Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
  • G01N21/645—Specially adapted constructive features of fluorimeters
  • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
  • G01N21/6458—Fluorescence microscopy
• • G—PHYSICS
  • G02—OPTICS
  • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
  • G02B21/00—Microscopes
  • G02B21/06—Means for illuminating specimens
  • G02B21/08—Condensers
  • G02B21/12—Condensers affording bright-field illumination
• • G—PHYSICS
  • G02—OPTICS
  • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
  • G02B21/00—Microscopes
  • G02B21/16—Microscopes adapted for ultraviolet illumination ; Fluorescence microscopes
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N1/00—Sampling; Preparing specimens for investigation
  • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
  • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
  • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
  • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2201/00—Features of devices classified in G01N21/00
  • G01N2201/06—Illumination; Optics
  • G01N2201/061—Sources
  • G01N2201/06193—Secondary in situ sources, e.g. fluorescent particles

Definitions

• the disclosed embodiments generally relate to techniques for imaging tissue samples. More specifically, the disclosed embodiments relate to a technique for imaging tissue samples that operates by illuminating stained tissue inside a tissue sample to produce fluorescent emissions, which function as a backlight that is absorbed by features in stained tissue located on the surface of the tissue sample.
• FFPE-based processing techniques are quite slow, minimally requiring multiple hours. Hence, it is typically necessary to wait overnight to obtain a diagnosis, and if transportation is involved, many days. Note that in surgical-guidance settings, it is advantageous to obtain results right away, to inform a surgeon about whether they successfully removed a tumor and/or identify the type of tumor. Also, in biopsy situations, if an answer can be obtained the same day, time-critical care can be dramatically accelerated.
• the disclosed embodiments provide a system that images a tissue sample.
• the system receives the tissue sample, which has been stained using absorbing and fluorescently emitting stains.
• the system illuminates the tissue sample with excitation light having a wavelength or wavelengths in a range that covers a portion of an absorption spectrum for both fluorescently emitting and absorbing stains, whereby the excitation light interacts with stained tissue located inside the tissue sample to both limit penetration depth and generate emitted dye fluorescence and tissue autofluorescence that provides a backlight, which is absorbed by features in stained tissue located on or near the surface of the tissue sample.
• the system uses an imaging device to capture an image of emitted fluorescence that emanates from the surface of the tissue sample.
• the excitation light has a wavelength or wavelengths that falls in a range between 320 nm and 800 nm.
• the image is captured through an emission filter that filters out the excitation light.
• the emission filter comprises one of the following: a long- pass emission filter; a multi-band-pass filter; and a notch filter.
• the absorbing and fluorescently emitting stains comprise hematoxylin and eosin.
• additional dyes or stains are used to label additional tissue components, wherein the additional dyes or stains include one or more of the following: acridine orange, toluidine blue, rhodamine, and propidium iodide.
• the excitation light includes light from multiple frequency ranges, which are applied either simultaneously or sequentially. [0014] In some embodiments, the excitation light includes light from multiple laser lines, which pass through a multi-line emission filter.
• the staining of the tissue sample involves simultaneously or sequentially performing immunofluorescence staining to provide information on location and abundance of specific molecular species.
• the tissue sample is located in a modified histology cassette or other sample holder, which includes a transparent window against which the tissue sample is compressed.
• this histology cassette can be implemented in a number of ways. For example, it can be large in size and it can include a large window. Moreover, it can be made with different window materials, such as Gorilla GlassTM or plastic. It can also be reusable or disposable.
• the imaging device includes one or more of the following: a monochrome camera; a color camera; and a multi- spectral image-capture device.
• FIG. 1 illustrates an imaging system in accordance with the disclosed embodiments.
• FIG. 2 presents a flow chart illustrating the process of imaging a tissue sample in accordance with the disclosed embodiments.
• FIG. 3 presents an image of a kidney vessel with associated glomeruli and tubules in accordance with the disclosed embodiments.
• FIG. 4 presents an image of a hepatocellular carcinoma and associated fibrosis in accordance with the disclosed embodiments.
• FIG. 5 presents an image of a mouse small bowel in accordance with the disclosed embodiments.
• FIG. 6 presents an image of a human liver in accordance with the disclosed embodiments.
• FIG. 7 presents an image of a human pancreas in accordance with the disclosed embodiments.
