TWI835337B - A rapid fresh digital-pathology method - Google Patents

Abstract

A rapid fresh digital-pathology (RFP) method for assessing an excised unfixed biological specimen stained with hematoxylin (H) or eosin (E) or both hematoxylin and eosin (HE) staining dyes. The RFP method is assisted by a rapid tissue staining (RTS) procedure which is performed on the excised unfixed biological specimen, involving a short fixation; an H-staining; a rinsing; a bluing; an E-staining; a rinsing; and finally, a covering of a stained specimen with a coverslip. The RFP method is further assisted by a multimodal nonlinear optical laser-raster-scanning approach to provide with a nonlinear multi-harmonic generation and/or a nonlinear multi-photon excitation fluorescence signal(s) for multichannel digitization and real-time digital display of H- or E- or HE-specific histopathological features while providing a centimeter-scale imaging area, a submicron digital resolution, and a sustained effective data throughput of at least 500 Megabits per second (Mbps).

Description

A rapid fresh digital pathology method

The present invention relates to a rapid fresh digital pathology method (RFP); more specifically, it is a A fast and fresh digital pathology method that can assist pathologists in the practical use of H&E without additional training. Histopathological evaluation is performed on excised unfixed biological specimens stained with hematoxylin (H) or eosin (E) or hematoxylin and eosin (HE) dyes.

Although the formalin-fixed paraffin-embedded (FFPE) pathology method offers extremely high reliability, However, it is not suitable for intraoperative tumor assessment (ITA) because it requires processing time of up to 1–2 days. The frozen section (FS)-pathology method is currently the global standard for ITA due to its shorter sample processing and evaluation time. However, cold sectioning involves freezing, slicing, and staining samples, which requires considerable manpower and ultimately limits the feasible rounds of ITA since a single round process consumes up to 30 minutes in duration. Furthermore, FS biopsies are susceptible to several artifacts that may affect assessment. Therefore, an alternative to FS is urgently needed to be developed, which must be section-free, portable, fast, accurate, artifact-free, suitable for fresh samples, as reliable as FFPE biopsy, and most importantly, must be based on global gold standard pathological staining. Agent-H&E stain enables the technology to be more reliable without requiring any deep learning and/or additional training for pathologists.

In modern computer-assisted digital pathology specifications, whole slide imaging (WSI) and stitched panoramic virtual sectioning (VS) are rapidly developing, which help to digitally store billions of images of FS/FFPE tissue pathology slides , so that assessment can be restarted at any time, and further enables pathologists to quickly and remotely assess WSI-VS from any location. However, in order to maintain a high degree of diagnostic reliability based on digital methods, it is important to maintain good resolution along the path of the imaging pixels from the sample to the digital display system. According to the Society for Digital Pathology guidelines, WSI-VS with standard ×20 magnification can generally be used for standard observation and interpretation based on 0.5 micron (µm) digital resolution; standard ×40 magnification meets Nyquat It has a digital resolution of 0.25µm, while maintaining a color depth of 24 bits. Such a situation would require an area of 1 × 1 square millimeter (mm ² ) to contain ≥384M bits, which would extend to ≥38.4 G bits or 1.6 G pixels for an area of 1 square centimeter (cm ² ). For such ×40 or even ×20 objectives with high numerical aperture (NA, close to or greater than 1), the system field of view (FOV) is usually limited to less than 1mm ² , which requires a series of artifact-free splicing processes to avoid Will affect the reliability of diagnosis.

In the context of surgical pathology ITA, the primary need is rapid evaluation to minimize operative time and ensure patient safety. Some existing technologies associated with ITA-capable digital imaging modalities use optical virtual slides that enable faster evaluation, as opposed to the physical slides involved in FS/FFPE. However, existing technologies may not be able to achieve half-micron digital resolution with billion-pixel sampling that meets the Nyquist requirements of the latest WSI standards, and/or may not be able to achieve real-time large field of view, artifact-free stitching/mosaicing capabilities, and/or sub-minute billion-pixel acquisition and digital display capabilities with uncompromised resolution on the pixel path from "sample to digital display". In addition, when different nuclear stains are used internationally instead of the gold standard, hematoxylin, pathologists may need specific interpretation training to make interpretations. This important factor may lead to reduced accuracy compared to FS/FFPE biopsies. However, in order to optically section three-dimensional biological specimens with opaque nuclei stained with hematoxylin dyes, a nonlinear multiharmonic generation method is required to visualize high-contrast nuclei.

