SYSTEM AND METHOD FOR PROVIDING LIVE SPECIMEN MONITORING INFORMATION IN A PARALLEL IMAGING SYSTEM - Patent application

Abstract

In some embodiments, the method provides a live view mode without scanning the micro-optical array to generate and optionally display sequential image(s), the micro-optical array including image pixels representing sample light received from the micro-optical elements in the array for different spatially distinct sample locations. The images may be of a size and resolution useful for obtaining information indicative of real-time sample conditions. Full image collection by scanning the micro-optical array may begin when the sample is sufficiently (self-) stabilized. In some embodiments, the method provides an image including a stabilization indicator without scanning the micro-optical array. A stabilization indicator representing an empirically derived quantitative assessment of the degree of stabilization may be determined (e.g., calculated) with respect to sample light received from one or more micro-optical elements each represented by one or more image pixels of the image.

Description

[Technical field]

[0001] Priority Application This application claims the benefit of U.S. Provisional Patent Application No. 63 / 229,258 (filed August 4, 2021), and U.S. Provisional Patent Application No. 63 / 232,120 (filed August 11, 2021), the disclosures of which are hereby incorporated by reference in their entireties.

[0002] The present disclosure generally relates to systems and methods that use parallel imaging systems to provide live sample monitoring information to a user, for example, with respect to sample positioning, movement, and / or stabilization. [Background technology]

[0003] Traditionally, tissues excised during surgery are evaluated post-operatively with pathological evaluations that are performed after the tissue is fixed. While such processes provide high quality evaluations, a significant amount of time is required to fix the tissue and obtain the pathological evaluation. Thus, important information about the sample is not known until a long time has passed after the surgical procedure is completed. Recently, parallel imaging systems have been developed that have the ability to image freshly excised tissue samples. Examples of such imaging systems are disclosed in U.S. Pat. Nos. 10,094,784 and 10,539,776, each of which is incorporated herein by reference in its entirety. Even with parallel imaging systems that can image tissue quickly, freshly excised tissue can be difficult to image because it is not fixed and thus tends to move (e.g., relax) even over short time scales. One option for dealing with such sample motion has been to simply wait during a period of time before imaging to allow the sample to equilibrate in its position. However, doing so significantly slows the imaging process relative to the length of time that the parallel imaging system can image the sample. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] U.S. Patent No. 10,094,784 [Patent Document 2] U.S. Pat. No. 10,539,776 Summary of the Invention [Means for solving the problem]

[0005] Using an imaging system to rapidly provide a user with sample monitoring information, such as sample positioning and (self-)stabilization, can significantly reduce the amount of time required to generate a high-quality image of the sample. For example, a sample may be monitored in real time using an imaging system that allows imaging as soon as sufficient (self-)stabilization is achieved, without the need to estimate the appropriate length of time for the sample to (self-)stabilize. Sufficiency may be determined automatically by the imaging system or by a user who subsequently provides input to initiate imaging. For example, certain methods may be used to provide test images at lower resolutions or by scanning over a partial scan pattern to generate partial images, but the test images themselves may (undesirably) require a significant amount of time to acquire. (Examples of methods for acquiring test scans are disclosed in U.S. Patent Application Serial No. 17 / 174,919, filed February 12, 2021, the disclosure of which is hereby incorporated by reference in its entirety.) The present disclosure further improves such "test image" methods by providing a method for acquiring images at high speed without scanning any objective lens (e.g., micro-optical element array) or the sample. The fixed micro-optical array allows light (e.g., fluorescence) from the sample to be rapidly collected and then received from the micro-optical array (e.g., at a detector) to form image(s) in real time. Such images may be relatively low resolution (compared to full resolution images produced by scanning), but may still provide valuable sample information to the user. That information may be used, for example, to perform real-time assessment of sample positioning, movement, and / or stabilization, which may assist in later generation of higher quality full resolution images (e.g., by scanning the micro-optical array across a scan pattern) without undue delay, which full resolution images are of higher quality due, at least in part, to the reduction or elimination of sample motion artifacts.Alternatively, or additionally, the information may provide live feedback to the user to assist the user in (re)positioning the sample in order to acquire one or more images of higher quality more quickly.

[0006] In general, when imaging the surface of a resected tissue sample as part of a clinical diagnostic procedure (e.g., surgical margin assessment), it is desirable to maximize the size of the imaging area of ​​the sample to minimize the risk of not imaging areas of high clinical interest (e.g., missing positive margins). This can be achieved by imaging different planes of the sample (e.g., sequentially) and also by ensuring that for a given image of a given sample plane, a greater percentage of that sample plane is in focus and thus imaged by the imaging system. It is also desirable to avoid the presence of artifacts induced by sample motion that may interfere with the interpretation of the image (e.g., by the user or by image processing or image recognition algorithms), and thus it is important to know if the sample is undergoing sample motion or is unstable prior to imaging.

[0007] The systems and methods disclosed herein utilize rapid image generation and display by imaging the sample with a parallel imaging system (e.g., using a micro-optical element array to collect and transmit sample light) without scanning (e.g., on either the array or the sample) to allow for a rapid initial assessment of the current state of the sample. Using a fast initial assessment, the desire to maximize the area of ​​the sample in focus and reduce sample motion artifacts in the full image can be achieved by avoiding starting imaging before the sample is sufficiently stable. Imaging time can be reduced when the micro-optical element array is fixed during imaging because the time required to separately collect light at multiple positions in the scan pattern is eliminated. Imaging without scanning can result in relatively low resolution images, for example, where adjacent image pixels correspond to sample light received from micro-optical elements in the array for different locations on the sample, the different locations being separated by a distance corresponding to the pitch of the micro-optical elements. That is, in some embodiments, images acquired without scanning can be obtained by a reconstruction process that assigns each image pixel a value (e.g., an intensity value) corresponding to the light collected by one micro-optical element in the array. (Other embodiments may use other methods, such as direct imaging with a detector (noted above for the need for a reconstruction process) or indirect imaging.) However, even at low resolution, such images provide useful information to a user or an image processing or image recognition algorithm. The possibility of generating such images, and in some embodiments displaying such images in real time, allows a user or an image processing or image recognition algorithm to quickly determine, for example, when a sufficiently large area of ​​the sample is in focus and / or when the sample has been (self-)stabilized to a sufficient degree before starting to collect high resolution images to generate images that are free or substantially free of interfering motion artifacts.

[0008] In some embodiments, the disclosed method provides a live view mode to a user and / or an image processing or image recognition algorithm. In some embodiments, without scanning (e.g., of the micro-optical element array), a sequential image(s) including image pixels representing sample light received from the micro-optical elements in the array for different spatially distinct locations of the sample is generated and selectively displayed to the user. Because the micro-optical element array may be relatively large in one or more spatial dimensions and include a large number of micro-optical elements, the image may be of a size and resolution useful for obtaining sample information indicative of the real-time condition of the sample. In this manner, current sample information for the sample can be obtained and monitored. The user may adjust the sample on the mounting surface (e.g., of a sample dish) based on the live view mode to change its position or increase its area in focus. The user may also determine that the sample is sufficiently (self-) stabilized and initiate full image collection by scanning the micro-optical element array as appropriate. In some embodiments, (self-)stabilization is determined by the image processing or image recognition algorithm, and then imaging by scanning begins automatically.

[0009] In some embodiments, the disclosed method provides an image including a stabilization indicator to a user and / or an image processing or image recognition algorithm. A stabilization indicator, representing an empirically derived quantitative assessment of the degree of stabilization, may be determined (e.g., calculated) for sample light received from one or more micro-optical elements represented by one or more image pixels (e.g., each image pixel or region of image pixels) of the image. The stabilization indicator for one or more image pixels may reflect how much the intensity of the sample light is changing for one or more image pixels over some period of time. A higher stabilization index value may indicate more fluctuation, which in turn implies that more sample motion is occurring in real time. The image may include an indication of the stabilization indicator for each of a plurality of regions, each corresponding to a cluster of micro-optical elements in the array. A decreasing stabilization index value over time may indicate that the sample is approaching (self-)stabilization. Although a live view mode may be useful, it may be difficult for a user to know how actively the sample is stabilized (e.g., relaxed or otherwise moving) based purely on a representation of the intensity of the sample light, even in real time. A stabilization indicator overlay may be presented in live view mode, for example to provide the user with additional information to help them more quickly and easily understand whether the sample is (self-)stabilized or not.

[0010] In some embodiments, a method is directed to providing live sample monitoring information to a user. The method may include generating (e.g., displaying), by a processor of a computing device, one or more images (e.g., frames of a video) of the sample in real time based at least in part on sample light (e.g., fluorescence) received from micro-optical elements (e.g., refractive lenses, Fresnel zone plates, reflective objective lenses, and refractive index (GRIN) lenses) in a micro-optical element array without scanning the array or the sample. In some embodiments, an imaging system includes a micro-optical element array, and no part of the imaging system moves (e.g., is not scanned) while generating (e.g., displaying) the one or more images.

[0011] In some embodiments, for each of the one or more images, adjacent pixels of the image represent portions of sample light (e.g., fluorescence) received from portions of the micro-optical elements for different locations of the sample, the different locations being separated by a characteristic distance (e.g., corresponding to the pitch of the micro-element array) relative to the array (e.g., the separation occurs at the center of the spot size of adjacent portions of the micro-optical elements). In some embodiments, each image pixel of the one or more images corresponds to sample light (e.g., fluorescence) received from a micro-optical element in the array.

[0012] In some embodiments, the array remains in a fixed position during production (e.g., also display). In some embodiments, the sample is not perturbed (e.g., not manipulated) during production.

[0013] In some embodiments, an image pixel corresponds individually to sample light (e.g., fluorescent light) received from each micro-optical element in the array, and in some embodiments, each of the image pixels corresponds to sample light received from one of the micro-optical elements in the array (e.g., and each of the micro-optical elements in the array corresponds to only one of the image pixels) (e.g., each of the image pixels corresponds to sample light received from each one of the micro-optical elements in the array).

[0014] In some embodiments, the method includes determining (e.g., automatically by a processor) whether an air bubble is represented in one or more of the one or more images. In some embodiments, determining whether an air bubble is represented includes automatically determining by a processor whether an area of ​​image pixels having zero pixel values ​​greater than a threshold area (e.g., an area corresponding to a size of a cluster of 50 or less, 25 or less, 10 or less, or 5 or less micro-optical elements in the array) is present in one or more of the one or more images (e.g., over a period of time, e.g., at least 1 second, at least 2 seconds, or at least 5 seconds). In some embodiments, determining whether an air bubble is represented includes automatically determining by a processor whether a perimeter of an area of ​​image pixels having zero pixel values ​​defined by image pixels having non-zero pixel values ​​is present in one or more of the one or more images (e.g., over a period of time, e.g., at least 1 second, at least 2 seconds, or at least 5 seconds). In some embodiments, the method includes adjusting the sample (e.g., by weighting and / or repositioning the sample) in response to determining that the bubble is not represented in one or more of the one or more images.

[0015] In some embodiments, the method includes determining (e.g., automatically by a processor) whether the sample has a sufficiently large area in focus in one or more of the one or more images. In some embodiments, determining whether the sample has a sufficiently large area in focus includes automatically determining, by a processor, whether an area of ​​image pixels with non-zero pixel values ​​is above a predefined threshold (e.g., a value set by a user, for example, based on the sample size). In some embodiments, determining whether the sample has a sufficiently large area in focus includes automatically determining, by a processor, whether a convex hull of the portion of image pixels with non-zero pixel values ​​has changed by 10% or less (e.g., 5% or less, or 1% or less), for example, over a period of time (e.g., at least 1 second, at least 2 seconds, or at least 5 seconds). In some embodiments, the method includes adjusting the sample (e.g., by weighting and / or repositioning the sample) in response to determining whether the sample has a sufficiently large area in focus in one or more of the one or more images.

[0016] In some embodiments, the method includes adjusting the sample during generation (eg, also during display) in response to one or more images.

[0017] In some embodiments, the sample is accessible to the user during generation (e.g., and also during viewing) (e.g., placed on a sample tray that allows (e.g., lateral) sample access during imaging).

