more mechanical scans that produce two or more image -strips, for example, the "forward scan" P _¾ to P.? (e.g., velocity profile 305) and the "return scan" P ₃ to P« {velocity profile 31 ), An image strip 315 can be acquired during the forward scan. Then, the stage can step or translate through, for example, about 400 μηι, which can foe less than the line length 485 μ.ΐη, or the width of a strip, in the X-direction, and another image strip 320 can be acquired on the return scan. Since the acceleration and deceleration profiles of the exemplary translation stage for forward and return scans can be different (e.g., as depicted in Figure 3 A), the image can be acquired within the constant velocit portion of both scans. This can avoid a distortion of the mosaic due to compression or elongation of pixels. Therefore, it can. be possible to select a scan, distance (D) for the Y-tfanslaiion. stage such that the region of constant velocity section (d) can be larger than the size of the tissue excision, as shown in Figure 3A, Accordin to an. exemplary embodiment, it can be possible to first determine the positions P«, P _}> P¾ and Ps b observing the velocity profiles of the Y-ttanslation stage through one cycle. When the coordinates can be known, it can be possible to scan the sample and acquire images.

(0049) A graph of exemplary oscilloscope traces of the timing procedure, for example, the front portion, of the scan, is shown in an exemplary signal diagram of Figure 4. for example, after the forward scan, can be initiated, the data acquisition card can monitor the stage positions Po, Pj, P , and P¾ in real time via the encoder and stage controller. When the stage reaches Pi , the "start" signal 405 can be asserted to arm the counter which monitors horizontal synchronization ("HSYNC) pulses 410. When, the HSYNC -counter receives its first HSYNC pulse 415, it can trigger another counter to generate a pixel, clock ("PCLK"), The . PCL ca sample the analog input video signal creating the first line of the image in the strip. A number of PCLK pulses to be generated by the counter can be predefined. W hen the counter reaches the predefined value, it can reset and wait for the next HSYNC pulse to repeat the process. This exemplary process can continue until the Y -translation stage reaches position P ₂ . At Fj the "stop" signal (see signal 505 of Figure 5) can be asserted, and the HSYNC counter can be stopped. However, the image acquisition can continue until the PCLK counter reaches its predefined value, thus completing the last line in the strip image. At the end of Y-translation stage travel at Pj.the HSYNC counter can be reset. The X- transialion stage can then step or translate in the -direction to a predetermined value, which can set the width of the strip. The return scan can he initiated from P¾ to Po, and can follow, for example, a similar mechanism to the forward scan. This exemplary cyclical process, forward scan and return scan, can be repeated until the entire tissue can be imaged.

Exemplary Acquisition of Mosaics

[6050] The exemplary acquisition time can be restricted by the speed of the optical

(e.g., polygon) scanning rate of, for example, -8.9 kHz. Since the exemplary lateral resolution can be about 0.33 adequate sampling can utilize the speed of the stage as being less than about 2.9 ram's (e.g., 0.33 μηι x 8.9 kHz). However, the stage can be scanned at approximately 8.5 mm s to reduce the mosaicing time and thus under-sample by a factor of approximately 6. This can result in a pixel size of approximately 1 pra in both X and Y directions. An exemplary resolution of about 1 μηι can be adequate for the interpretation of images by surgeons and pathologists f 15 ). When the Y-scan is completed, the X-stage can move, for example, about 400 pro. laterally, leaving approximately a 17% overlap between any two strips. It can be possible to repeat this exemplary procedure until, for example, the sample can be fully scanned.

[005XJ Th fluorescence images can be captured with, for example, a last DAQ card

(e.g., PCi-61 15 and Labview, National instruments,, Austin TX), The captured image strips can be stitched according to the exemplary-methods and procedures according to exemplary embodiments of the present, disclosure,

£xeinp.kg.Stitching

|ββ»2 An exemplary stitching procedure according to an exemplary embodiment of the present disclosure (e.g., which can be written in or utilize Matiab, R201 1 a, Mathworks, Inc.) can be provided to automatically register image strips in the order in which they can be collected. Strips can he registered pair-wise, and then stitched together into one mosaic, A phase correlation method can be selected for speed and ease of implementation, as can be determined by Fast Fourier Transform ('TFT"). An exemplary correlation between the images can be maxim zed when features in the subject images can be properly overlapped. However, the phase correlation can be biased towards solutions near zero offset between the two images, and so the bias can be removed in the x-direciion by dividing the cross

correlation by a triangle function. The removal of the bias can emphasize the noise near the edges of the cross correlation. Thus, for example, about 30 pixels near the edge of the cross- correlation can be excluded from the search region, as solutions in that region of maximum offset may not be expected. Along the y-direction, as solutions near zero offset can be expected, the removal of the bias can be unnecessary, and can likely serve to emphasize the noise. Generally, a window function can be applied to an image before performing a phase correlation algorithm to reduce high-frequency edge effects. However, according to an exemplary embodiment of ihe present disclosure, the illumination falloff due to optical vignetting across the strips can serve this purpose, and thus, the illumination falloff can be corrected after an exemplary registration procedure can be applied.