• the data structures and code described in this detailed description are typically stored on a computer-readable storage medium, which may be any device or medium that can store code and/or data for use by a computer system.
• the computer-readable storage medium includes, but is not limited to, volatile memory, non-volatile memory, magnetic and optical storage devices such as disk drives, magnetic tape, CDs (compact discs), DVDs (digital versatile discs or digital video discs), or other media capable of storing computer-readable media now known or later developed.
• the methods and processes described in the detailed description section can be embodied as code and/or data, which can be stored in a computer-readable storage medium as described above.
• a computer system reads and executes the code and/or data stored on the computer-readable storage medium, the computer system performs the methods and processes embodied as data structures and code and stored within the computer-readable storage medium.
• the methods and processes described below can be included in hardware modules.
• the hardware modules can include, but are not limited to, application-specific integrated circuit (ASIC) chips, field-programmable gate arrays (FPGAs), and other programmable-logic devices now known or later developed. When the hardware modules are activated, the hardware modules perform the methods and processes included within the hardware modules.
• ASIC application-specific integrated circuit
• FPGAs field-programmable gate arrays
• FIBI Fluorescence Imitating Brightfield Imaging
• OLED organic-light-emitting-diode
• FIBI FIBI workflow
• a typical FIBI workflow can include capture of digital images of small biopsies generated near point of care for evaluation within minutes, perhaps remotely by pathology experts, accompanied by real-time immunofluorescence assays, and followed, if indicated, by rapid RNA sequencing or other molecular assays using histologically guided panels, on nucleic acids quickly extracted from the fresh specimen.
• This makes it possible to compress elaborate diagnostic workups that can involve multiple separate biomarker evaluations down to two to three steps that can be routinized and deployed even in minimally equipped settings.
• the ultimate result is rapid, definitive and precise diagnosis and therapy guidance achieved with small biopsies, with decrease in cost, delay, and patient anxiety.
• FIBI can also be used to improve the quality and yield of biospecimen banks that can empower future research activities.
• FIBI facilitates the rapid histomorphological examination of a slide-free, thick tissue specimen via the light absorption properties of chemical dyes that affect the depth of light penetration.
• a well-known histologic dye hematoxylin
• additional light- absorbing dyes can prove useful as well.
• Hematoxylin absorbs a broad spectrum of light; this phenomenon causes a reduced penetration of excitation light into the tissue, thereby limiting the imaging volume to something slightly thicker than a conventional histology slide.
• the use of eosin provides additional value in two ways.
• the diffused fluorescence signal contributed by eosin illuminates “from behind” the specimen surface, thereby mimicking the transillumination light in regular brightfield microscopy.
• the fluorescence signal from eosin in places where it accumulates in higher concentrations, generates the familiar pink-red staining seen in conventional hematoxylin and eosin (H&E).
• Hematoxylin is a widely used dye for histology and was the first dye we observed this behavior in.
• various excitation wavelengths (405 nm, 440 nm, 500 nm) result in an image that is remarkably “thinner” in terms of imaging volume than images generated with the same excitation wavelengths but without hematoxylin.
• any dye that has the following properties can be useful to FIBI to facilitate the reduction of imaging volume: (1) the dye must not be fluorescent; (2) the dye must absorb at the wavelength being used to excite the sample; and (3) the dye must stain a majority of tissue components to some degree.
• eosin stain In traditional histology, a second stain is employed to provide color contrast and improved spatial context to the viewer. This is traditionally an eosin stain (often eosin Y), which is viewed in brightfield as an absorbing dye contributing a pink color to stained portions of the specimen. However, it is also fluorescent. When eosin is used in combination with hematoxylin and thick specimens are excited with excitation light, potentially at a variety of wavelengths, an image is generated that is remarkably close to traditional thin-section histology, both in content and contrast.
• eosin stain often eosin Y
• Eosin has some unusual properties that give it an advantage for FIBI over other conventional fluorescent counterstains.
• One of these properties is the relatively weak binding of eosin to tissue components; when a sample is stained with eosin and mounted in an aqueous media, the stain tends to continue to leach out of the tissue and into surrounding spaces, large and small. Conventionally, this has been an annoyance, but for FIBI this helps contribute to a diffuse backlighting fluorescence. Fluorescence images normally have a dark background, contrasting with brightfield imaging techniques that have a white background. Pathologists are well- versed in interpreting brightfield images but outside of special circumstances, they generally do not feel comfortable interpreting relatively unfamiliar fluorescence-mode images.