[0018] In some embodiments, the method includes initiating imaging of the sample based on the one or more images (e.g., based on determining that one or more of the one or more images are sufficient to indicate that the sample is stabilized (e.g., self-stabilized)), where imaging the sample includes scanning the micro-optical array. In some embodiments, the method includes initiating imaging automatically, by a processor, in response to determining that one or more of the one or more images are sufficient to indicate that the sample is stabilized (e.g., self-stabilized). In some embodiments, determining that one or more of the one or more images are sufficient to indicate that the sample is stabilized occurs automatically by a processor. In some embodiments, determining that one or more of the one or more images are sufficient to indicate that the sample is stabilized includes determining, by a processor, that no air bubbles are represented in one or more of the one or more images. In some embodiments, determining that one or more of the one or more images are sufficient to indicate that the sample is stabilized includes determining, by a processor, that the sample has a sufficiently large area that is in focus in one or more of the one or more images.

[0019] In some embodiments, one or more of the images are grayscale image(s). In some embodiments, one or more of the images are pseudocolor image(s) (e.g., the pixels of the image(s) are displayed in a purple / pink color scale, e.g., to mimic a hematoxylin and eosin stained light microscope image). In some embodiments, the hue, saturation, brightness, or a combination thereof (e.g., gray value) of the image pixels corresponds to the relative intensity of the received sample light.

[0020] In some embodiments, the method includes determining, by the processor, a stabilization indication of the sample light for each of at least a portion of the micro-optical elements in the array (e.g., all of the micro-optical elements) based on comparing the sample light received from the micro-optical elements over an observation period, and the one or more images include a graphical indication (e.g., an icon, shading, graphic, or color) of the stabilization indication. In some embodiments, the stabilization indication is dynamic over the observation period. In some embodiments, the stabilization indication changes over the observation period based on changes in the sample light received from the micro-optical elements.

[0021] In some embodiments, the method includes determining, by the processor, a stabilization indicator by comparing a change in intensity of sample light received from the micro-optical elements over a calculation period (e.g., a period that is a subset of the observation period). In some embodiments, comparing the change in intensity of sample light includes determining, by the processor, a minimum intensity and a maximum intensity of sample light received from each of the micro-optical elements over the calculation period (e.g., a predefined number of detection frames set by a user, for example). In some embodiments, the minimum intensity and maximum intensity are each determined from a weighted average (e.g., exponentially weighted average) (e.g., weighted time average) for the micro-optical elements over the calculation period (e.g., one or more weighting parameters are set by a user) (e.g., the weighted average is calculated using the intensity of sample light received from the micro-optical elements over two or more consecutive periods). In some embodiments, the stabilization indicator is the difference between the maximum intensity and the minimum intensity.

[0022] In some embodiments, each of the one or more images includes a region that includes a respective graphical indication (e.g., an icon, shading, graphic, or color) of stabilization indices for all micro-optical elements corresponding to a region. In some embodiments, the regions each correspond to a respective cluster of at least 9 micro-optical elements (e.g., at least 16 micro-optical elements, at least 25 micro-optical elements, at least 49 micro-optical elements, or at least 64 micro-optical elements). In some embodiments, the method includes determining, by the processor, at each of the regions, an average of the stabilization indices of the micro-optical elements corresponding to the region, and generating, by the processor, a graphical indication of the region based on the average. In some embodiments, generating the graphical indication includes determining, by the processor, whether the average exceeds one or more thresholds (e.g., multiple thresholds) (e.g., received by the processor as input from a user), such graphical indication indicating whether the average exceeds the one or more thresholds (e.g., based on transparency, brightness, saturation, hue, or a combination thereof).

[0023] In some embodiments, one or more of the one or more images include image pixels based in part on a first sample light (e.g., fluorescence) received from the micro-optical elements in the array during the observation period combined with a graphical indication of the stabilization indication, in some embodiments, the graphical indication of the stabilization indication in one or more of the one or more images is based on the first sample light and a second sample light received prior to the first sample light.

[0024] In some embodiments, at least a portion of each of the one or more images includes a region that includes a graphical indication (e.g., an icon, indication, shape, or color) of a respective stabilization indicator for that region. In some embodiments, the method includes determining, by the processor, a stabilization indicator for one of the one or more images based on one or more of the one or more images prior to one of the one or more images.

[0025] In some embodiments, at least a portion of each of the one or more images includes a region that includes a respective graphical indication of the motion of the sample relative to that region.

[0026] In some embodiments, the graphical indication is a color (eg, green or yellow or red) within the region (eg, the graphical indication is based on transparency, brightness, saturation, hue, or combinations thereof for the region).

[0027] In some embodiments, the graphical indications are overlaid on image pixels that correspond to sample light (eg, fluorescent light) received from micro-optical elements in the array.

[0028] In some embodiments, the method includes displaying, by the processor, the one or more images as they are generated. In some embodiments, the method includes repeatedly collecting sample light received from the micro-optical element over a period of time such that the one or more images are generated and displayed at a rate of at least 4 images / second (e.g., at least 10 images / second, at least 20 images / second).

[0029] In some embodiments, the generating (e.g., and displaying) occurs in real time such that the generating (e.g., and displaying) is delayed by the time required for processing (e.g., there is no time offset).

[0030] In some embodiments, each image pixel of the one or more images corresponds to sample light received from the micro-optical element over a period of 0.25 seconds or less (e.g., 0.1 seconds or less, 0.05 seconds or less, 0.025 seconds or less, 0.01 seconds or less, or 0.005 seconds or less). In some embodiments, the period is 0.005 seconds or less.

[0031] In some embodiments, the sample is a freshly excised tissue sample (eg, fluorescently tagged with a stain).

[0032] In some embodiments, the method includes receiving the sample light with a detector, and generating (e.g., displaying) the one or more images includes processing signals from the detector by a processor. In some embodiments, the one or more images are displayed on a display (e.g., via one or more graphical user interfaces). In some embodiments, the display, processor, and micro-optical array are included in an imaging system (e.g., a mobile imaging system) (e.g., located in a hospital room, e.g., a surgical suite).

[0033] In some embodiments, the micro-optical elements of the array have a lateral optical resolution of 10 μm or less (eg, 5 μm or less, 2 μm or less, or 1 μm or less).

[0034] In some embodiments, an imaging system includes a processor (e.g., the processor) and one or more non-transitory computer-readable media (e.g., a display and / or a micro-optical element array), the one or more media storing instructions that, when executed by the processor, cause the processor to perform the methods disclosed herein.

[0035] In some embodiments, a method is directed to providing live sample monitoring information to a user. The method may include generating (e.g., displaying) one or more images (e.g., frames of a video) of a sample in real time based at least in part on sample light (e.g., fluorescence) received from micro-optical elements (e.g., refractive lenses, Fresnel zone plates, reflective objective lenses, and refractive index (GRIN) lenses) in a micro-optical element array. In some embodiments, for each of the one or more images, adjacent pixels of the image represent portions of sample light (e.g., fluorescence) received from portions of the micro-optical elements for different locations of the sample, the different locations being separated by a characteristic distance (e.g., corresponding to the pitch of the micro-optical element array) relative to the array (e.g., the separation occurs at the center of the spot size of adjacent portions of the micro-optical elements). In some embodiments, neither (i) the array nor (ii) the sample is scanned during generation (e.g., during display).

[0036] Any two or more of the features described in this specification, including this summary section, may be combined to form an embodiment not specifically and explicitly described herein.

[0037] At least some of the methods, systems, and techniques described herein may be controlled by executing instructions stored on one or more non-transitory machine-readable storage media on one or more processing devices. Examples of non-transitory machine-readable storage media include read-only memory, optical disk drives, memory disk drives, and random access memories. At least some of the methods, systems, and techniques described herein may be controlled using a computing system, which consists of one or more processing devices and a memory that stores instructions executable by the one or more processing devices to perform various control operations.

[0038] definition In order that this disclosure may be more readily understood, certain terms used herein are defined below. Additional definitions for the following terms, as well as other terms, may be found throughout the specification.

[0039] In this application, unless otherwise clear from the context or specifically stated, (i) the term "a" may be understood to mean "at least one," (ii) the term "or" may be understood to mean "and / or," (iii) the terms "comprising" and "including" may be understood to encompass the listed components or steps, whether presented by themselves or with one or more additional components or steps, (iv) the terms "about" and "approximately" may be understood to allow for standard variations as would be understood by one of ordinary skill in the art, and (v) when ranges are provided, the endpoints are included. In certain embodiments, the term "approximately" or "about" refers to a range of values ​​that includes 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less than 1% in either direction (above or below) of the stated reference value, unless otherwise specified or otherwise apparent from the context (unless such number would exceed 100% of possible values).

[0040] "Image": As used herein, the term "image" includes any visual representation, such as, for example, a two-dimensional or three-dimensional image of a tissue (or other sample), e.g., a photograph, a video frame, a streaming video, and any electronic, digital, or mathematical analog of a photograph, a video frame, or a streaming video. In some embodiments, one or more images generated and / or displayed by the methods disclosed herein may be displayed continuously, with the images having a particular frame rate, such as a video, even if the frame rate is lower than the frame rate of a standard video format (e.g., 30 or 60 Hz). Any system or device described herein, in certain embodiments, includes a display for displaying the image or any other result generated by the processor. Any method described herein, in certain embodiments, includes a step of displaying the image or any other result generated by the method. Any system or device described herein, in certain embodiments, outputs the image to a remote receiving device (e.g., a cloud server, a remote monitor, or a hospital information system (e.g., a picture archiving and communication system (PACS)) or to an external storage device that can be connected to the system or device. In some embodiments, a fluorescence imaging system, a luminescence imaging system, and / or a reflectance imaging system are used to generate the image. In some embodiments, the image is a two-dimensional (2D) image. In some embodiments, the image is a three-dimensional (3D) image. In some embodiments, the image is a reconstructed image. In some embodiments, the image is a confocal image. The image (e.g., a 3D image) may be a single image or a set of images. In some embodiments, whether sample motion has occurred is reflected by the presence of one or more sample motion artifacts in an image (e.g., a full image or a test image). The one or more sample motion artifacts may be detectable by image processing performed by the imaging system. In some embodiments, determining whether one or more sample motion artifacts are present determines (e.g., determines) whether sample motion has occurred.

[0041] User: As used herein, a user is any person using an imaging system disclosed herein. A user may be, for example, without limitation, a surgeon, surgical staff (e.g., nurses or doctors in an operating room), a lab technician, a scientist, or a pathologist. When an action is described as being performed by a surgeon, it is understood that in some embodiments, a user who is not a surgeon performs an equivalent function.

[0042] Real-time: As used herein, an image may be generated and / or displayed in "real-time." Generally, actions that occur in real-time occur without intentional delay. There may be some amount of time required to process signals (e.g., from a detector) and / or collect light (e.g., to illuminate the sample and receive back-emitted sample light from the sample). For example, in some embodiments, image generation includes providing illumination light through an optical module that includes a micro-optical element array, collecting back-emitted sample light from the sample through the optical module, receiving the sample light at a detector, and processing signals from the detector to determine a pixel value (e.g., a grayscale value) for each image pixel of the image generated based on the intensity of the sample light for each of the micro-optical elements in the array. Thus, the "frame rate" at which an image can be generated and displayed may be limited by such processing and / or collection times. For example, an effective frame rate may be at least 4 frames (images) per second (e.g., at least 10 frames / second, at least 15 frames / second, at least 20 frames / second, or at least 30 frames / second).

[0043] Sample: As used herein, a "sample" may be any material that is desired to be characterized. In some embodiments, the sample is a biological sample. In some embodiments, the sample is a tissue, such as a human tissue. In some embodiments, the tissue is fresh (e.g., unfixed). In some embodiments, the tissue is freshly excised. For example, a tissue sample may be excised during a surgical procedure and optionally imaged using the methods disclosed herein during the procedure. Similarly, "sample light" is light from the sample. Sample light may be, for example, reflected light, refracted light, diffracted light, or back-emitted light. In some embodiments, the sample light is fluorescent. Sample light that is fluorescent may be back-emitted light from the sample emitted from one or more fluorescent tags that are applied to the sample by staining (e.g., selectively staining a feature(s) of interest in the sample).

[0044] Stabilization: As used herein, "stabilization" refers to the reduction (e.g., elimination) of sample movement (e.g., over a period of time). Stabilization may be self-stabilization, e.g., resulting from relaxation of the sample. Unless otherwise clear from the context, reference to "stabilization" not preceded by "self-" or "(self-)" should be understood to indicate that embodiments are contemplated in which the described stabilization is self-stabilization. Stabilization may also be achieved using a tool manipulated by a user, such as forceps or a sample weighting tool. Stabilization may occur when any remaining sample movement falls below a detectable threshold (e.g., sample movement occurs only on a time scale significantly longer than the sampling period at which the sample light is received from the micro-optical element array). Thus, the stabilization index may represent an empirically derived quantitative assessment of the degree of stabilization present at a particular time or over a particular period of time, as determined, for example, by changes in the intensity of the sample light received from the micro-optical elements in the array. Thus, a higher stabilization index value may indicate relatively more sample movement, as inferred from a larger change in the intensity of the received sample light.

