+

WO2018175565A1 - Système et procédé pour commander la profondeur d'imagerie dans les tissus à l'aide de la microscopie de fluorescence sous excitation aux ultraviolets après coloration avec des agents fluorescents - Google Patents

Système et procédé pour commander la profondeur d'imagerie dans les tissus à l'aide de la microscopie de fluorescence sous excitation aux ultraviolets après coloration avec des agents fluorescents Download PDF

Info

Publication number
WO2018175565A1
WO2018175565A1 PCT/US2018/023541 US2018023541W WO2018175565A1 WO 2018175565 A1 WO2018175565 A1 WO 2018175565A1 US 2018023541 W US2018023541 W US 2018023541W WO 2018175565 A1 WO2018175565 A1 WO 2018175565A1
Authority
WO
WIPO (PCT)
Prior art keywords
tissue
image
specimen
tissue sample
wavelength
Prior art date
Application number
PCT/US2018/023541
Other languages
English (en)
Inventor
Richard Levenson
Stavros Demos
Original Assignee
Lawrence Livermore National Security, Llc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US15/466,195 external-priority patent/US9964489B2/en
Application filed by Lawrence Livermore National Security, Llc filed Critical Lawrence Livermore National Security, Llc
Publication of WO2018175565A1 publication Critical patent/WO2018175565A1/fr

Links

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
    • 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
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/47—Scattering, i.e. diffuse reflection
    • G01N21/4795—Scattering, i.e. diffuse reflection spatially resolved investigating of object in scattering medium
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/00584—Control arrangements for automatic analysers
    • G01N35/00722—Communications; Identification
    • G01N35/00871—Communications between instruments or with remote terminals