It is worth noting that some previously published physical sectioning-free fast ITA methods can cope with For use in certain specific surgical pathology applications. Stimulated Raman microscopy provides useful chemical information to examine cellular abnormalities; however, it often suffers from low signal-to-noise ratio (SNR) and may not be suitable for rapid detection of centimeter-sized specimens. Although optical interference tomography provides high-speed imaging, it still suffers from poor spatial resolution, poor signal-to-noise ratio, and poor contrast. Fluorescence optical microscopy, conjugate focus microscopy, and UV surface excitation microscopy are noteworthy, effective methods that enable optical sectioning; but may not be suitable for dyes that are not fluorescent, such as the gold standard hematoxylin , especially if the biological sample is inherently thick and opaque. In short, when performing optical sectioning and high-resolution, high-throughput laser scanning on HE-stained whole biological samples, the existing technology cannot meet the actual needs of all users.

This invention introduces a method called Rapid Fresh Digital Pathology or RFP for short. method, a digital ITA solution for whole-specimen surface imaging (WSSI) that provides 4x faster sample evaluation compared to global FS biopsy standards, with RFP compared to standards H and E similar to conventional FS- or FFPE-biopsies Dye compatible.

In order to achieve the above object, the present invention is a method for performing histopathology on excised unfixed biological samples stained with hematoxylin (H) or eosin (E) or hematoxylin and eosin (HE) stains. RFP methods evaluated include optical virtual slicing through multimodal nonlinear optical laser raster scanning to collect the generated nonlinear multiharmonic and/or nonlinear multiphoton excitation fluorescence signals for H or E or HE Multi-channel digitization and real-time digital display of specific histopathological characteristics; laser raster scanning single image field of view (FOV) of at least 1 square millimeter, effective digital lateral resolution less than 1 micrometer (μm), FOV resolution ratio greater than 1000 ; and the cumulative imaging area of a single image or a combination of multiple images in the range of 1 mm ² to 400 mm ² , with a continuous effective data input and output volume of at least 500M bits per second (Mbps).

The resected unfixed biological sample is an opaque intact volume of tissue without After fixation, freezing, physical sectioning and tissue transparency; it can come from liver tissue, breast tissue, pancreatic tissue, brain tissue, thymus tissue, prostate tissue, colon tissue, lymphoid tissue and solid tissue, etc.

In the RFP method, the Rapid Tissue Staining (RTS) process allows the resection of unfixed tissue. Samples were stained with H, E, or HE. Performs an RTS process on excised, unfixed biological specimens to place them into tissue chambers or tissue containers. Perform RTS procedures on resected unfixed biological samples, in which Use fixative to perform the fixation process; H staining procedure using Gill's hematoxylin solution and/or Mayer's hematoxylin solution; Use distilled water and/or cleaning fluid for the cleaning process; Blue treatment with ammonia solution; Use eosin solution to perform the E staining process; Perform a rinsing process with alcohol solutions and/or cleaning solutions; Cover the excised unfixed biological specimen with a coverslip. The RTS process consumes less than 6 minutes of HE staining time.

Multimodal nonlinear optical laser raster scanning is able to complete point scanning of a 10 × 10 mm ^area in less than 2 minutes while maintaining a pixel size of less than 170 nanometers (nm). The RFP method performs laser raster scanning of optical sections on the surface of a 1 cm ² sample. The staining time is less than 6 minutes and the scanning time is less than 2 minutes. Therefore, the total evaluation time provided for the resected unfixed biological samples is less than 8 minutes. minute.

Multimodal nonlinear optical laser raster scanning utilization (1) Fiber-based pulsed laser sources with emission spectra in the 1000 nm to 1100 nm range, where third harmonic generation (THG) signals from H staining are collected, and/or two-photons from E staining are collected Excited fluorescence (TPEF) signal; (2) Alternatively, a pulsed laser source of chromium forsterite (Cr:F) with an emission spectrum in the range of 1200 nm to 1300 nm, in which the THG signal is collected from H staining, and/or the three-photon excitation fluorescence is collected from E staining. Light (3PEF) signal.

The specific requirements for focusing objectives used in multi-modal nonlinear optical laser raster scanning are as follows: Objective lens with a numerical aperture of at least 0.7.

Multimodal nonlinear optical laser raster scanning utilizing resonant scanners or polygon scanning For fast-axis scanning, the device must provide a line rate of at least 4 kHz.

The cumulative imaging area obtained by combining multiple images is assisted by a two-dimensional electronic control platform unit. The electronic control platform unit is composed of at least two electronic linear translation platforms that are vertically connected to each other.