[0045] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0046] The drawings herein are presented for purposes of illustration and not limitation. The foregoing and other objects, aspects, features, and advantages of the present disclosure will become more apparent and may be better understood by referring to the following description in conjunction with the accompanying drawings, in which: [Brief description of the drawings]

[0047] [Figure 1A] FIG. 2 is a plan view illustrating an exemplary rectangular optical chip including an array of microlenses arranged in a square lattice, according to an exemplary embodiment of the present disclosure. [Figure 1B] FIG. 2 is a plan view illustrating an exemplary rectangular optical chip including an array of microlenses arranged in a square lattice, according to an exemplary embodiment of the present disclosure.

[0048] [Figure 1C] 1C is a cross-sectional view of a portion of the optical chip shown in FIGS. 1A and 1B according to an exemplary embodiment of the present disclosure.

[0049] [Figure 2A] 1 is a schematic diagram of an exemplary imaging system illustrating illumination of a tissue sample, according to an exemplary embodiment of the present disclosure.

[0050] [Figure 2B] FIG. 2B is a schematic diagram of an exemplary imaging system according to FIG. 2A illustrating detection of back-emitted light from a sample by a detector according to an exemplary embodiment of the present disclosure.

[0051] [Figure 3A] FIG. 1 is a process diagram of a method for determining whether a sample is moving using a stationary micro-optical element array, according to an exemplary embodiment of the present disclosure. [Figure 3B]FIG. 1 is a process diagram of a method for determining whether a sample is moving using a stationary micro-optical element array, according to an exemplary embodiment of the present disclosure. [Figure 3C] FIG. 1 is a process diagram of a method for determining whether a sample is moving using a stationary micro-optical element array, according to an exemplary embodiment of the present disclosure.

[0052] [Figure 4A] FIG. 2 is a process diagram of a method for generating and selectively displaying images in real time without scanning, according to an exemplary embodiment of the present disclosure. [Figure 4B] FIG. 2 is a process diagram of a method for generating and selectively displaying images in real time without scanning, according to an exemplary embodiment of the present disclosure. [Figure 4C] FIG. 2 is a process diagram of a method for generating and selectively displaying images in real time without scanning, according to an exemplary embodiment of the present disclosure. [Figure 4D] FIG. 2 is a process diagram of a method for generating and selectively displaying images in real time without scanning, according to an exemplary embodiment of the present disclosure.

[0053] [Figure 4E] FIG. 1 is an illustration of a method for calculating a stabilization index according to an exemplary embodiment of the present disclosure.

[0054] [Figure 5A] 14A-14C are images illustrating the use of live view mode to monitor a sample area that is in focus over time as it grows due to repositioning by a user, according to an exemplary embodiment of the present disclosure. [Figure 5B] 14A-14C are images illustrating the use of live view mode to monitor a sample area that is in focus over time as it grows due to repositioning by a user, according to an exemplary embodiment of the present disclosure. [Figure 5C]14A-14C are images illustrating the use of live view mode to monitor a sample area that is in focus over time as it grows due to repositioning by a user, according to an exemplary embodiment of the present disclosure. [Figure 5D] 14A-14C are images illustrating the use of live view mode to monitor a sample area that is in focus over time as it grows due to repositioning by a user, according to an exemplary embodiment of the present disclosure.

[0055] [Figure 6A] 13 is an image illustrating the use of live view mode to monitor the presence of a bubble with a sample that shrinks due to repositioning by a user, according to an exemplary embodiment of the present disclosure. [Figure 6B] 13 is an image illustrating the use of live view mode to monitor the presence of a bubble with a sample that shrinks due to repositioning by a user, according to an exemplary embodiment of the present disclosure. [Figure 6C] 13 is an image illustrating the use of live view mode to monitor the presence of a bubble with a sample that shrinks due to repositioning by a user, according to an exemplary embodiment of the present disclosure. [Figure 6D] 13 is an image illustrating the use of live view mode to monitor the presence of a bubble with a sample that shrinks due to repositioning by a user, according to an exemplary embodiment of the present disclosure. [Figure 6E] 13 is an image illustrating the use of live view mode to monitor the presence of a bubble with a sample that shrinks due to repositioning by a user, according to an exemplary embodiment of the present disclosure.

[0056] [Figure 7A] 13A-13C are images illustrating the use of live view mode with a semi-transparent stabilization indicator view overlay to monitor sample motion and stabilization over time, which decreases over time due to relaxation of the sample, according to an exemplary embodiment of the present disclosure. [Figure 7B]13A-13C are images illustrating the use of live view mode with a semi-transparent stabilization indicator view overlay to monitor sample motion and stabilization over time, which decreases over time due to relaxation of the sample, according to an exemplary embodiment of the present disclosure. [Figure 7C] 13A-13C are images illustrating the use of live view mode with a semi-transparent stabilization indicator view overlay to monitor sample motion and stabilization over time, which decreases over time due to relaxation of the sample, according to an exemplary embodiment of the present disclosure. [Figure 7D] 13A-13C are images illustrating the use of live view mode with a semi-transparent stabilization indicator view overlay to monitor sample motion and stabilization over time, which decreases over time due to relaxation of the sample, according to an exemplary embodiment of the present disclosure.

[0057] [Figure 7E] 1 illustrates a live view mode of a sample without a stabilization indicator mode overlay, according to an exemplary embodiment of the present disclosure.

[0058] [Figure 8A] 1 is an exemplary screen capture of a graphic user interface showing a live view mode image with stabilization indicator overlay and summary statistics, according to an exemplary embodiment of the present disclosure.

[0059] [Figure 8B] 1 is an exemplary screen capture of a graphic user interface showing a live view mode image with stabilization indicator overlay and time-resolved summary statistics, according to an exemplary embodiment of the present disclosure.

[0060] [Figure 8C] 1 is an exemplary screen capture of a graphic user interface showing a grayscale live view mode image with a stabilization index overlay and user selectable stabilization index weighting parameters and thresholding according to an exemplary embodiment of the present disclosure.

[0061] [Figure 8D] 1 is an exemplary screen capture of a graphic user interface showing a live view mode image in pseudocolor (mimicking histological staining) with stabilization index overlay, user-selectable stabilization index weighting parameters, and thresholding according to an exemplary embodiment of the present disclosure.

[0062] [Figure 9] FIG. 1 is a block diagram of an exemplary network environment for use in the methods and systems described herein, in accordance with an exemplary embodiment of the present disclosure.

[0063] [Figure 10] 1A-1C are block diagrams of an exemplary computing device and an exemplary mobile computing device for use in exemplary embodiments of the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0064] It is contemplated that the systems, devices, methods, and processes of the present disclosure encompass variations and modifications developed using information from the embodiments described herein. Modifications and / or variations of the systems, devices, methods, and processes described herein may be made by those skilled in the art.

[0065] Throughout the description, when articles, devices, and systems are described as having, including, or comprising particular components, or processes and methods are described as having, including, or comprising particular steps, it is believed that in addition there are articles, devices, and systems according to particular embodiments of the disclosure that consist essentially of or consist of the recited components, and there are processes and methods according to particular embodiments of the disclosure that consist essentially of or consist of the recited processing steps.

[0066] It should be understood that the order of steps or order for performing certain actions is immaterial, so long as it does not result in loss of operability. Moreover, two or more steps or actions may be conducted simultaneously.

[0067] Headings are provided for the convenience of the reader and are not intended to be limiting with respect to the claimed subject matter.

[0068] Examples of Arrays of Micro-Optical Elements and Imaging Systems In some embodiments, the imaging system used for imaging with or without scanning (e.g., depending on the type of image acquired) includes an array of micro-optical elements, which may include one or more of a refractive lens, a Fresnel zone plate, a reflective objective lens, and a refractive index (GRIN) lens. The array of micro-optical elements may be scanned across a scan pattern during imaging, for example, by a scanning stage including an actuator. The scan pattern may have a size corresponding to the size of a unit cell of a micro-optical element in the array of micro-optical elements (e.g., may be a square of approximately equivalent size). As such, each micro-optical element in the array of micro-optical elements may scan an area corresponding to its unit cell to generate an image corresponding in size (e.g., having a size of the same order of magnitude) as the array of micro-optical elements. The scan pattern may include a series of consecutive positions (e.g., positions arranged in an array, such as a regular array) that are moved continuously during imaging. The array of consecutive positions defining the scan pattern may generally be an M×N array (M=N or M≠N). Illumination light can be provided to the sample through an array of micro-optical elements at a subset (e.g., all) of the series of consecutive positions. For example, when the imaging system is a fluorescence microscope such as a confocal microscope, back-emitted light can be collected from the sample with an array of micro-optical elements at a subset (e.g., all) of the series (e.g., array) of consecutive positions.

[0069] In some embodiments, the imaging system is located in an operating room and used during a surgical procedure (e.g., during a diagnostic procedure or during treatment of a diagnosed disease), In some embodiments, multiple systems are used and / or multiple methods are performed during surgery.

[0070] The array of micro-optical elements may be disposed on a surface of the optical chip. For example, the micro-optical elements may be disposed on a surface of a substrate of the optical chip. In some embodiments, the optical chip includes an array of micro-optical elements attached to a holder around the periphery of the array (e.g., not disposed on a substrate). In general, the periphery of the optical chip may have any shape. In some embodiments, the optical chip is rectangular (e.g., square or non-square). For example, in some embodiments, the array of micro-optical elements is integral with the substrate of the optical chip. The array of micro-optical elements may be non-integral but attached to the substrate of the optical chip. The array of micro-optical elements may include at least 25,000 microlenses (e.g., with a radius of curvature (ROC) with 200 μm to 300 μm). Absorbing and / or reflecting layers may be provided on the optical chip between the micro-optical elements in the array (e.g., to act as apertures). The optical chip may be made of fused silica. The micro-optical elements may be arranged in a regular array on the optical chip (e.g., a square lattice). In some embodiments, the array of micro-optical elements has a pitch of 100 μm to 500 μm (e.g., 200 μm to 300 μm). In some embodiments, the optical chip has a non-regular array of micro-optical elements, e.g., with different pitches in the x and y directions. In some embodiments, the optical chip has a high aperture number for high resolution imaging and more efficient background removal.

[0071] In some embodiments, the array of micro-optical elements is not part of an optical chip, for example, in some embodiments, the array of micro-optical elements is, for example, an array of separate objective lenses mounted in fixed relative positions (e.g., to each other or to a physical support).

[0072] In some embodiments, the array of micro-optical elements is a regular array, and the pitch of the micro-optical elements in the array in a first direction is equal to the pitch of the micro-optical elements in the array in a second direction perpendicular to the first direction. For example, the micro-optical elements may be arranged in a square lattice. In some embodiments, each micro-optical element in the array of micro-optical elements has at least one convex surface. For example, each micro-optical element may be a plano-convex lens or a biconvex lens. The convex surface of each micro-optical element may have a shape obtained by the rotation of a conical section (e.g., with a radius of curvature of 200 μm to 300 μm). In some embodiments, each micro-optical element in the array of micro-optical elements focuses light onto an area (spot) that is smaller than the pitch of the array (e.g., the pitch). In some embodiments, the micro-optical elements in the array of micro-optical elements collectively focus onto a common focal plane. For example, each element of the micro-optical element array may focus onto a single point on the common focal plane.

[0073] 1A and 1B show two schematic views of an exemplary optical chip 100 including an array of micro-optical elements 102 that can be used in the systems and / or to perform the methods disclosed herein. FIG. 1A shows a plan view of the entire optical chip 100 (individual micro-optical elements and optional reflective / absorbent layers are not shown in FIG. 1A). The optical chip 100 has a rectangular cross-section with dimensions W and L (i.e., W≠L). In some embodiments, W=L. The optical chip 100 has a high degree of parallelism, with the edges of the optical chip 100 having a parallelism better than about ±0.250 mrad (e.g., less than or equal to ±0.125 mrad). FIG. 5B shows a portion of the optical chip 100 including a portion of the array of micro-optical elements 102. The array of micro-optical elements disposed on the surface of the optical chip 100 may include at least 1,000 micro-optical elements, at least 5,000 micro-optical elements, at least 10,000 micro-optical elements, at least 20,000 micro-optical elements, at least 30,000 micro-optical elements, at least 50,000 micro-optical elements, at least 60,000 micro-optical elements, or at least 100,000 micro-optical elements. The array of micro-optical elements 102 is highly parallel to the edge of the optical chip 100. The array 102 is parallel to the edge of the optical chip better than about ±0.250 mrad (e.g., less than or equal to about ±0.125 mrad). The array 102 is a regular array. In some embodiments, the array of micro-optical elements is irregular. The dashed box 112a illustrates an example of a unit cell of a micro-optical element in the array 102. Dashed box 112b illustrates an example of a unit cell of a micro-optical element in array 102 drawn with a different origin than dashed box 112a. In general, the choice of origin is arbitrary. The crosshairs on each micro-optical element of array 102 indicate the center of each of the micro-optical elements.