Definitions

  • the present disclosure relates to systems and methods for structural and molecular imaging of human and animal tissues using fluorescence microscopy under short wavelength, such as ultraviolet light, excitation and more particularly to a system and method which is able to optically section thick tissue samples to obtain high-resolution images of the near-surface tissue microstructure without requiring formalin fixation and paraffin embedding (FFPE) followed by microtome sectioning, or freezing and sectioning of the tissue sample, and which further is able to be used with conventional or novel fluorescing stains and labels, to allow effective analysis of tissue for diagnosis, tissue composition, and/or surgical guidance, such as monitoring surgical margin areas of a biopsy specimen for the presence of tumor cells.
  • FFPE formalin fixation and paraffin embedding
  • Biopsy quality control is another area that the present disclosure aims to address. It is important that biopsies, especially small, relatively non-invasive needle biopsies, contain the tissue of interest. For renal diagnosis, needle biopsies must contain glomeruli; for cancer diagnosis, of course the lesion must be properly sampled, and so on. In the case of samples that have to be partitioned for various purposes— histopathology, flow cytometry, nucleic acid extraction, for example, it is desirable that each aliquot of tissue has the cells or structures of interest. Moreover, it is important in some cases to have an estimate of what percentage of tumor verses stroma may be represented. Having a non-destructive method of rapidly examining the biopsy tissue to ensure that the appropriate content is present would be useful.
  • the second main physical mechanism or characteristic is the use of native fluorophores within the cell compartments of the tissue being analyzed.
  • native fluorophores such as tryptophan, collagen, elastin, NADH
  • images based on the emission of contrast agents can be attained and can be combined with those of native fluorophores to provide additional molecular information.
  • the method may also comprise exciting, with an ultraviolet (UV) light source, the one or more different exogenous fluorophores with a first wavelength of UV light between about 200 nm and about 290 nm.
  • the method may further comprise collecting with an optical system, emissions from each of the one or more different exogenous fluorophores at a second wavelength different from the first wavelength of UV light, that being from about 350 nm to about 950 nm, and being generated in response to the first wavelength of UV light, to produce an image for analysis.
  • UV ultraviolet
  • Figure 2B is a montage of 10X-fields of eosin-stained lamb kidney
  • Figure 3A is an image of conventionally fixed and stained mouse heart muscle, viewed with a standard transmission light microscope.
  • Figure 3B shows fresh mouse heart muscle tissue stained with both eosin and the nuclear stain, DAPI, and excited with an LED with excitation light centered at 275 nm. The image was taken at 10X magnification with the system depicted, and the eosin and DAPI bands were separately collected using appropriate emission bandpass filters. The resulting images were recolored to simulate traditional bright-field transmission H&E staining. The insert indicates that nuclear features can be visualized.
  • Figure 6 shows the same thin tissue sample as shown in Figure 5 but imaged using the methodology of the present disclosure
  • Figures 7 and 8 further illustrate the dramatic improvement in image contrast in helping to distinguish collagen, a distal tubule and a proximal tubule provided by the Thin MUSE methodology of the present disclosure (Figure 7), versus the same image obtained through conventional H&E staining ( Figure 8);
  • Figure 20 shows the same tissue sample as Figure 19 but with the image having been produced using the Thin MUSE methodology, to illustrate the significantly enhanced nuclear definition over the image of Figure 19;
  • fluorescing stains and probes in combination with a methodology that does not require freezing and physical sectioning of tissue samples enables rapid imaging, typically enabling a wide-field-of-view and high-resolution image to be built up in a minute or less.
  • staining of the tissue with directly labeled antibodies or nucleic acid probes (which can be rapidly hybridized, such as with RNA fluorescence in situ hybridization ("Turbo RNA FISH.") will also be possible, and specific and non-specific staining could be readily distinguished by using a targeted and non-targeted probe simultaneously.
  • Turbo RNA FISH. RNA fluorescence in situ hybridization
  • pathologist-acceptable image quality is achievable. The image quality may even be suitable for primary diagnosis work.
  • the contrast agent used should be able to provide staining of subcellular and intracellular compartments of fresh tissue specimens (with or without brief exposure) to conditioning solutions that optimize staining, by for example, changing the pH, ionic strength, permeability of cells and subcellular structures, hydration state, solvent, protein and nucleic acid structure and cross-linking, antigen availability, and the like, to enable visualization of tissue microstructure and organization suitable for histopathologic diagnosis or characterization.
  • the contrast agents should absorb in the UV spectral range used for excitation and emit at a longer wavelength such as in the visible spectrum.
  • the contrast agents should stain the tissue upon physical exposure as fast as possible to minimize the processing time.
  • the contrast agent should not substantially alter or damage the macro- or microstructure of the tissue. The time of exposure of the tissue to the solution containing the contrast agent may be optimized.
  • a large specimen can be imaged with this general approach using various methods such as: a) stitching high-resolution area-illuminated images of smaller sections, as shown in Figure 2B; b) scanning UV light point-by-point using a scanning method; or c) using a line of UV light for line-scanning.
  • Compressive sensing using, for example, structured illumination and single-pixel detectors may also be suitable.
  • methods that can maintain resolution while viewing large fields-of-view can be employed, such as high-NA low-power lenses, high-pixel count cameras, and computational approaches that use information from multiple images to generate high- quality renderings.
  • the scanning time depends on a number of parameters related to the instrumentation such as the sensitivity of the detection system, the numerical aperture of the lens system, the transmission efficiency of any filters, the excitation intensity, the quantum efficiency of the cameras and/or detectors and concentration of the contrast agents. There are also limiting factors such as the maximum excitation intensity before photo-bleaching of the contrast agent, or actual tissue photodamage or tissue ablation.
  • the required spatial resolution plays a key role in the scanning speed. The signal-to-noise ratio during image acquisition should remain sufficiently high so that image quality is not impaired. As multiple images of different emission bands of each site may be needed, using multiple cameras or other methods for parallel image acquisition will enable faster scanning speeds. Alternatively, a standard full-color (RGB) sensor may also be used to decrease the number of exposures required, or various techniques for snap-shot multispectral imaging can be employed.
  • RGB full-color
  • the depth of the imaging zone can be controlled using the excitation wavelength of the signal from the UV illumination excitation source 12.
  • This imaging method involves using UV excitation to provide shallow penetration depth. But if the exact depth of the imaging zone must be controlled, the excitation wavelength must be precisely tuned to the proper wavelength.
  • the depth of the imaging zone is generally decreasing or remains approximately the same as the wavelength of the excitation light is tuned to shorter values. This is true from about 370 nm down to about 240 nm. Below about 240 nm there is a sharp decrease of the penetration depth as the wavelength is further decreased. Therefore, it is possible to choose the proper excitation wavelength in order to achieve a predetermined penetration depth. This is illustrated in Figure 2A.
  • the penetration depth will be less than it would be with the illumination excitation source 12 arranged at 90 degrees relative to the surface of the tissue sample 14.
  • the penetration depth decreases as the incidence angle 26 increases (i.e., moves towards 90 degrees relative to the surface of the tissue sample 14).
  • Additional information from the images can be obtained using multiple oblique illumination sources arranged radially around the optical axis, including shape- from-shading analysis, as suggested by the image shown in Figure 2B.
  • the image of Figure 3B was acquired in about one minute- including the staining of the tissue specimen. Since the image of Figure 3B is a digital image, it can be transmitted immediately via wired or wireless digital transmission subsystems to a remote diagnostic facility or hospital. If a wireless communication link is employed, the images could be relayed virtually immediately to a pathologist located anywhere in the world. In addition, the tissue specimen remains intact, apart from superficial staining or other surface modifications from brief exposure to various solutions because no physical sectioning apart from possible bisecting to provide a flat face for imaging is required for the analysis to be performed. [0083] In Figure 3B a larger concentration of nuclei 102 are visible in the image due to imaging a thicker tissue section.
  • the present system and method thus enables much more rapid analysis and evaluation of tissue specimens in way that has limited impact on the integrity of the tissue specimen, which can be available for downstream processing, including standard FFPE-based histology, extraction of genetic material or other procedures.
  • the Thin MUSE methodology described herein can significantly mimic or improve on other histochemical stains for specific structural or molecular features such as certain collagen types, basement membrane, elastin, infectious organisms including amebae and fungi, lipids, Nissl substance, mucin and others.
  • the benefits of the Thin MUSE methodology described herein include the potential for standardized tissue preparation. It can be expected that contrast will be different on FFPE-processed slides compared to simply fixed or fresh thick tissues imaged with the MUSE methodology described herein, due to changes induced by the entire conventional histology process, including dehydration and sequential exposure to alcohols and anhydrous solvents such as xylene.
  • the tissue section after microtomy can be positioned directly on a UV-transparent support, such as a plastic or fused-silica slide, in which case the coverslip can be made of glass.
  • a UV-transparent support such as a plastic or fused-silica slide
  • the coverslip can be made of glass. This is less preferable because it is more difficult to perform high-resolution imaging through an optical material as thick as a typical glass slide ( ⁇ 1 mm) as opposed to a coverslip (often 170 microns).