• FIBI images combine the best of both; the background and staining pattern is similar to conventional H&E-stained thin sections, allowing for a quick acclimation period.
• FIBI works with thick, fresh or fixed, but unsectioned tissue, it can be accomplished within minutes of acquiring a tissue specimen. Steps such as dehydration, paraffin-embedding, sectioning, and mounting on slides are not required, cutting hours off the time needed to acquire high-quality images.
• These images can be viewed directly, or can be quickly processed using a variety of tools (either algorithmic or machine-leaming-based) to generate images as good as or better than can be visualized on conventional H&E slides.
• OCT Optical Coherence Tomography
• MUSE UV surface excitation
• MUSE is a technology that is closely related to FIBI. (See Farzad Fereidouni, Ananya Datta Mitra, Stavros Demos, Richard Fevenson, "Microscopy with UV Surface Excitation (MUSE) for slide-free histology and pathology imaging," Proc.
• FIBI provides a number of advatanges over MUSE, which are described below.
• FIBI uses brighter, less expensive excitation sources.
• FIBI can employ convenient FED sources in the visible range that are both brighter and cheaper than the UV FEDs required for MUSE.
• Brighter excitation with brighter emission means faster imaging.
• Multiple excitation wavelengths can be deployed with FIBI, which can provide additional spectral contrast. This is not as conveniently achieved with MUSE optics.
• MUSE can deploy other UV sources beyond LEDs if increased brightness or other excitation properties are required, but these will be considerably more expensive.
• FIBI is immediately compatible with immunofluorescence reagents.
• histology stains such as hematoxylin and eosin
• probes can extend beyond standard antibodies to include nanobodies, peptides, nucleic acids, and other entities that can bind to defined targets. If these are labeled with fluorescent probes of sufficient abundance and brightness, they can be visualized even in the presence of the FIBI background stains using only labeled primary reagents. If necessary, primary and secondary antibodies or other visualization techniques can be deployed.
• FIBI provides an epifluorescence optical light path versus oblique illumination for higher numerical aperture lenses and imaging flexibility. With this arrangement there are no constraints based on working distance. It is also easy to switch from lens to lens if desired and employing lenses with higher NA, because the excitation geometry is independent of lens choice. Higher magnification lenses are easier to deploy with FIBI than with MUSE. The highest resolution described to date is enabled by the use of a high numerical aperture (NA) lOx lens. Better resolution would require higher NA lenses, which then require shorter working distances between lens and specimen. This, however, can be problematic with the current MUSE design, which deploys oblique, off-axis excitation rather than standard epifluorescence optical light paths.
• NA numerical aperture
• oblique illumination is that the excitation light does not have to travel through the objective lens, which in most cases cannot transmit light in the 280-nm UV range.
• MUSE imaging is usually limited to that provided by the relatively long-working distance high-quality lOx lens used to date, which is currently excellent, as we can use a high- NA (0.45) lOx objective.
• FIBI is compatible with existing fluorescent microscopes for rapid viewing through eyepieces, or single frame or large-field-of-view imaging with a motorized stage.
• Fresh tissue preparation involves the following steps. Prior to staining, prepare a sample with the dimensions 1 cm x 1 cm x 0.5 cm (the length and width and depth of the specimen can range anywhere from 0.1 to 10 cm or larger in any dimension. Optional: rapidly fix the specimen in either approximately 40 ml of ethanol or PBS for 30s in a microwave (600 W).
• staining protocols which include serial staining (i.e., the tissue is exposed to a number of dyes one after the other, with additional washing steps), or combined solutions, in which all the dyes are combined into a single solution.
• serial staining i.e., the tissue is exposed to a number of dyes one after the other, with additional washing steps
• combined solutions in which all the dyes are combined into a single solution. The latter is more efficient and can be modified by adding additional dyes for more informative color contrast after the described single stain.
• Some single- or single+ procedures (1) 30s rinse in diH20, 30s combination mixture (2 ml of Mayer’s hematoxylin diluted in 95% ethanol (0.5 mg/ml), 2 ml of alcoholic eosin (1 mg/ml), 1 ml of Scott’s bluing reagent), 2 x 30s rinse in diH20.