[0074] FIG. 1C shows a cross-sectional view of a portion of an exemplary optical chip 100. The optical chip 100 includes a substrate 106 and an array of micro-optical elements. Each micro-optical element 102 is a convex micro-lens. The convex micro-lenses 102 are integral with the substrate 106, such that the substrate 106 and the micro-lenses 102 together are one continuous material. For example, they may be formed simultaneously during manufacturing. The thickness (H) of the optical chip 100 may be considered as the distance between the top of the micro-optical element and the opposite surface of the substrate, as shown. The thickness of the optical chip may be less than 2.0 mm (e.g., less than or about 1.5 mm). The optical chip may have a total thickness variation and / or total flatness deviation of less than 20 μm (e.g., less than 15 μm, less than 10 μm, or less than 5 μm). The optical chip 100 is coated with a reflective layer 104 of chrome. The reflective layer 104 is disposed in the inter-lens areas between the micro-optical elements 102. It is understood that a reflective layer disposed in an inter-lens area may extend partially over one or more lenses near the periphery of the lens(es), as shown in Figures 1A and 1B. When the reflective layer 104 extends partially over the micro-optical elements near the periphery of the micro-optical elements, the diameter 110 of the micro-optical elements is larger than the aperture 108 of the reflective layer formed by the reflective layer 104.

[0075] FIG. 2A is a schematic diagram of an exemplary imaging system 200 illustrating the behavior of the optical system of the exemplary system during illumination of a tissue sample. The imaging system 200 may include features described herein and / or may be used to perform the methods disclosed herein. FIG. 2B is a schematic exemplary imaging system 200 illustrating detection of back-emitted light from the sample by a detector. Referring now to FIG. 2A, a laser 218 providing light with a wavelength between 450 nm and 490 nm provides an illumination beam to a focusing lens 216. The illumination beam passes through the focusing lens 216 and a first aperture 214 before being directed by a dichroic mirror 214. The dichroic mirror reflects the illumination beam onto a collimating lens 202. The illumination beam is collimated by the collimating lens 202, and the collimated illumination beam propagates to an optical chip 222. The optical chip includes an array of micro-optical elements. The micro-optical elements in the array of micro-optical elements can be refractive lenses, Fresnel zone plates, reflective objective lenses, GRIN lenses, or micro-lenses. In certain embodiments, the optical chip includes an array of refractive micro-lenses. The micro-optical elements focus light from a collimated illumination beam onto a sample through an imaging window. In this case, the sample 228 is placed on a disposable sample holder 226 that is mounted directly on the imaging window 224. In some embodiments, the sample is placed on the imaging window (e.g., on a sample dish) (e.g., without touching the imaging window) during imaging. In some embodiments, the sample holder 226 is not present and the sample is placed directly on the transparent imaging window during imaging. The use of a sample dish may reduce or eliminate the need to clean (e.g., sterilize) the transparent imaging window when changing samples. FIG. 25 shows a sample dish 2504 mounted on a transparent imaging window 2502 with a sample 2520 placed therein as an example of an imaging system 2500 that can be used and / or is used with the sample dish 2502. Similarly, the imaging system 200 may be modified or designed.

[0076] Referring again to FIG. 2A, the optical tip 222 is connected to a support of the scan stage 220. The scan stage 220 uses a controller and actuators connected to the support to move the optical tip 222 along a scan pattern during imaging. Each micro-optical element of the optical tip 222 generates a tight focus (e.g., a small spot, e.g., a unique point) of light from a collimated illumination beam on or within the sample during imaging on a common focal plane (imaging plane) on or within the sample. The scan pattern in which the optical tip 222 is moved can be one-dimensional or two-dimensional.

[0077] 2B is a schematic diagram of an exemplary imaging system 200 illustrating the behavior of the optical system shown in FIG. 2A during detection. Light from a collimated illumination beam focused on a sample 228 by an array of micro-optical elements in an optical chip 222 generates light (e.g., fluorescence or luminescence) in the sample 228, which is emitted back toward the optical chip 222 through an imaging window 224. The back-emission light is then collected by the micro-optical elements in the array in the optical chip 222 and directed to the detector 212. When the back-emission light is within the transmission band of the mirror, it passes through a dichroic mirror 204. The back-emission light then passes through a second aperture 206 and is collimated by an imaging lens 208. The collimated back-emission light passes through an emission filter 210 before landing on the detector 212. Detector 212 is a CMOS camera that includes an array of detector elements (e.g., camera pixels) that each receive back-emitted light from the micro-optics in the array of optical elements in the optical chip 222. To block ambient light (e.g., stray light) from entering the detector 212, an opaque housing may be placed around the optical path of the back-emitted light passing through filter 210.

[0078] In some embodiments, images of the micro-optical element array are captured by a detector (e.g., a detector array such as a CMOS or CCD camera). The detector frames can be processed to generate images of the sample, where each image pixel represents the signal from a unique and different micro-optical element in the array. In these images, two adjacent pixels represent the intensity collected from two points in the sample, separated by a distance corresponding to the pitch of the micro-element array.

[0079] In some embodiments, the imaging system may be designed and calibrated such that one micro-optical element is imaged onto exactly one detector element, in some such embodiments, the detector frame without further processing already constitutes an image of the sample where one pixel represents the signal from a unique and distinct micro-optical element in the array.

[0080] In some embodiments, one micro-optical element is imaged across many detector elements (e.g., >4, >9, >16, >25, >100 detector elements). In some such embodiments, the intensity collected by a unique micro-optical element can be calculated from the values ​​of the many detector elements across which this micro-optical element is imaged (e.g., by adding or interpolating the values ​​of the detector elements) to reconstruct an image in which each image pixel represents the signal from a unique and different micro-optical element in the array.

[0081] The imaging system may be used for in-operation room imaging of fresh tissue excised during surgery (e.g., cancer surgery). In some embodiments, the imaging system is operable to image a portion of a sample in less than 10 minutes (e.g., less than 5 minutes, less than 3 minutes, or less than 2 minutes). In some embodiments, the system is operable to image a portion of a sample in less than 2 minutes (e.g., less than 90 seconds, or less than 1 minute). In some embodiments, the portion of a sample is at least 10 cm 2 (e.g. at least 12 cm 2 , at least 15 cm 2 , or at least 17 cm 2In some embodiments, the sample has a volume of 10 cm x 10 cm x 10 cm or less, and the system is configured to image the entire exterior surface of the sample with an imaging time of 45 minutes or less (e.g., 30 minutes or less).

[0082] The imaging system that can be used to perform the methods disclosed herein is generally a point-scanning imaging system. That is, in some embodiments, each micro-optical element in the micro-optical element array images a unique point (as opposed to, for example, a small field of view). In some embodiments, the imaging system is a confocal imaging system (e.g., a confocal microscope). By way of example, a confocal imaging system allows high-resolution imaging of a sample by scanning a micro-optical element array (e.g., included in an optical chip) over a scan pattern. The live view mode and / or stabilization indicator mode can be used before scanning to determine sample information, such as a qualitative assessment of sample self-stabilization, to further improve image quality during scanning (e.g., to improve image quality due to reduction of sample motion artifacts that are more likely and / or more likely to occur before self-stabilization), as described further below.

[0083] In general, the imaging system can generate images from light collected by the micro-optical array (e.g., back-emitted sample light) with or without scanning using any suitable method. In some embodiments, the imaging system generates images for characterizing the sample by scanning the micro-optical array in a lateral scan pattern (e.g., a 2D scan pattern), for example, as described in the embodiments disclosed in U.S. Pat. No. 10,094,784. The detector and sample can remain in fixed relative positions during imaging while the sample and the micro-optical array move relative to each other. A reconstruction process can be used to reconstruct an image using information derived from the light collected at each position of the lateral scan pattern and the known position information of the micro-optical array. Even when the micro-optical array is not scanned (remains stationary), a similar reconstruction process can be used when performing sample monitoring to determine whether sample motion is occurring. That is, the imaging system can be constructed to apply a reconstruction process during sample motion monitoring similar to the reconstruction process used during subsequent imaging. In some embodiments, the reconstruction process assigns an image pixel a value (e.g., an intensity value) corresponding to the light collected by a micro-optical element in the array. However, such a reconstruction process is not necessary to practice the embodiments disclosed herein, regardless of whether such a reconstruction process is used for subsequent imaging. For example, in some embodiments, sample motion monitoring is performed using direct imaging from the detector. Other indirect imaging methods may also be used.

[0084] Imaging systems (e.g., confocal microscopes) that can be used according to (e.g., to perform) certain embodiments of the present disclosure are described in U.S. Patent Nos. 10,094,784 and 10,539,776, each of which is hereby incorporated by reference in its entirety. Sample dishes that can be used with certain embodiments of the present disclosure are described in U.S. Patent No. 10,928,621, the disclosure of which is hereby incorporated by reference in its entirety. The sample can be stained prior to imaging. For example, the sample can be stained using the staining solution disclosed in U.S. Patent Application No. 16 / 806,555, filed March 2, 2020, the disclosure of which is hereby incorporated by reference in its entirety.

[0085] Sample monitoring using fixed micro-optical element arrays For example, for a parallel imaging system including an array of micro-optical elements, imaging of a large sample area can be achieved without any movement of the optical elements (or sample). The intensity of the sample light received from the micro-optical elements in the array can be detected to generate an image including image pixels that individually correspond to the micro-optical elements. Depending on the ratio of detector elements to micro-optical elements in the imaging system, each image pixel can represent signals from multiple detector elements. The intensity variation over time is greater for samples that are moving significantly (e.g., compared to the image resolution and / or imaging speed) than for samples that are not moving significantly (e.g., compared to the image resolution and / or imaging speed). A threshold amount can be set, for example, based on a typical intensity change between adjacent pixels of an image (e.g., for a given sample type), below which the intensity variation of the image pixel(s) would indicate that no sample motion is occurring (e.g., compared to the image resolution and / or imaging speed). The typical intensity change can be known and / or determined based on the image parameters (e.g., resolution) and / or sample characteristic(s). The threshold amount may be preset or determined during monitoring, for example as a percentage of intensity variation over an initial period of time.

[0086] Such changes may also be used to determine (e.g., set) an intensity of the sample light below a pixel value at which a pixel value for a corresponding image pixel of the image (e.g., a gray value in a grayscale image) is set to zero. That is, if only a minimum intensity of the sample light is received for a particular micro-optical element, the intensity may not be sufficient to distinguish it from the background such that a pixel value of zero is assigned. Similarly, the intensity of the sample light may also be thresholded to bin small ranges of intensity to distinguish distinct hues, luminances, saturations, or combinations thereof (e.g., distinct gray values ​​in a grayscale) for the image pixel. For example, the detector signal may be normalized or scaled to the determined average intensity change. In some embodiments, such as implementations of a confocal imaging system, the optical system in the imaging system uses one or more apertures to remove out-of-focus background intensity.

[0087] 3A-3C are process diagrams of a method 300 for determining whether a sample is moving. In step 302, image pixels that individually correspond to micro-optical elements in an array of micro-optical elements are monitored while the micro-optical elements remain in a fixed position. The intensity of the image pixel is based on the amount of back-emitted light received by the detector that is being collected through the corresponding micro-optical element. In step 304, it is determined whether sample motion is occurring, which in this example is determined at least in part based on whether the variation in the intensity of the image pixel is below a threshold amount during a period of time. In some embodiments, multiple image pixels are monitored simultaneously (e.g., each corresponding to a respective micro-optical element in the array of micro-optical elements, e.g., each micro-optical element is at least ¼, at least ½, or all of the micro-optical elements in the array) to determine whether sample motion is occurring. Determining whether sample motion is occurring may be based at least in part on a respective variation of each image pixel not exceeding a threshold amount, a variation in the average intensity of each image pixel not exceeding a threshold amount, or a variation in the average intensity of each image pixel not exceeding a threshold amount. The period may correspond to the acquisition time of the full image to be acquired. In optional step 306, upon determining that the variation in intensity of the image pixel(s) does not exceed the threshold amount during a period of time, an image of the sample is acquired (e.g., automatically). In optional step 308, the user is notified (e.g., automatically) (e.g., via a graphical user interface, e.g., by a pop-up notification) whether sample motion is occurring based on the determination in step 304. The system may inform the user about the stabilization status of the sample to support the user in determining the best time to start image acquisition. In some embodiments, the user may be notified by a single automatically triggered event when the sample motion meets a predetermined rule (e.g., when the sample motion becomes small enough not to generate visible motion artifacts in the full image to be acquired).In some embodiments, the user is continuously informed of the current state of sample motion by a continuously updated indicator (e.g., a graphical or textual indicator) that may be reduced to a single scalar (e.g., a single color or symbol if graphical, or a single value (e.g., scale) if textual) for the entire sample. In some embodiments, the user is continuously informed of the current state of sample motion by a continuously updated array of indicators that locally represent the state of sample motion (e.g., displayed as a color-coded miniature map of the sample).