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention concerne un procédé d'analyse d'un échantillon de tissu mince et conçu pour être supporté sur une lame. L'échantillon de tissu peut être placé sur une lame et exposé à un ou plusieurs fluorophores exogènes différents excitables dans une plage d'environ 300 nm à 200 nm, présentant une bande d'émission utile variant d'environ 350 nm à 900 nm et comprenant un ou plusieurs colorants fluorescents ou sondes moléculaires marquées par fluorescence qui s'accumulent dans les composants tissulaires ou cellulaires. Les fluorophores peuvent être excités par une première longueur d'onde de lumière UV comprise entre environ 200 nm et 290 nm. Un système optique collecte les émissions provenant des fluorophores à une seconde longueur d'onde, différente de la première longueur d'onde, émissions qui sont générées en réponse à la première longueur d'onde de lumière UV, pour produire une image à des fins d'analyse.
PCT/US2018/023541 2017-03-22 2018-03-21 Système et procédé pour commander la profondeur d'imagerie dans les tissus à l'aide de la microscopie de fluorescence sous excitation aux ultraviolets après coloration avec des agents fluorescents WO2018175565A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US15/466,195 2017-03-22
US15/466,195 US9964489B2 (en) 2014-09-16 2017-03-22 System and method for controlling depth of imaging in tissues using fluorescence microscopy under ultraviolet excitation following staining with fluorescing agents