f i ^} 9S3| According to an exemplary embodiment of the present disclosure, the falloff can be corrected by averaging each strip along its length, normalizing that average to one, and then dividing each Sine in that strip by the normalized average falloff along the strip. Because the laser illumination can be constant throughout the acquisition of the mosaic, the signal level of each strip can be properly related to its neighbors, and therefore divi ding by the normalized average falloff can preserve the signal level of each strip with respect to its neighbors. The entire mosaic can be resettled to attain a ull range of brightness levels.

[ΘΘ54| In the exemplary overlap region between strips, the strips can be blended by a weighted average of the overlapping pixels determined by the pixel distance to the edge of the strip. Pixels close to th edge of the strip can be weighted less than pixels far from the edge of the strip. An exemplary result can be, for example, a seamless mosaic,

[ΘΘ55] To reduce the total time for the acquisition and stitching of the mosaics, an exemplary embodiment of the stitchin : procedure can be run simultaneously with the exemplary acquisition. For example, a each strip is acquired, it can be saved into a directory, which can be polled, for example, every few second (e.g., three) by the exemplary stitching procedure. ^' When two or more strips have been collected, they can be registered and blended together into the mosaic, which can be displayed on the screen so that the operator can view the progress of the exemplary acquisition. As each new strip is collected, it can be registered to the previous strip, and blended into the mosaic. Therefore, the exemplar registration and blending of the mosaic stri ps do not add addi tional time to the total mosaic acquisition time. Exemplary R mits

|ΘΘ56| Figure 6 shows an exemplary image of a strip mosaic o f a skin excision from

Mohs surgery. An exemplary measured time for scanning a 30-strip 12 x 10 mm' mosaic can be about 130 seconds. Stitching and blending of the 30 strips after completing the acquisition can take about 54 seconds. Therefore, the total time for 12 x. 10 mm ^" mosaic can be approximately 3 minutes. Features such as, for example, sebaceous glands 605, epidermis 610, eccrine ducts 615 and hair follicle 620 can be seen in Figure 6. The mosaic dimensions can be .13,035 pixels in the X-direeliosi and ! 0788 pixels in the Y -direction with, a pixel depth of 8 bits. The magnified areas can be digital zooms from, the original image showing detail and resolution of the mosaic. The features of Figure 6 can be compared to the pathology of Figure 7, which illustrates a frozeafiematoxylk and eosin {'Ti&E'>stained pathology of excised tissue from Mohs surgery for sebaceous glands 705, epidermis 710, eccrine ducts 715 and hair follicle 720, where the wide-field microscopy images correspond to a. tissue slice adjacent to the tissue slice of Figure 6.

[0057] Using an exemplary embodiment of the present disclosure, which can utilize exemplary stage profiles, it can be possible to scan, e.g., about 18 mm in the Y-direetion (D in Figure 3 A} to acquire data for, for example, about 10 mm (d in Figure 3A). The exemplary Y-sean and the exemplary lateral movement in X-direction can take

approximately 3 seconds. Such time can be further reduced using an exemplary embodiment of a stage profile, for example, with reduced D with shorter acceleration ramps.

[ΘΘ58] For example, at about 8,5 rum/s scan speed in Y, it can be possible to achieve approximately 1.8 mm scan in about 2.1 seconds, and leave another approximately 0.5 seconds for lateral movement. This can leave about 2.5 seconds total time for a strip. The additional half of a second per strip can be due to the DAQ card setup times. This time delay can also be eliminated or reduced using electronics according to certain exemplary

embodiment of the present disclosure.

|ΘΘ ] Figure 6 shows the exemplary systematic Y-offset along the X-direction. This offset can be due to the optical scanning- axis (X) not being perpendicular to the mechanical scannin axis (Y). Since this possible error can be a constant, it can be simply corrected using an exemplary embodime t of the stitching procedure according to the present

disclosure,

Ιθθβθ] Exemplary strip mosaicing conl cal microscopy procedures can oiler an imaging technology platform for real time detection of tumor margins directly in fresh tissue during surgery and from biopsies. Large amounts of tissue can be examined for, for example, tumor margins rapidly enough to be of practical use in surgical and clinical settings. The exemplary imaging can potentially be developed into an adjunct for pathology, to facilitate more complete and accurate removal of tumor.

|ΘΘ61 j Exemplary embodiments of Strip mosaicing procedures implemented on tissue from Mohs surgery in skin cancer described and shown herein can be used in surgical settings. Such exemplary embodiments can be applied in and for other tissues, not only for surgical settings but also in clinics for screening of biopsies.