[0088] In some embodiments of method 300, as shown in FIG. 3B, in step 310, the intensity is used to determine whether the sample has moved locally over a threshold amount during a period of time. In step 312, the user is notified that the sample has moved over a threshold amount. In step 314, an image is acquired in response to an explicit request from the user. In high time pressure applications, the user may wish to be empowered with the ability to initiate acquisition at whatever moment(s) the user feels appropriate (e.g., based on continuous notification of the current state of sample motion).

[0089] FIG. 3C illustrates an additional exemplary process flow for method 300.

[0090] In some embodiments, upon determining that the intensity of one or more image pixels has fluctuated by no more than a threshold amount during a period of time, an image of the sample is acquired (e.g., automatically, e.g., without user input). In some embodiments, the threshold amount is a predetermined (e.g., predefined) threshold amount, and the method includes presetting the threshold amount based on a resolution (e.g., a selected resolution) of the image to be acquired before starting monitoring. In some embodiments, the threshold amount is a predetermined (e.g., predefined) threshold amount, and the method includes presetting the threshold amount based on one or more characteristics of the sample. In some embodiments, the threshold amount is no more than 20% or no more than 10%. Generally, as sample motion slows or stops, intensity fluctuations are reduced because there is generally no significant discontinuity in intensity between adjacent pixels and pixel drift due to sample motion slows. Using an absolute threshold amount of no more than 20% or no more than 10% may be sufficient in some embodiments to reduce or remove noticeable sample motion artifacts from subsequently acquired images. In some embodiments, the period is at least 2 seconds and no more than 90 seconds, or at least 0.1 seconds and no more than 2 seconds (e.g., at least 0.25 seconds and no more than 1 second), In some embodiments, the period is at least 5 seconds and no more than 30 seconds.

[0091] Monitoring the intensity of the image pixel(s) may include making discrete measurements of the back-emission light received over separate short periods. For example, the intensity at a first time may be based on the back-emission light received at a detector (e.g., a CCD or CMOS camera) through the micro-optical element(s) during a first short period (e.g., 10 ms or less, 5 ms or less, 3 ms or less, 2 ms or less, or less than 1 ms), and the intensity at a second time may be based on the back-emission light received at the detector through the micro-optical element(s) during a second short period that is equal in length of time to the first short period. There may be a period of delay (e.g., at least 1 ms to 1 s or less, or 100 ms or less) between the first and second short periods. A longer period of delay generally means that the method is more sensitive to migration, but also reduces the actual or potential time savings compared to simply waiting a sufficient time to ensure sample stabilization (e.g., equilibration). In addition, longer periods of delay, if provided, may cause user confusion when viewing the graphical output of the monitoring. Thus, in some embodiments, the delay period ranges from 0.25 seconds to 0.75 seconds (e.g., about 0.5 seconds). In some embodiments, the delay period is 5 seconds or less (e.g., 3 seconds or less, 2 seconds or less, or 1 second or less).

[0092] Determining whether the sample is moving may include processing (e.g., comparing) the intensity at a first time with the intensity at a second time. In some embodiments, the delay period must be carefully chosen. If the delay period is too small, small movements of the sample will be imperceptible on this time scale, but will result in movement artifacts visible in the full image acquired subsequently. On the other hand, if the delay period is too large, movement of the sample occurring early in the observation period, even if the sample has stabilized in the meantime, will still lead to the idea that the sample is still moving, thus resulting in a waste of time. By selecting a delay period that allows the user to be provided with a "real-time" frame rate of the image, the user can observe the fluctuations as they occur to make a determination whether the sample is stable or not. The fluctuations in intensity over time may be based on discrete measurements of intensity made at a series of times during monitoring.

[0093] The intensity fluctuation may be calculated simply by taking the absolute value of the difference in the intensity of a pixel at two instants separated by a period of the delay. Such an approach provides only sparse sampling and may thus not be sensitive to intensity fluctuations occurring between the two sampled instants (e.g., the intensity may change and return to roughly the same value). The intensity fluctuation may be calculated more sensitively by recording the image pixel values ​​(representing the sample light intensity for the micro-optical element) at multiple instants and taking the intensity difference between the maximum and minimum values ​​recorded over a period of time. Such an intensity fluctuation metric may also be normalized by dividing the value by the elapsed time between the maximum and minimum values. The intensity fluctuation may be calculated more sensitively by recording the pixel values ​​at multiple instants and taking the cumulative absolute difference in intensity between all successive values ​​recorded over a period of time. Such an intensity fluctuation metric may be normalized by dividing it by the delay period over which it is calculated. This approach offers the advantage of making the image pixel values ​​(representing the sample light intensity for the micro-optical element) more sensitive to sample motion that varies non-monotonic in time. However, it has the disadvantage of being more sensitive to noise in the intensity signal. Therefore, it may be desirable to smooth the intensity signal, for example with a moving average filter, before calculating the intensity fluctuations in this way. For example, for continuously recorded intensity values, averaging (e.g. with a moving window filter) over at least 25 values ​​at intervals of about 1-5 ms may be desirable.

[0094] When monitoring the pixel value of a single image pixel, depending on the nature of the sample, it is relatively likely that there is not enough spatial frequency modulation and / or contrast tissue structure at the pixel to provide sufficient sensitivity to sample motion. Therefore, it may be advantageous to consider multiple image pixels when assessing whether sample motion has occurred or is occurring. For example, an intrinsic intensity variation metric may be calculated for an area comprised of multiple image pixels (e.g., the intensity variation at each pixel of a region of image pixels may be averaged to give an average intensity variation for those pixels). These regions may be constructed from isotropic binnings of image pixels (e.g., 2x2 image pixels, 3x3 image pixels, 4x4 image pixels, 6x6 image pixels, 8x8 image pixels, 16x16 image pixels grouped together) or from anisotropic binnings (e.g., 1x2 image pixels, 3x4 image pixels, 6x8 image pixels, 1x12 image pixels). Because sample motion is sometimes localized to a relatively small area, combining too many image pixels together in a given area may be counterproductive, especially if the pixels are located relatively far away from each other.

[0095] Live View Mode In some embodiments, the method provides live sample monitoring information to a user. In some embodiments, such methods also include generating and optionally displaying one or more images in real time, where the image(s) are generated based on sample light received from the micro-optical elements in the micro-optical element array without scanning the array or the sample. Thus, image(s) can be generated as soon as light is received, without the need to receive light from multiple positions in a scan pattern before image(s) can be generated. This approach can substantially reduce the time required to receive a sufficient signal to generate an image.

[0096] In certain embodiments, light of sufficient intensity to produce a useful image can be received from the micro-optical element at a detector with an exposure time of <250 milliseconds (ms), generating an image frame rate of at least 4 frames / sec that can be displayed to a user. (For some users, a frame rate of at least 4 frames / sec is necessary to respond in real time to changes in sample position, movement, and / or stability. Shorter exposure times (e.g., <10 ms, <5 ms, or <2 ms) can allow for higher frame rates that provide information to the user in a manner that is more sensitive to sample motion, for example. Shorter exposure times also mean that each image corresponds to a more instantaneous "snapshot," such that comparison of such images can provide a more sensitive assessment of sample motion that may have occurred. The sample light received from the micro-optical element while remaining in a fixed position during the exposure time can be detected by a detector (e.g., a CMOS or CCD camera). An image can be generated that includes image pixels (an example of a "live view" mode) that represent in real time the relative intensity of sample light received at a detector element(s) corresponding to a particular micro-optical element in the array over an exposure time. When the sample and the micro-optical element array are fixed during imaging, each micro-optical element in the array images a different (e.g., distinct) location on the sample, the different locations being spatially separated by a characteristic distance (e.g., the pitch of the micro-optical elements in the array) relative to the micro-optical element array. Of course, a given image pixel may represent changing locations of the sample over time if the sample is moving (e.g., due to natural relaxation), possibly resulting in a variation in the intensity of a given image pixel between successive images.

[0097] In some embodiments, the imaging system may be designed and calibrated such that (e.g., when not scanning) one micro-optical element is imaged onto exactly one detector element. In some such embodiments, the detection frame (without further processing) already constitutes an image of the sample where one pixel represents the signal from a unique and distinct micro-optical element in the micro-optical element array. In some embodiments, one micro-optical element is imaged across many detector elements (e.g., >4, >9, >16, >25, >100 detector elements). For example, a micro-optical element array may have on the order of tens of thousands of micro-optical elements, while a detector of corresponding size may include millions or tens of millions of detector elements (e.g., a 10+ megapixel camera). In some such embodiments, the intensity collected by a unique micro-optical element may be calculated from the values ​​of the many detector elements on which this micro-optical element is imaged (e.g., by adding or interpolating the values ​​of the detector elements) to generate an image where one image pixel represents the signal from the unique and distinct micro-optical elements in the array as determined from multiple detector elements. An image pixel may represent the sum or average of the intensity of the sample light received at the detector element corresponding to a particular micro-optical element.

[0098] In general, the higher the optical resolution of the micro-optical element, the more sensitive the live view mode. In some embodiments, the optical resolution of the micro-optical element is preferably substantially equal (e.g., within 10%) to or smaller than the sample structure (e.g., tissue sample microstructure), e.g., preferably having a lateral point spread function <10 μm, <5 μm, <2 μm, or <1 μm. In the case of a smaller optical resolution, the spatial resolution of the image pixels of the image generated when not scanning is improved and tends to indicate that movement or stabilization is occurring more clearly, providing the user with a better understanding of the current state of the sample when observing in live view mode.

[0099] 4A-4C show an exemplary method 400 for generating and optionally displaying one or more images to provide a user with live sample monitoring information. In step 402, sample light is received from a micro-optical element array. With reference to FIG. 4B, step 402 may include sub-step 402a of illuminating the sample with illumination light using an optical module including an array of micro-optical elements, step 402b of receiving sample light from the sample from the micro-optical element array (e.g., through the optical module) at a detector over a period of time, and step 402c of processing the signal from the detector to determine the intensity of the sample light over a collection period (e.g., detection frames captured at a given exposure time). With reference back to FIG. 4A, in step 404, one or more images are generated in real time based on the sample light received from the micro-optical elements. For example, in step 404, one or more images can be generated while sub-steps 402a-402c are performed for new sample light from a new period of time, resulting in (nearly) continuous receiving and processing of sample light. FIG. 4C illustrates an exemplary subroutine of step 404, including substep 404a of generating individual image pixels for each image, each of which represents the intensity of sample light received from one of the micro-optical elements in the detector (e.g., in one or more respective detector elements). Referring back to FIG. 4A, in step 406, one or more images are optionally displayed. Step 406 may occur simultaneously with step 402 and / or step 404. In step 408, imaging, including scanning the micro-optical element array, is initiated (e.g., automatically) based on one or more of the one or more images. For example, imaging by scanning may be initiated when one or more images indicate (e.g., to a user or as determined by an image processing or image recognition algorithm) that the sample is sufficiently stable (e.g., over a period of time).A sample may be quantitatively determined to be sufficiently stabilized based on a stabilization index (e.g., as described in the following paragraphs), in which the sample has a specific sufficiently large area that is in focus (e.g., does not change significantly, such as by more than 10% over a period of time) and / or does not contain air bubbles (e.g., over a period of time).

[0100] Producing one or more images may include calculating the absolute number and / or percentage of micro-optical elements that return sample light above a predefined intensity threshold. If a micro-optical element returns sample light below the threshold, the corresponding image pixel may have a zero pixel value. If it returns sample light above the threshold, the corresponding image pixel may have a non-zero pixel value. Detecting areas of the image that correspond to the background (e.g., out-of-focus image areas of the sample area) (e.g., using an operator based on the Laplacian) and calculating the absolute number and / or percentage of micro-optical elements that do not face the background may be part of determining and displaying the size of the imaged surface of the sample plane. In some embodiments, the micro-optical elements may not return sample light or may not return sample light below the detection threshold of the detector such that the corresponding image pixel has a pixel value of zero.