The following description of preferred embodiments is provided to understand the features and structure of the present invention. Please refer to Figures 1 to 4, which are respectively a schematic diagram representing the RFP method, a flow diagram representing the RFP method assisted by a multi-modal nonlinear optical laser grating scanning method, a workflow diagram representing the RTS method, and a schematic diagram representing an image example obtained using the RFP method. As shown in the figure: The present invention is an RFP method [1] for performing tissue pathology assessment on resected unfixed biological specimens stained with hematoxylin (H) or eosin (E) or hematoxylin and eosin (HE) stains, comprising: (a) optical virtual sections by multimodal nonlinear optical laser grating scanning [15] to provide specific tissue pathology features for multi-channel digitization and real-time digitization display of nonlinear multiharmonic and/or nonlinear multiphoton excited fluorescence signals H or E or HE [17], [27]; (b) a laser grating scanning single image field of view (FOV) of at least 1 ^mm2 [161], with an effective digital lateral resolution of less than 1 micrometer (μm) [163] and FOV resolution[164] greater than 1000; and (c) a cumulative imaging area[162] of 1 mm ² to 400 mm ² for a single image or a combination of images, with a continuous effective data throughput[165] of at least 500 Mbits per second (Mbps).

The resected unfixed biological specimen [11] is an opaque complete volume of tissue without fixation. Determination, freezing, physical sectioning, and tissue transparency can be obtained from liver tissue, breast tissue, pancreatic tissue, brain tissue, thymus tissue, prostate tissue, colon tissue, lymphoid tissue, and solid tissue, etc.

In the RFP method [1], the RTS [3] process can be performed on resected unfixed biological samples. Perform H or E or HE staining [13]. The RTS [3] process is performed on excised, unfixed biological specimens, which are placed into tissue chambers or tissue containers [12].

In Figures 2 and 3, the RFP [1] approach assisted by an RTS process of less than 6 minutes The excised tissue is placed in the space formed by the iSpacer with the microscope slide. The total time of the RTS [3] process is less than 6 minutes, including short-term fixation [131], two-stage H staining of cell nuclei [132], intermediate rinsing [133], bluening [134], and E staining [135] , final cleaning [136]. Once the RTS [3] process is complete, a coverslip is added [137] and imaged under a multimodal nonlinear optical laser raster scanning system [33] .

The RFP method [1] laser raster scanning of surface optical sections of a ¹ cm sample area consumes less than 6 minutes of staining time and less than 2 minutes of scanning time, so the total evaluation time of the resected unfixed biological specimen is less than 8 minutes.

It is worth noting that the multi-modal nonlinear optical laser grating scanning system [14] is used to detect The non-linear multi-harmonic and/or non-linear multi-photon excitation fluorescence signals generated by the measurement are used for multi-channel digitalization and real-time digital display of specific histopathological characteristics of H, E or HE [17].

The characteristics of the multi-modal nonlinear optical laser raster scanning system [14] include: (1) The laser raster scanning single image FOV [161] is at least 1 mm ² , the effective digit lateral resolution [163] is less than 1 μm and FOV resolution greater than 1000 [164]; (2) The total imaging area of a single or multiple images within the range of 1 mm ² to 400 mm ² [162], and the continuous effective data input and output volume [165] is at least per 500M bits per second (Mbps); (3) Perform and complete point scanning of a 10 × 10 mm ² area in less than 2 minutes [166] while maintaining a pixel size of less than 170 nanometers (nm). And it is worth noting that the multi-image combination process is performed through real-time mosaic stitching [16], [26] for centimeter-level laser scanning without post-processing.

The focusing objective lens used in multi-modal nonlinear optical laser grating scanning [23] has specific requirements. Find an objective lens with a numerical aperture of at least 0.7.

A multimodal nonlinear optical laser grating scanning system [14] utilizes a resonant scanner or multiple Edge scanners [22] perform fast-axis scanning and must provide a line rate of at least 4 kHz.

The cumulative imaging area obtained by combining multiple images is assisted by a two-dimensional electronic control platform unit [24] The electronic control platform unit is composed of at least two electronic linear translation platforms that are vertically connected to each other.

Multimodal nonlinear optical laser grating scanning system [14] utilizes (a) A fiber-based pulsed laser source [21] with an emission spectrum ranging from 1000 nm to 1100 nm, in which signals from two-photon excited fluorescence (TPEF) derived from H staining and/or E staining are collected [25 ];or (b) Pulsed laser source of chromium forsterite (Cr:F) [21] with an emission spectrum ranging from 1200 nm to 1300 nm, where third harmonic generation (THG) signals originating from H staining are collected and/or Three-photon excited fluorescence (3PEF) signal derived from E staining [25].