(ΘΘ62! Beyond imaging on excised tissue, the exemplary system, method and computer-accessible medium, according to exemplary embodiments o f the present disclosure, can be implemented directly on patients to delineate tumor margins, either preoperative!}-' or intraoperatively in surgical wounds. Preliminary feasibility of such techniques has been reported for mosaicing on skin in vivo (See e.g., References 1 and 17} and also in shave- biopsied wounds in which residual tumor (e.g., basal and squamous ceil carcinoma) margins can be delineated (See e.g., Reference ! 8). Such exemplary embodiments can utilize exemplary mosaking procedures, boih on excised tissue at die bedside and infxa-operativeiy on the patient, which can prove useful for detection of tumor margins in. a rapid, efficient and cost-effective mariner.

[ΘΘ63] Th exemplary system, method and computer-accessible medium, according to exemplary embodiments of the present disclosure, can have a focused line that can include a cylindrical lens and objective lens (e.g., or any other arrangement to provide the exemplary focused line). For example, the line can be focused on tissue. The tissue can be translated back and forth, and light in reflectance and/or fluorescence can be collected onto and/or detected by a single, a pair or a further plurality of linear detectors. According to exemplary system, method and computer-accessible medium, such exemplary light can also be collected on or by, or detected by, a single line of pixels on a 2d detector arrangement, it can also be possible to exclude scanners altogether. The dual reflectance and fluorescence defection can facilitate an exemplar implementation of various digital staining procedures.

j0064 Figure 9 shows a block diagram of an exemplary embodiment of a system according to the present disclosure. For example, exemplary procedures in accordance with the present disclosure described herein can be performed by a processing arrangement and/or a computing arrangement 902. Such processing/computing arrangement 902 can be, for example, entirely or a part of, or include, but not limited to, a computer/processor 904 that can include, for example, one or more microprocessors, and use instructions stored on a computer-accessible medium (e.g., RAM. ROM, hard drive, or other storage device).

[0065] As shown in Figure 9, fo example, a computer-accessible medium 906 (e.g., as described herein above, a storage device such as a hard disk, floppy disk, memory stick, CD-ROM, RAM, ROM, etc., or a collection thereof) can be provided (e.g., in communication with the processing arrangement 902). The computer-accessible medium 906 can contain executable instructions 908 thereon, in addition or alternatively, a storage arrangement 910 can be provided separately from the computer-accessible medium 906, which can provide the instructions to the processing arrangement 902 so as to configure the processing arrangement to execute certain exemplary procedures, processes and methods, as described herein above, for example. [0066] Farther, the exemplary processing arrangement 902 can be provided with or include an input/output arrangement 914, which can include, for example, a wired network, a wireless network, the internet, an intranet, a data collection, probe, a sensor, etc. As shown in Figure 9, the exemplary processing arrangement 902 can be in communication with an exemplary display arrangement 912, which, according to certain exemplary embodiments of the present disclosure, can be a touch-screen configured for inputting information to the processin arrangement in addition to outpacing information from the processing

arrangement, for example, further, the exemplary display 912 and/or a storage arrangement. 910 can be used to display and/or store data in a user-accessible format and or user-readable format. £0067 j The foregoing merely illustrates the principles of the disclosure. Various modifications and alterations to the described embodiments will be apparent to those skilled in the art in view of the teachings herein.. It will thus be appreciated that those skilled in the art will be able to devise numerous systems, arrangements, and procedures which, although not explicitly shown or described herein, embody the principles of the disclosure and can be thus within the spirit and scope of the disclosure. In addition, all publications and references referred to above can be incorporated herein by reference in their entireties, it should be understood that the exemplary procedures described herein can be stored on any computer accessible medium, including a hard chive, ^' RAM, ROM, removable disks, CD-ROM, memory sticks, etc., and executed by a processing arrangement and/or computing arrangement which can be and/or include a hardware processors, microprocessor, mini, macro, mainframe, etc., including a plurality and/or combination, thereof, in addition, certain terms used in the present disclosure, including the specification, drawings and claims thereof, can be used synonymously in certain instances, including, but not. limited to, for example, data, and information. It should be understood that, while these words, and/or other words tha can be synonymous to one another, can be used synonymously herein, that there can foe instances when such words can be intended Co not be used synonymously. Further, to the extent that the prior art knowledge has not. been explicitly incorporated by reference herein above, it can be explicitly bein incorporated herein, in its entirety. All publications referenced can be incorporated herein by reference in their entireties.

EXEMPLARY REFERENCES

The following references are hereby incorporated by reference m their entirety.