[0101] 5A-5D show an example of the use of live view mode of a sample achieved according to the methods disclosed herein. In this example, an area of ​​the sample that is in focus is monitored in live view mode. Each image pixel represents the intensity of light received from an individual micro-optical element in the array for a distinct location in the sample over a short period of time (e.g., 1-3 milliseconds (ms)) prior to image generation. The area defined by the dashed outline is t 05D ) indicates a zero pixel value for all image pixels within a region in (shown in FIG. 5A ) (representing no sample light collected and thus no sample light received within the area of ​​the sample corresponding to that region of the image). The zero pixel values ​​for the image pixels may indicate that the sample is out of focus in the areas corresponding to those image pixels (e.g., light that would have been detected at the corresponding detector element(s) has been removed by the aperture(s)). At subsequent successive times shown in FIGS. 5B-5D , the area in focus increases, such that the area defined by the dashed outline becomes progressively larger and filled in over time. Thus, over time, a progressively larger area of ​​image pixels have non-zero pixel values, and the convex hull of image pixels having non-zero pixel values ​​grows at a progressively slower rate. The sample may be considered to have a sufficiently large area to be in focus (e.g., to warrant the start of imaging by the scan) in one or more images based on the area of ​​image pixels having non-zero pixel values ​​and / or the rate of change of the convex hull. The increasing areas in focus shown over the time series of Figures 5A-5D may be the result of a user adjusting (e.g., manipulating) the sample to reposition the sample to have areas in focus. Figures 5A-5D are grayscale images with image pixels representing a range of sample light intensities received from the micro-optical element for different locations in the sample.

[0102] 6A-6E show an example of the use of live view mode of a sample achieved according to the methods disclosed herein. In this example, the live view mode is monitored to determine if a bubble(s) is present in the sample. Each image pixel represents the intensity of light received from an individual micro-optical element in the array for a distinct location in the sample over a short period of time prior to image generation. The area defined by the dashed outline represents the intensity of light received from an individual micro-optical element in the array for a distinct location in the sample over a short period of time prior to image generation. 0(shown in FIG. 6A ) shows zero pixel values ​​for all image pixels within a region in the image (representing no collected sample light and thus no sample light received within the area of ​​the sample corresponding to that region of the image). Such image pixels with zero pixel values ​​are surrounded (e.g., at least partially) by image pixels with non-zero pixel values, indicating the presence of an air bubble. In FIG. 6A , two air bubbles are present, each indicated by a white outline highlighting the perimeter of the area of ​​image pixels with zero pixel values, the perimeter being defined by image pixels with non-zero pixel values ​​(e.g., at least 70% of the image pixels on the perimeter have non-zero pixel values). Image processing or image recognition algorithms can be applied to automatically determine whether any such regions are present in an image or over time (e.g., across multiple images). Over time, as shown in FIGS. 6B-6E , the live view mode shows the shifting, shrinking, and eventual disappearance of the area of ​​image pixels with zero pixel values ​​surrounded by a perimeter that includes primarily (e.g., at least 70%) image pixels with non-zero pixel values. A user may consider a sample ready for scanning when the live view shows no remaining air bubbles or specifically requires that no air bubbles be present prior to scanning. In some embodiments, the processor may automatically determine that no air bubbles are present (e.g., using image processing or image recognition algorithms). An area threshold (e.g., set by a user) may be used to distinguish air bubbles from regions of the sample that would never yield image pixels with non-zero pixel values ​​(e.g., not fluorescently tagged). Figures 6A-6E are grayscale images including image pixels representing a range of sample light intensities received from the micro-optical element for different locations in the sample.

[0103] Image with stabilization indicators The live view mode allows the user to view real-time sample information that can be used to monitor sample positioning and sample motion, and (self-)stabilization, among other characteristics of the sample, as disclosed in the previous paragraph. In general, in the live view mode, a more moving sample, whether due to relaxation or other mechanisms, appears to result in more variation in the intensity of image pixels over a period of time. An experienced user may be able to determine when such variation is small enough to indicate that a full image subsequently acquired by scanning the micro-optical element array across a scan pattern is of sufficiently high quality (e.g., sufficiently devoid of sample motion artifacts), which is useful (e.g., in determining whether one or more features, such as one(s) indicative of cancer, are present in the image). However, an inexperienced user, or even a user with some experience, may not have or may be able to develop such skills. Therefore, in certain embodiments, it is advantageous to present a quantitative assessment of sample stabilization over a period of time, i.e., a stabilization index.

[0104] The stabilization index or indices may be presented to the user by a graphical indication (e.g., an icon, shading, graphic, or color) on the image. Thresholding may be applied to the stabilization indices calculated for different image pixel regions to allow the image to be shaded or colored for easy reading by the user (e.g., using a null, yellow, red, or null, yellow, orange, red color scheme). Using a graphical indication of one or more stabilization indices, the user may be able to easily read the image to determine when to begin imaging. Such a determination may also be made automatically by the processor using the stabilization index values ​​for one or more images.

[0105] Many different stabilization indices may be calculated and a graphical indication(s) may be presented to the user to provide a quantitative assessment of sample stabilization. In some embodiments, all of the stabilization indices are calculated for each of a subset of image pixels of the image (e.g., each image pixel within a region of image pixels). In some embodiments, the stabilization indices are determined by comparing changes in the intensity of sample light received from the micro-optical elements over a period of time for at least a portion of the micro-optical elements in the array. The stabilization indices may be dynamic / changing over time (e.g., changing between successive images) because the intensity of sample light received from the micro-optical elements may vary non-uniformly over a period of time and the signal from the sample light used to determine the stabilization indices may correspond to different periods in different instances (e.g., using a moving period of fixed duration).

[0106] Referring to FIG. 4A, in some embodiments, step 404 of generating one or more images based on sample light received from the micro-optical elements in the array without scanning may include performing a subroutine shown in FIG. 4D to calculate a stabilization index. In step 404a, the sample light is collected over a number of discrete periods (e.g., consecutive periods where one period ends when another period begins) using the micro-optical element array. In step 404b, the collected sample light is received from the micro-optical element array at a detector. In step 404c, the signal from the detector is processed to determine the intensity of each micro-optical element during each period. That is, the micro-optical element array is used to capture a series of detection frames, one frame per period. In step 404d, the detection frames are used to determine a weighted average (e.g., an exponential moving average) of the intensity. Equations 1 and 2 are examples of calculating an exponential moving average. For t=1 (i.e., for the first frame), I'(m,t)=I(m,1) (Equation 1) For t>1 (i.e., for every subsequent frame), I'(m,t)=α*I(m,t)+(1-α)*I'(m,t-1) (Eq. 2) α is a user-configurable parameter between 0 and 1, e.g., 0.1. The number of detection frames used in determining the weighted average and / or stabilization index may also be a user-configurable parameter N. In step 404e, the minimum value (I' min (m)) and maximum value (I' max (m)) is calculated for each micro-optical element over a period of time, for example over the last N detection frames. Then, in step 404f, a stabilization index is calculated as I' max (m) and I' min (m) and the difference (S=I' max (m)-I' min (m)) in real time. Figure 4E provides a visual demonstration of such a calculation.

[0107] Although one particular example of a stabilization index is detailed in the previous paragraph, different formulas can be used to determine the change in intensity, including one or more of difference, ratio, floor, and upper limit. A weighted time average, such as a weighted exponential average, can be used to calculate the stabilization index. Furthermore, the determined stabilization index corresponds to an individual micro-optical element in the array, e.g., for an individual image pixel. Providing an individual stabilization index for each of the image pixels does not make image interpretation (e.g., by a user) any easier than a normal live view mode. Thus, in some embodiments, a stabilization index is determined for a region of the image pixel (a region corresponding to a cluster of micro-optical elements). An easy to interpret graphical indication (e.g., an icon, shading, graphic, or color) can then be included in the image showing the stabilization index of the micro-optical element corresponding to that region. The cluster can be at least 9 micro-optical elements (e.g., at least 16 micro-optical elements, at least 25 micro-optical elements, at least 49 micro-optical elements, or at least 64 micro-optical elements). The indication may be based, for example, on a minimum, maximum, or average stabilization index of the sample light received from the micro-optical elements in the cluster.

[0108] 7A-7D show examples of images generated and displayed to a user of a sample with a semi-transparent stabilization indicator overlay, where the indication of the stabilization indicator is for an area of ​​image pixels. The stabilization indicator is overlaid over the live view mode, but in some embodiments the image includes only an indication of the calculated stabilization indicator (without any live view mode). 0 In FIG. 7B (t), much of the sample is moving (having low stabilization) as evidenced by the large percentage of image pixel regions with a semi-transparent red overlay resulting from a high Stabilization Index value (and thus indicating relatively large sample motion), which is calculated by determining the change in intensity of sample light received over a period of time for these locations. At the periphery of the high motion regions, there are several small areas with a moderate amount of sample motion, as indicated by the semi-transparent yellow overlay. The regions of image pixels with yellow indications correspond to areas of the sample that are relatively more stable, and thus have a lower Stabilization Index value than the regions of image pixels with red indications. 1 ), Figure 7C(t 2 ), and Fig. 7D(t 3 ) the sample becomes more and more stable, resulting in a smaller and smaller stabilization index value and a larger and larger area of ​​the sample, which in turn results in smaller and smaller regions of image pixels overlaid with red and yellow indications (fewer and fewer clusters of micro-optical elements receiving sample light, indicating a significant change in sample motion and less sample motion). Even if the user is unable to determine whether the pixel value variance for an image pixel is significantly decreasing over time in live view mode, the graphical indications in FIGS. 7A-7D are easy to interpret. 3 Sample at, or t 3The sample immediately after can be imaged by scanning the micro-optical array with fewer sample motion artifacts, if any. Figure 7E shows a live view mode of a sample without the stabilization indicator mode overlay (e.g., immediately before imaging by scanning the micro-optical array).

[0109] Image display In some embodiments, the image is displayed in real-time as it is generated. In some embodiments, the image is automatically processed by image processing or image recognition algorithms and may not therefore be displayed separately, at least not in real-time. The displayed image may be displayed in one or more graphical user interfaces. The one or more graphical user interfaces may allow user input that modifies the image. For example, a user may be able to show or hide a stabilization indicator view (e.g., an overlay), show or hide summary statistics for one or more stabilization indicators for the image(s), or show or hide a live view mode.

[0110] In some embodiments, it may be preferable to hide the stabilization indicator view (e.g., overlay) when positioning the sample. During positioning, the sample will move significantly, resulting in very high stabilization indicator values ​​over a large area of ​​the sample (e.g., throughout the sample). Thus, the stabilization indicator view will not provide useful information during that time. In fact, it may be distracting to a user attempting to determine the state in which the sample is positioned. Thus, a computing device (e.g., included in an imaging system) may hide the stabilization indicator view during the sample positioning period (e.g., by user input) and then enable the stabilization indicator view (e.g., by further user input) to track the stabilization of the sample after positioning is complete. Once one or more image stabilization indicators indicate that sufficient stabilization has occurred, image acquisition using a scan pattern (e.g., of a micro-optical array) may begin (e.g., automatically).

[0111] One or more graphical user interfaces (e.g., used to display the generated images to the user in real time) may be provided to allow the user to provide various inputs. In some embodiments, the graphical user interface allows the user to provide parameters used to calculate the stabilization index (e.g., weight parameter(s) of the weighted average) for the image pixels. In some embodiments, the graphical user interface allows the user to provide inputs for tagging a single image or multiple images from the live view mode or the stabilization index view (e.g., overlaid over the live view mode) with location and / or orientation information. In some embodiments, the graphical user interface allows the user to provide inputs for thresholding the stabilization index (e.g., a particular threshold stabilization index value that serves as a threshold, bin size, or characteristic of the indication (e.g., color and / or transparency)). In some embodiments, the graphical user interface allows the user to adjust the brightness and / or contrast of the generated and / or displayed image(s) in real time. In some embodiments, the graphical user interface allows a user to select (e.g., switch between) a grayscale view and a pseudocolor view (e.g., showing shades of purple that mimic a histologically stained sample) for the live view mode.