In Figure 4, an example image was obtained by performing RFP [1] on fresh human glioma tissue. Some specific histopathological features can be seen in the centimeter-sized images and their magnified images. (A) is a 1.3 Gigapixel color-converted RFP image. The scale bar is 0.5 mm and the FOV is 6.4×5.6 mm ² . After a total imaging time of 44 seconds, it has an effective pixel rate of approximately 30 M/s and an effective scan rate of greater than 1 mm ² /s. (B)-(I) are cropped and enlarged Regions of interest (ROI) labeled R1-8 in (A), where the number of pixels in each ROI is 6700×3300 and the scale bar is 150 μm. (J)-(Q) are cropped and enlarged Regions of interest (ROI) labeled R9-16 in (B)-(I), where the number of pixels in each ROI is 3200 × 2500 and the scale bar is 70 μm. The distribution, shape, and size of cell nuclei and other structural details such as blood vessels are visualized in (B)-(Q). White dashed curves mark vascular structures. White dashed circles show examples of areas of significant increased cell number. The atypical cell structure in some atypical blood vessels in (K), (M), (N), and (Q) is obvious, indicating the phenomenon of microvascular proliferation.

In the present invention, a method called Rapid Fresh Digital Pathology or RFP for short is introduced. method, a digital ITA solution for whole-specimen surface imaging that provides a 4x increase in evaluation speed compared to global FS biopsy standards, and the RFP is compatible with standard H and E stains similar to traditional FS/FFPE biopsies. Notably, this technology demonstrated 100% tumor interpretation accuracy in a no-training blind test involving a total of 50 human brain tissue samples (normal and glioma), demonstrating comparable accuracy to FS/FFPE. and 100% sensitivity and specificity.

In the present invention, the total time for dyeing, scanning and displaying an area of ^1cm2 is about 8 minutes, of which the rapid dyeing process takes less than 6 minutes, and the subsequent scanning and displaying process takes about 2 minutes. Through subsequent optimization of staining parameters, the cumulative evaluation time can be further reduced.

In the present invention, the RFP method is particularly applicable to resected fresh human brain tissue samples. To study tumor tissue. Especially for soft and fragile fresh brain samples, the RTS process uses traditional H&E stains. It is worth noting that RFP has great potential and can be applied to other types of biological samples, such as but not limited to breast, prostate, skin, etc., to provide fast, accurate and reliable ITA. The preferred embodiments disclosed herein are not intended to unnecessarily limit the scope of the invention. Therefore, any simple modifications or changes that fall within the scope of the claims and the scope disclosed in the patent specification are within the scope of the present invention.

[1]:RFP method

[11]: Resection of unfixed biological specimens

[12]: Place in tissue chamber or tissue container

[13]: Perform H or E or HE staining

[131]: Fixed for short time

[132]:H staining

[133]:rinse

[134]:Blue

[135]:E staining

[136]:rinse

[137]:Add coverslip

[14]: Multi-modal nonlinear optical laser grating scanning system

[15]: Multi-modal nonlinear optical laser raster scanning for optical virtual slicing

[16]: Multi-image combination through real-time mosaic stitching

[161]:View

[162]:Imaging area

[163]: Significant digit lateral resolution

[164]:FOV resolution

[165]: Valid data input and output amount

[166]: Point scan

[17]: Multi-channel digitization and real-time digitization display the specific histopathological characteristics of H or E or HE

[21]: Pulse laser source

[22]:Scanner

[23]: Focusing objective lens

[24]:Electronic control platform unit

[25]: Multi-modal nonlinear optical laser grating scanning system

[26]: Real-time mosaic stitching

[27]RTS[3]: Multi-channel digitization and real-time digitization display the specific histopathological characteristics of H or E or HE

[33]: Multi-modal nonlinear optical laser grating scanning system

Figure 1 is a schematic diagram showing the rapid fresh digital pathology (RFP) method. Figure 2 shows the flow of the RFP method assisted by the multi-modal nonlinear optical laser raster scanning method. Schematic diagram. Figure 3 is a schematic workflow diagram showing the rapid tissue staining (RTS) method. Figure 4 is a schematic diagram of an image example obtained using the RFP method.