• Sample positioning The sample can be placed onto a large image stage, or alternatively, can be introduced into modified histology cassettes, with the removable lid or bottom support altered to contain a transparent window against which the specimen is gently compressed by, e.g., plastic foam when the lid is closed.
• the benefit of this arrangement is that it is easy to barcode and track the cassette and enclosed specimen, which can also be diverted into conventional FFPE processing by simply replacing the transparent lid (if used) with a conventional perforated one.
• sample holders can be as large as 10 x 10 cm, and they can use different sample-compression techniques.
• Additional excitation and imaging techniques include side-launch with standing wave illumination, oblique illumination, and even cell- phone-enabled optics with the cell phone lens and camera.
• excitation sources can include LEDs, halogen or other conventional fluorescence excitation lamps, laser diodes, or other sources.
• light emitted by the specimen can be directed to a monochrome sensor with or without a series of filters for multispectral image data collection, an RGB color sensor with a Bayer pattern for snapshot collection, enhanced snapshot cameras with three non-standard color filters, or four or more for multispectral data capture.
• the sample can be illuminated sequentially with different excitation wavelengths, or alternatively, the dichroic and other filtering optics can have multiple band passes to allow for simultaneous excitation with a number of sources with single image capture.
• samples can be observed directly through eyepieces on conventional or moderately adapted fluorescence microscopes.
• a color-shaping emission filter to adjust the perceived color composition to be, e.g., less green and more pink.
• FIG. 1 illustrates an exemplary imaging system 100 for the FIBI technique in accordance with the disclosed embodiments.
• Imaging system 100 includes an illumination source 120 (e.g., a 405 nm UV LED), which produces excitation light for fluorescence.
• Stained tissue sample 102 is affixed to an XYZ stage 108, which, for example, can have a travel range of 50 mm and 25 mm in x and y directions, and also a limited travel range in the z direction for focusing purposes.
• stained tissue sample 102 is located in a histology cassette, which includes a transparent window against which the tissue sample is compressed.
• imaging mechanism 118 comprises a scientific-grade color camera (Ximea 9MP) that uses a 200-mm tube lens 114 (Thorlab ILT 200).
• FIG. 2 presents a flow chart illustrating an exemplary process for imaging a tissue sample using the FIBI technique in accordance with the disclosed embodiments.
• the system receives the tissue sample, which has been stained using absorbing and fluorescently emitting stains (step 202).
• the system illuminates the tissue sample with excitation light having a wavelength or wavelengths in a range that covers a portion of an absorption spectrum for both fluorescently emitting and absorbing stains, whereby the excitation light interacts with stained tissue located inside the tissue sample to both limit penetration depth and generate emitted fluorescence, which provides diffuse backlighting that is absorbed by stained tissue elements located on or very close to the surface of the tissue sample (step 204).
• tissue components can express their own fluorescent signals and provide additional spatial content.
• the system directs the emitted fluorescence that emanates from the surface of the tissue sample through an emission filter that filters out the excitation light (step 206.)
• the system then allows the image to be viewed through an eyepiece or uses an imaging device to capture an image of the filtered emitted fluorescence (step 208).
• image-processing operations can include, but are not limited to: sharpening, magnification, computational superresolution, denoising, etc., and can be implemented using either traditional image -processing functions or through AI-based tools that can be trained to produce the desired image results.
• Color- mode-conversion functionality in which the native coloration seen in FIB I images is converted to faithfully mimic conventional H&E appearance constitutes an important component of such a set of operations.
• AI tools can be somewhat slow to apply to large images, and may not provide convenient real-time conversion.
• One strategy that we have developed is to train an AI system to perform a FIBI-to-H&E conversion, and then use the resulting converted image to provide color data, which is pixel-matched with the original-color FIB I image to train a non linear matrix color conversion operator that can be applied in near-real-time.
• Images can be analyzed or separated into multiple signal layers, which can highlight the presence and appearance of tissue components such as elastin, collagen, and the like, using a large variety of multispectral analysis techniques, which can include, but are not limited to: end-member linear unmixing, phasor analysis, convex hull analysis and non-parametric AI-enabled methods.

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• 2020
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