[0112] 8A-8D show examples of graphical user interfaces including a live view mode of a sample, each with a stabilization index overlay. In the graphical user interface(s) of FIG. 8A, image 802 is a grayscale image representing the fluorescence intensity of the sample light received from the micro-optical elements in the array. Some image pixels are bright and some are dark, indicating a variation in intensity over the exposure time used to collect the sample light. Image 802 also includes a stabilization index overlay indicating that at the time the image was generated and displayed, some sample motion was occurring, mostly on the right side of the image. User interface 804 shows summary statistics for image 802. The summary statistics include the percentage of sample area imaged (the percentage of the total area that is in focus and therefore available for imaging with the fixed micro-optical element array being imaged), the percentage of area of ​​significant motion (sample motion is currently high, which corresponds to a high stabilization index value), and the percentage of area of ​​substantial motion (sample motion is significant, but less than the area of ​​significant motion, which corresponds to an intermediate stabilization index value). The interface 806 allows a user to tag the image 802 with location and / or orientation information, as well as initiate a full image collection by initiating a scan of the micro-optical array. For example, a user may view the image 802 and determine that the amount of sample motion indicated by the stabilization indicator overlay is small enough that a high quality full scan image can be produced, and thus click the "Collect" button to begin the scan.

[0113] FIG. 8B is similar to FIG. 8A, except that summary statistics are shown with time resolution, which may facilitate a user to observe trends in the percentage of imaged sample area, the percentage of significant motion area, and the percentage of substantial motion area. Longer periods of time with smaller or minimal changes in these statistics would indicate better sample stabilization. In some embodiments, it is preferred that the percentage of significant motion area and / or the percentage of substantial motion area trend toward zero or are within a small amount (e.g., 1-5%) of zero before initiating full imaging with a scan.

[0114] FIG. 8C is similar to FIGS. 8A and 8B, except that interfaces 808, 810 are provided to allow a user to input parameters used to generate image 802. Interface 808 includes input of parameters associated with the stabilization indicator overlay shown on image 802, and a button to show / hide the interface. Parameters that can be modified by the user include the transparency of the stabilization indicator indication (e.g., can be changed by the user to make the underlying live view mode easier or harder to see), binning (e.g., the size of the cluster of micro-optical elements to which the region of the indication corresponds, currently set to 4×4), and the stabilization indicator threshold that determines the color (null, yellow, or red) each region (4×4) is darkened / colored with. Interface 810 includes parameters used to calculate the stabilization index value for each region, including weighting parameters and the number of detection frames to determine minimum and maximum intensity.

[0115] FIG. 8D is similar to FIG. 8C, except that image 802 is not a grayscale image, but rather an image in which the image pixels contained in image 802 in live view mode have a pseudocolor that mimics a histological stain, in this case purple.

[0116] An image generated from sample light received from a micro-optical element array without scanning may include image pixels, each representing a respective micro-optical element in the array. Thus, the image may be of relatively low resolution, since the number of micro-optical elements in the array may be low relative to typical image resolutions. The image may be displayed to a user using a display (e.g., of an imaging system) that has a high maximum resolution (e.g., may be a 1080p or 4K monitor). Thus, to make the image a reasonable physical size on the display, multiple display pixels may be used to display individual image pixels. As long as uniform scaling is used, no distortion to the image occurs. Interpolation may be used instead of or in addition to scaling to display the image on a high-resolution display.

[0117] Computer System, Computing Device, and Network Implementations Exemplary embodiments of the systems and methods disclosed herein have been described above with reference to computations performed locally by a computing device. However, computations performed over a network are also contemplated. FIG. 9 illustrates an exemplary network environment 900 for use with the methods and systems described herein. In a brief overview, a block diagram of an exemplary cloud computing environment 900 is shown and described with reference to FIG. 9. The cloud computing environment 900 may include one or more resource providers 902a, 902b, 902c (collectively, 902). Each of the resource providers 902 may include computing resources. In some implementations, the computing resources may include any hardware and / or software used to process data. For example, the computing resources may include hardware and / or software capable of executing algorithms, computer programs, and / or computer applications. In some implementations, exemplary computing resources may include application servers and / or databases with storage and retrieval capabilities. Each of the resource providers 902 may be connected to any other resource providers 902 in the cloud computing environment 900. In some implementations, the resource providers 902 may be connected through a computer network 908. Each of the resource providers 902 may be connected through the computer network 908 to one or more computing devices 904a, 904b, 904c (collectively, 904).

[0118] The cloud computing environment 900 may include a resource manager 906. The resource manager 906 may be connected to the resource providers 902 and the computing devices 904 through a computer network 908. In some implementations, the resource manager 906 may facilitate the provisioning of computing resources by one or more resource providers 902 to one or more computing devices 904. The resource manager 906 may receive a request for a computing resource from a particular computing device 904. The resource manager 906 may identify one or more resource providers 902 that have the capability to provide the computing resource requested by the computing device 904. The resource manager 906 may select a resource provider 902 to provide the computing resource. The resource manager 906 may facilitate a connection between the resource provider 902 and the particular computing device 904. In some implementations, the resource manager 906 may establish a connection between a particular resource provider 902 and a particular computing device 904. In some implementations, the resource manager 906 may redirect a particular computing device 904 to a particular resource provider 902 with the requested computing resource.

[0119] 10 illustrates an example of a computing device 1000 and a mobile computing device 1050 that can be used in the methods and systems described in this disclosure. The computing device 1000 is intended to represent various types of digital computers, such as laptops, desktops, workstations, personal digital assistants, servers, blade servers, mainframes, and other suitable computers. The mobile computing device 1050 is intended to represent various types of mobile devices, such as personal digital assistants, mobile phones, smartphones, and other similar computing devices. The components illustrated herein, their connections and relationships, and their functions are meant to be exemplary only and not limiting.

[0120] The computing device 1000 includes a processor 1002, a memory 1004, a storage device 1006, a high-speed interface 1008 connecting to the memory 1004 and multiple high-speed expansion ports 1010, and a low-speed interface 1012 connecting to a low-speed expansion port 1014 and the storage device 1006. Each of the processor 1002, the memory 1004, the storage device 1006, the high-speed interface 1008, the high-speed expansion port 1010, and the low-speed interface 1012 are interconnected using various buses and may be mounted on a common motherboard or in other manners as desired. The processor 1002 can process instructions for execution within the computing device 1000, including instructions stored in the memory 1004 or the storage device 1006, to display graphical information for a GUI on an external input / output device, such as a display 1016 coupled to the high-speed interface 1008. In other implementations, multiple processors and / or multiple buses may be used, as desired, along with multiple memories and multiple types of memories. Also, multiple computing devices may be connected (e.g., as a server bank, group of blade servers, or multiprocessor system) with each device providing a portion of the required computations. Also, multiple computing devices may be connected (e.g., as a server bank, group of blade servers, or multiprocessor system) with each device providing a portion of the required computations. Thus, as the term is used herein, when functions are described as being performed by a "processor," this encompasses embodiments in which the functions are performed by any number of processors (e.g., one or more processors) of any number of computing devices (e.g., one or more computing devices). Furthermore, when functions are described as being performed by a "processor," this encompasses embodiments in which the functions are performed by any number of processors (e.g., one or more processors) of any number of computing devices (e.g., one or more computing devices) (e.g., in a distributed computing system).

[0121] The memory 1004 stores information within the computing device 1000. In some implementations, the memory 1004 is a volatile memory unit or multiple volatile memory units. In some implementations, the memory 1004 is a non-volatile memory unit or multiple non-volatile memory units. The memory 1004 may also be another form of computer-readable medium, such as a magnetic disk or optical disk.

[0122] The storage device 1006 has the capability of providing mass storage for the computing device 1000. In some implementations, the storage device 1006 may be or include a computer-readable medium, such as a floppy disk device, a hard disk device, an optical disk device, or a tape device, a flash memory, or other similar solid-state memory device, or an array of devices including devices in a storage area network or other configuration. The instructions may be stored on an information carrier. The instructions, when executed by one or more processing devices (e.g., the processor 1002), perform one or more methods, such as those described above. The instructions may also be stored in one or more storage devices (e.g., the memory 1004, the storage device 1006, or a memory on the processor 1002), such as a computer-readable medium or a machine-readable medium.

[0123] The high-speed interface 1008 manages bandwidth-intensive operations for the computing device 1000, while the low-speed interface 1012 manages less bandwidth-intensive operations. Such allocation of functions is merely exemplary. In some implementations, the high-speed interface 1008 is coupled to the memory 1004, the display 1016 (e.g., via a graphics processor or accelerator), and a high-speed expansion port 1010 that may accept various expansion cards (not shown). In an embodiment, the low-speed interface 1012 is coupled to the storage device 1006 and the low-speed expansion port 1014. The low-speed expansion port 1014, which may include various communication ports (e.g., USB, Bluetooth, Ethernet, wireless Ethernet), may be coupled, for example, via a network adapter, to one or more input / output devices, such as a keyboard, pointing device, scanner, or a networking device, such as a switch or router.

[0124] Computing device 1000 may be implemented in a number of different forms, as shown in the figure. For example, it may be implemented as a standard server 1020, or multiple times in a group of such servers. In addition, it may be implemented in a personal computer, such as a laptop computer 1022. It may also be implemented as part of a rack server system 1024. Alternatively, components from computing device 1000 may be combined with other components in a mobile device (not shown), such as mobile computing device 1050. Each such device may include one or more of computing device 1000 and mobile computing device 1050, and the entire system may be composed of multiple computing devices in communication with each other.

[0125] The mobile computing device 1050 includes a processor 1052, a memory 1064, an input / output device such as a display 1054, a communication interface 1066, and a transceiver 1068, among other components. The mobile computing device 1050 may also be provided with a storage device, such as a microdrive or other device, to provide additional storage. Each of the processor 1052, memory 1064, display 1054, communication interface 1066, and transceiver 1068 are interconnected using various buses, and some of the components may be mounted on a common motherboard or in other manners, if desired.

[0126] The processor 1052 can execute instructions within the mobile computing device 1050, including instructions stored in the memory 1064. The processor 1052 can be implemented as a chipset of chips including discrete and multiple analog and digital processors. The processor 1052 can provide coordination of other components of the mobile computing device 1050, such as control of a user interface, control of applications run by the mobile computing device 1050, and control of wireless communications by the mobile computing device 1050.

[0127] The processor 1052 may communicate with a user via a control interface 1058 and a display interface 1056 coupled to a display 1054. The display 1054 may be, for example, a TFT display (thin film-transistor liquid crystal display) or an OLED (organic light-emitting diode) display, or other suitable display technology. The display interface 1056 may include appropriate circuitry to drive the display 1054 to provide graphical and other information to the user. The control interface 1058 may receive commands from a user and translate them for submission to the processor 1052. Additionally, an external interface 1062 may provide communication with the processor 1052 to enable short-range communication of the mobile computing device 1050 with other devices. The external interface 1062 may provide, for example, wired communication in some implementations or wireless communication in other implementations, and multiple interfaces may also be used.

[0128] The memory 1064 stores information within the mobile computing device 1050. The memory 1064 may be implemented as one or more of a computer-readable medium or media, a volatile memory unit or units, or a non-volatile memory unit or units. Expansion memory 1074 may also be provided and connected to the mobile computing device 1050 via an expansion interface 1072, which may include, for example, a SIMM (single in-line memory module) card interface. The expansion memory 1074 may provide extra storage space for the mobile computing device 1050 or may also store applications or other information for the mobile computing device 1050. In particular, the expansion memory 1074 may include instructions that perform or complement the processes described above and may also include secure information. Thus, for example, expansion memory 1074 may be provided as a security module for mobile computing device 1050 and programmed with instructions that permit secure use of mobile computing device 1050. Additionally, secure applications may be provided via SIMM cards along with additional information, such as placing identifying information on the SIMM card in a manner that cannot be hacked.

[0129] The memory may include, for example, flash memory and / or NVRAM memory (non-volatile random access memory), as described below. In some embodiments, the instructions are stored on an information carrier and, when executed by one or more processing devices (e.g., processor 1052), perform one or more methods, such as those described above. The instructions may also be stored in one or more storage devices, such as a computer-readable or machine-readable medium (e.g., memory 1064, expansion memory 1074, or memory on processor 1052). In some embodiments, the instructions may be received in a propagated signal, through transceiver 1068 or external interface 1062.