[1]:RFP method

[11]: Resection of unfixed biological specimens

[12]: Place in tissue chamber or tissue container

[13]: Perform H or E or HE staining

[131]: Fixed for short time

[132]:H staining

[133]:rinse

[134]:Blue

[135]:E staining

[136]:rinse

[137]:Add coverslip

[14]: Multi-modal nonlinear optical laser grating scanning system

[15]: Multi-modal nonlinear optical laser raster scanning for optical virtual slicing

[16]: Multi-image combination through real-time mosaic stitching

[161]:View

[162]:Imaging area

[163]: Significant digit lateral resolution

[164]:FOV resolution

[165]: Valid data input and output amount

[166]: Point scan

[17]: Multi-channel digitization and real-time digitization display the specific histopathological characteristics of H, E or HE

Claims (11)

A rapid fresh pathology (RFP) method for histopathology of resected unfixed biological specimens stained with hematoxylin (H) or eosin (E) or hematoxylin and eosin (HE) stains Optical virtual sectioning via multimodal nonlinear optical laser raster scanning to collect nonlinear multiharmonic and/or nonlinear multiphoton excited fluorescence signals for multichannel digitization and real-time digitization Showing specific histopathological features of H or E or HE, wherein the multi-modal nonlinear optical laser raster scan completes point scanning of a 10×10 mm2 area in less than 2 minutes while maintaining less than Pixel size of 170 nanometers (nm); laser raster scanning single image field of view (FOV) of at least 1 square millimeter (mm ² ), effective digital lateral resolution less than 1 micron (μm), and FOV resolution ratio greater than 1000; The cumulative imaging area of a single image or a combination of multiple images ranges from 1mm ² to 400mm ² , and the continuous effective data input and output is at least 500M bits per second (Mbps). The RFP method according to item 1 of the patent application, is characterized in that the resected unfixed biological sample is an opaque complete volume of tissue, which has not been fixed, frozen, physically sectioned, or tissue transparent; and can be obtained from Liver tissue, breast tissue, pancreatic tissue, brain tissue, thymus tissue, prostate tissue, colon tissue, lymphoid tissue and solid tissue, etc. According to the RFP method described in item 1 of the patent application, the resected unfixed biological specimen is stained with H, E, or HE through a rapid tissue staining (RTS) process. According to the RFP method described in item 3 of the patent application, the RTS process is performed on the resected unfixed biological sample and puts it into a tissue space or tissue container. According to the RFP method described in item 3 of the patent application, the RTS process is performed on the resected unfixed biological sample, including the following steps: Use fixative for short-term fixation; use Gill's hematoxylin solution and/or Mayer's hematoxylin solution for H staining; rinse with distilled water and/or cleaning solution; use ammonia solution for blue treatment; use eosin solution for E staining; rinsing with alcohol solution and/or cleaning solution; and covering the excised unfixed biological specimen with a coverslip. According to the RFP method described in items 3 and 5 of the patent application, the RTS process consumes a total HE staining time of less than 6 minutes. According to the RFP method described in item 1 of the patent application, laser raster scanning of surface optical sections is performed on a sample area of 1 square centimeter. The staining time consumed is less than 6 minutes and the scanning time is less than 2 minutes. Therefore, it is suitable for resection. Unfixed biological samples provide a total evaluation time of less than 8 minutes. According to the RFP method described in item 1 of the patent application, the multi-modal nonlinear optical laser grating scanning utilizes: an optical fiber-based pulse laser source with an emission spectrum in the range of 1000nm to 1100nm, wherein the collection source is A third harmonic generation (THG) signal from H staining and/or a two-photon excited fluorescence (TPEF) signal derived from E staining; alternatively, a pulsed laser source of chromium forsterite (Cr:F) with an emission spectrum The range is 1200 nm to 1300 nm, where third harmonic generation (THG) signals originating from H staining and/or three-photon excited fluorescence (3PEF) originating from E staining are collected. According to the RFP method described in item 1 of the patent application, the specific requirement for the focusing objective lens used in the multi-modal nonlinear optical laser grating scanning is an objective lens with a numerical aperture of at least 0.7. According to the RFP method described in item 1 of the patent application, the multi-modal nonlinear optical laser grating scanning uses a resonant scanner or a polygon scanner to perform fast-axis scanning, which must provide a linear rate of at least 4 kHz. According to the RFP method described in item 1 of the patent application, the cumulative imaging area obtained by combining the multiple images is assisted by a two-dimensional electronic control platform unit. The electronic control platform unit is composed of at least two electronic devices that are vertically connected to each other. Composed of linear translation platform.