1. Rajadhyaksha M, Menaker G, Dwyer PI, Flotte TI, Gonzalez S. Confocai

examination of non-melanoma cancers in skin excisions to potentially guide Mohs micrographic surgery without frozen histopatholog . Journal of Investigative Dermatology 117: 1 137-1 .143, 2001.

2. R. Haque, R, Contreras, M. P. McNicoll, B. C. Eekherg and D. B. Petitti "Surgical margins and ^' survival after head and neck cancer surgery," BMC Ear Nose Throat Disord 6: 2 (2006)

3. L. Jacobs, "Positive margins: The challenge continues for breast surgeons," Annals of Surgical Oncology 5(5): .1271-1272 (2008)

4. Pate! YG, Nehal KS, Aranda 1, Li Y, Halpern AC, Rajadhyaksha M. Confocai reflectance raosaiemg of basal cell carcinomas in Mohs surgical skin excisions. J. Biomedical Optics 12: 034027, 2007.

5. A -Arashi MY, Salomatina E, Yarosiavsky AN. Multimodal confocai microscopy for diagnosing nonmelanoma skin cancers. Lasers Surg Med 39:696-705, 2007.

6. Tilli MT, Cabrera MC _« Parrish AR, Torre KM, Sidawy M , Gallagher AL,

Mafcarioa E, Polio. SA, Liu C, Forth FA. Real-time imaging and characterisation of human breast tissue by reflectance confocai microscopy. J. Biomed. Optics 12(5): 051901, 2007.

7. Kaiig D, Suter MJ, Boudoux C, Yoo H, Yacliimski PS, Piirscelli WP, Nishioka NS, ino-Kenudson M, Lauwers GY, Bounia BE, Tearney GJ. Comprehensive imaging of gastroesophageal biopsy samples by spectrally encoded confocal microscopy. Gastroimest Endosc. 20.10 Jan;7 i(i):35-43.

Jain M„ Shukla N. Manzoor M, Nadolny S, Mukherjee S, Modified full-field optical coherence tomography; A novel tool for rapid histology of tissues. J Pathol Inform. 2 28, 201 1 .

Gareau DS, Li Y, Huang B, Eastman ZM, Nehai KS, Rajadhyaksha M. Confocal raosaicing microscopy in Mohs skin excisions: feasibility of rapid surgical pathology. J. Biomedical Optics 13: 05400 L 2008.

Gareau DS, Pate.1 YG, Li Y, Aranda 1, Halpem AC, Nehai KS, Rajadhyaksha. M. Confocal mosalcing microscopy in skin excisions: a demonstration of rapid surgical pathology. J. Microscopy 233: 149-159, 2009,

Gareaii D. S., The ieasibiiity of digitally stained multimodal confocal mosaics to simulate liistopathology. Journal of Biomedical Optics 14; 034050, 2009.

Birti J, Spain J, Nehai K, Haxehvood V, Di arzio C, Rajadhyaksha M, Confocal mosaicing microscopy of human skin ex vivo: spectral analysis for digital staining to Simulate tnsioSogy-Iike appearance. J. Biomedical Optics 16: 076008, 2011.

Gareau DS, Karen J , Dusza SW, Tudisco M, Nehai KS, Rajadhyaksha M.

Sensitivity and specificity for detecting basal cell carcinomas, in Mohs excisions with fluorescence confocal mosaicing microscopy. J. Biomedical Optics. 1 : 034 12, 2009. Karen IK, Gareau DS, Dusza S W. Tudisco M, Rajadhyaksha M, Nehal K.S.

Detection of basal ceil carcinomas in Mobs excisions with fluorescence confocal mosaicing microscopy. British J. Dermatol. 160: 1242-1250, 2009.

Abeytunge S. Li Y, Larson B, Toledo-Crow R, Rajadhyaksha M. Rapid confocal imaging of large areas of excised tissue with strip mosaicing. J. Biomedical Optics .16: 050504, 201 1.

V. Becker, T. Vercauteren, C. H, von Weyhe.ro, C, Wmz, R. M. Schmid and A. Meining, "High-resohuion miniprobe-hased confocal microscopy in eombmation with video mosaicing (vviih video)," G«stri>intes(ma! Endoscopy 66: 1001-1007 (2007)

K. E. Loewke, D. B. Camarii!o, C. A. Jobst and J. K, Salisbury, "Real-Time Image Mosaicing for Medical Applications," Medicine Meets Virtual Reality JS 125(304- 309 (2007)

Scope A, ahmood U, Gareau DS, Kenkre , Lieb J, Nehal S, Rajadhyaksha M. In vivo reflectance confocal microscopy of shave biopsy wounds: feasibility of intraoperative mapping of cancer margins. British J. Dermatology 163: 1218-1228, 2010.