[0130] The mobile computing device 1050 may communicate wirelessly via a communications interface 1066, which may include digital signal processing circuitry, if necessary. The communications interface 1066 may provide communications under various modes or protocols, such as GSM voice calls (Global System for Mobile Communications), SMS (Short Message Service), EMS (Enhanced Messaging Service), or MMS messaging (Multimedia Messaging Service), CDMA (Code Division Multiple Access), TDMA (Time Division Multiple Access), PDC (Personal Digital Cellular), WCDMA (Wideband Code Division Multiple Access), CDMA2000, or GPRS (General Packet Radio Service), among others. Such communications may occur via a transceiver 1068, for example, using radio frequencies. In addition, short-range communications may occur using Bluetooth, Wi-Fi, or other such transceivers (not shown), etc. In addition, a GPS (Global Positioning System) receiver module 1070 may provide the mobile computing device 1050 with additional navigation and location-related radio data that may be used by applications running on the mobile computing device 1050 as needed.

[0131] The mobile computing device 1050 may also communicate audibly using an audio codec 1060, which may receive spoken information from a user and convert that information into usable digital information. Similarly, the audio codec 1060 may generate audible sounds for the user, such as via a speaker in a handset of the mobile computing device 1050. Such sounds may include sounds from a voice telephone call, may include recorded sounds (e.g., voice messages, music files, etc.), and may also include sounds generated by applications running on the mobile computing device 1050.

[0132] The mobile computing device 1050 may be implemented in a number of different forms, as shown in the figure, For example, it may be implemented as a mobile phone 1080. It may also be implemented as part of a smartphone 1082, a personal digital assistant, or other similar mobile device.

[0133] Various implementations of the systems and techniques described herein can be realized in digital electronic circuitry, integrated circuits, specially designed ASICs (application-specific integrated circuits), computer hardware, firmware, software, and / or combinations thereof. These various implementations may include implementations in one or more computer programs executable and / or readable on a programmable system including at least one programmable processor, which may be special-purpose or general-purpose, coupled to receive data and instructions from, and transmit data and instructions to, a storage system, at least one input device, and at least one output device.

[0134] These computer programs (also known as programs, software, software applications, or code) include machine instructions for a programmable processor and may be implemented in high-level procedural programming languages, and / or object-oriented programming languages, and / or assembly / machine languages. As used herein, the terms machine-readable medium and computer-readable medium refer to any computer program product, apparatus, and / or device (e.g., magnetic disks, optical disks, memory, programmable logic circuits (PLDs)) used to provide machine instructions and / or data to a programmable processor, including a machine-readable medium that receives machine instructions as a machine-readable signal. The term machine-readable signal refers to any signal used to provide machine instructions and / or data to a programmable processor.

[0135] To provide interaction with a user, the systems and techniques described herein can be implemented on a computer having a display device (e.g., a CRT (cathode ray tube) or LCD (liquid crystal display) monitor) for displaying information to a user, and a keyboard and pointing device (e.g., a mouse or trackball) by which the user can provide input to the computer. Similarly, other types of devices can be used to provide interaction with a user. For example, feedback provided to the user can be any form of sensory feedback (e.g., visual feedback, auditory feedback, or tactile feedback), and input from the user can be received in any form, including acoustic input, speech input, or tactile input.

[0136] The systems and techniques described herein can be implemented in a computing system that includes back-end components (e.g., a data server, etc.), or that includes middleware components (e.g., an application server), or that includes front-end components (e.g., a client computer having a graphical user interface or a web browser through which a user can interact with embodiments of the systems and techniques described herein), or any combination of such back-end, middleware, or front-end components. The components of the system can be interconnected by any form or medium of digital data communication (e.g., a communications network). Examples of communications networks include a local area network (LAN), a wide area network (WAN), and the Internet.

[0137] A computing system may include clients and servers. Clients and servers are generally remote from each other and typically interact through a communication network. The relationship of client and server arises by virtue of computer programs running on the respective computers and having a client-server relationship to each other.

[0138] Specific embodiments of the present disclosure have been described above. However, it should be specifically noted that the present disclosure is not limited to these embodiments, but rather additions and modifications to those explicitly described in the present disclosure are also intended to be included within the scope of the present disclosure. Furthermore, it should be understood that the features of the various embodiments described in the present disclosure are not mutually exclusive and may exist in various combinations and permutations, which may be possible without departing from the spirit and scope of the present disclosure even if such combinations or permutations are not expressed. Although the present disclosure has been described in detail with particular reference to specific embodiments thereof, it will be understood that variations and modifications may occur within the spirit and scope of the invention as claimed.

Claims

1. 1. A method for providing live sample monitoring information to a user, comprising: generating, by a processor of a computing device, one or more images of the sample in real time based at least in part on sample light received from micro-optical elements in the micro-optical element array without scanning the array or the sample. Including, The method.

2. 2. The method of claim 1, wherein for each of the one or more images, adjacent pixels of the image represent portions of the sample light received from portions of the micro-optical elements for different locations on the sample, the different locations being separated by a characteristic distance relative to the array.

3. The method of claim 1 or 2, wherein the array remains in a fixed position during the generation.

4. The method of claim 1 , wherein the sample is unperturbed during the generation.

5. The method of claim 1 , wherein each image pixel of the one or more images corresponds to sample light received from a micro-optical element in the array.

6. The method of claim 2 , wherein the image pixels individually correspond to sample light received from each micro-optical element in the array.

7. The method of claim 2 , wherein each of the image pixels corresponds to sample light received from one of the micro-optical elements in the array.

8. The method of claim 1 , comprising determining whether an air bubble is represented in one or more of the one or more images.

9. 9. The method of claim 8, wherein determining whether an air bubble is represented includes automatically determining, by the processor, whether an area of image pixels having zero pixel values greater than a threshold area exists in the one or more of the one or more images.

10. 10. The method of claim 8 or 9, wherein determining whether an air bubble is represented comprises automatically determining, by the processor, whether a perimeter of an area of image pixels having zero pixel values defined by image pixels having non-zero pixel values is present in the one or more of the one or more images.

11. 9. The method of claim 8, comprising adjusting the sample in response to determining that no air bubbles are represented in the one or more of the one or more images.

12. 10. The method of claim 1, comprising determining whether the sample has a sufficiently large area that is in focus in one or more of the one or more images.

13. 13. The method of claim 12, wherein determining whether the specimen has the sufficiently large area in focus comprises automatically determining, by the processor, whether an area of image pixels with non-zero pixel values exceeds a predetermined threshold.

14. 14. The method of claim 12 or 13, wherein determining whether the specimen has the sufficiently large area in focus comprises automatically determining, by the processor, whether a convex hull of the portion of the image pixels with non-zero pixel values changes by 10% or less over a period of time.

15. 13. The method of claim 12, comprising adjusting the specimen in response to determining whether the specimen has a sufficiently large area that is in focus in the one or more of the one or more images.

16. The method of claim 1 , comprising adjusting the specimen during the generation in response to the one or more images.

17. The method of claim 1 , wherein the sample is accessible to a user during the generation.

18. The method of claim 1 , further comprising: initiating imaging of the sample based on the one or more images; and imaging the sample comprises scanning the micro-optical element array.

19. 20. The method of claim 18, comprising automatically initiating the imaging by the processor in response to determining that one or more of the one or more images is sufficient to indicate that the specimen is stabilized.

20. 20. The method of claim 19, wherein determining that the one or more of the one or more images is sufficient to indicate that the sample is stabilized occurs automatically by the processor.

21. 21. The method of claim 20, wherein determining that the one or more of the one or more images is sufficient to indicate that the sample is stabilized comprises determining, by the processor, that no air bubbles are represented in the one or more of the one or more images.

22. 22. The method of claim 20 or 21, wherein determining that the one or more of the one or more images is sufficient to indicate that the sample is stabilized comprises determining, by the processor, that the sample has a sufficiently large area that is in focus in the one or more of the one or more images.

23. The method of claim 1 , wherein the one or more images are grayscale image(s).

24. The method of claim 1 , wherein the one or more images are pseudocolor image(s).

25. The method of claim 2 , wherein the hue, saturation, luminance, or a combination thereof of the image pixels corresponds to the relative intensity of the received sample light.

26. 2. The method of claim 1, further comprising determining, by the processor, a stabilization indicator of the sample light for each of at least some of the micro-optical elements in the array based on comparing the sample light received from the micro-optical elements over an observation period, wherein the one or more images include a graphical indication of the stabilization indicator.

27. 27. The method of claim 26, wherein the stabilization index is dynamic over the observation period.

28. 28. The method of claim 26 or 27, wherein the stabilization indicator changes over the observation period based on changes in the sample light received from the micro-optical element.

29. 27. The method of claim 26, comprising determining, by the processor, the stabilization indicator by comparing a change in intensity of the sample light received from the micro-optical element over a calculation period.

30. 30. The method of claim 29, wherein comparing the changes in intensity of the sample light includes determining, by the processor, a minimum and a maximum intensity of the sample light received from each of the micro-optical elements over the calculation period.

31. 31. The method of claim 30, wherein the minimum intensity and the maximum intensity are each determined from a weighted average for the micro-optical element over the calculation period.

32. 32. The method of claim 30 or 31, wherein the stabilization index is the difference between the maximum intensity and the minimum intensity.

33. The method of claim 1 , wherein each of the one or more images includes a region that includes a graphical indication of the stabilization indicia for all micro-optical elements corresponding to the region.

34. 34. The method of claim 33, wherein the regions each correspond to a respective cluster of at least nine micro-optical elements.

35. determining, by the processor, for each of the regions, an average of the stabilization metrics of the micro-optical elements corresponding to the region; generating, by the processor, the graphical indication of the region based on the average; 35. The method of claim 33 or 34, comprising:

36. 36. The method of claim 35, wherein generating the graphical indication includes determining, by the processor, whether the average exceeds one or more thresholds, such graphical indication indicating whether the average exceeds the one or more thresholds.

37. 27. The method of claim 26, wherein one or more of the one or more images includes image pixels based in part on first sample light received from micro-optical elements in the array during the observation period combined with the graphical indication of the stabilization indicator.

38. 38. The method of claim 37, wherein the graphical indication of the stabilization indicator in the one or more of the one or more images is based on the first sample light and a second sample light received before the first sample light.

39. The method of claim 1 , wherein at least a portion of each of the one or more images includes a region that includes a respective graphical indication of a respective stabilization indicator for the region.

40. 40. The method of claim 39, comprising determining, by the processor, the stabilization indicator for the one of the one or more images based on one or more of the one or more images prior to the one of the one or more images.

41. 10. The method of claim 1, wherein at least a portion of each of the one or more images includes a region that includes a respective graphical indication of a movement of the sample relative to the region.

42. 27. The method of claim 26, wherein the graphical indication is a color within the region.

43. 27. The method of claim 26, wherein the graphical indication is overlaid on an image pixel corresponding to sample light received from a micro-optical element in the array.

44. The method of claim 1 , comprising displaying, by the processor, the one or more images as they are generated.

45. 45. The method of claim 44, comprising repeatedly collecting the sample light received from the micro-optical element over a period of time such that the one or more images are generated and displayed at a rate of at least 4 images per second.

46. The method of claim 1 , wherein the generating is performed in real time such that the generating is delayed by the time required for processing.

47. The method of claim 1 , wherein each image pixel of the one or more images corresponds to sample light received from the micro-optical element over a period of 0.25 seconds or less.

48. 48. The method of claim 47, wherein the period of time is 0.005 seconds or less.

49. The method of claim 1 , wherein the sample is a freshly excised tissue sample.

50. The method of claim 1 , comprising receiving the sample light with a detector, and generating the one or more images comprises processing, by the processor, a signal from the detector.

51. The method of claim 1 , wherein the one or more images are displayed on a display.

52. 52. The method of claim 51, wherein the display, the processor, and the micro-optical element array are included in an imaging system.

53. The method of claim 1 , wherein the micro-optical elements of the array have a lateral optical resolution of 10 μm or less.

54. The method of claim 1 , wherein the micro-optical element array is included in a point-scanning imaging system.

55. 10. An imaging system comprising a processor and one or more non-transitory computer-readable media storing instructions that, when executed by the processor, cause the processor to perform the method of claim 1.

56. 1. A method for providing live sample monitoring information to a user, comprising: generating one or more images of the sample in real time based at least in part on the sample light received from the micro-optical elements in the micro-optical element array; for each of the one or more images, adjacent pixels of the image represent portions of the sample light received from portions of the micro-optical elements for different locations of the sample, the different locations being separated by a characteristic distance relative to the array. The method.

57. 57. The method of claim 56, wherein neither (i) the array nor (ii) the sample is scanned during said generating.

58. 58. The method of claim 56 or 57, wherein the micro-optical element array is included in a point-scanning imaging system.

59. 56. The imaging system of claim 55, wherein the imaging system is a point-scanning imaging system.

Images