Publications (1)

Publication Number Publication Date
WO2018175565A1 true WO2018175565A1 (fr) 2018-09-27

Family

ID=63586561

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2018/023541 WO2018175565A1 (fr) 2017-03-22 2018-03-21 Système et procédé pour commander la profondeur d'imagerie dans les tissus à l'aide de la microscopie de fluorescence sous excitation aux ultraviolets après coloration avec des agents fluorescents

Country Status (1)

Country Link
WO (1) WO2018175565A1 (fr)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110160958A (zh) * 2019-05-23 2019-08-23 佛山科学技术学院 一种光学相干层析成像装置、成像系统以及成像方法
WO2021067726A1 (fr) * 2019-10-03 2021-04-08 The Regents Of The University Of California Imagerie en fond clair imitant la fluorescence
CN112698503A (zh) * 2020-08-11 2021-04-23 江苏省海洋水产研究所 一种显微染色鉴别肝肠胞虫的方法及其专用装置
CN114279991A (zh) * 2021-12-30 2022-04-05 宜宾五粮液股份有限公司 白酒品牌鉴定的方法
CN115746840A (zh) * 2022-11-28 2023-03-07 陕西科技大学 一种黄色碳量子点石油醚荧光探针溶液、制备方法及应用
WO2023061068A1 (fr) * 2021-10-12 2023-04-20 The Hong Kong University Of Science And Technology Tomographie par sectionnement à excitation par ultraviolets rapide translationnelle assistée par apprentissage profond
CN117070213A (zh) * 2023-08-21 2023-11-17 扬州大学 一种基于荧光碳点的金胺o响应型比率型荧光探针及可视化试纸条及其用途

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090052021A1 (en) * 2005-09-26 2009-02-26 Hideo Mogami Microscopic Cell Observation and Inspection System Using a Plurality of Observation Methods
US20110117025A1 (en) * 2008-05-20 2011-05-19 Ralph Sebastian Dacosta Device and method for fluorescence-based imaging and monitoring
WO2011072401A1 (fr) * 2009-12-18 2011-06-23 University Health Network Système et procédé d'imagerie par fluorescence subsuperficielle
US20110224574A1 (en) * 2008-11-13 2011-09-15 Sadler John W Methods and systems for tissue processing and imaging
US20160077007A1 (en) * 2014-09-16 2016-03-17 Lawrence Livermore National Security, Llc System and method for controlling depth of imaging in tissues using fluorescence microscopy under ultraviolet excitation following staining with fluorescing agents

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090052021A1 (en) * 2005-09-26 2009-02-26 Hideo Mogami Microscopic Cell Observation and Inspection System Using a Plurality of Observation Methods
US20110117025A1 (en) * 2008-05-20 2011-05-19 Ralph Sebastian Dacosta Device and method for fluorescence-based imaging and monitoring
US20110224574A1 (en) * 2008-11-13 2011-09-15 Sadler John W Methods and systems for tissue processing and imaging
WO2011072401A1 (fr) * 2009-12-18 2011-06-23 University Health Network Système et procédé d'imagerie par fluorescence subsuperficielle
US20160077007A1 (en) * 2014-09-16 2016-03-17 Lawrence Livermore National Security, Llc System and method for controlling depth of imaging in tissues using fluorescence microscopy under ultraviolet excitation following staining with fluorescing agents

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110160958A (zh) * 2019-05-23 2019-08-23 佛山科学技术学院 一种光学相干层析成像装置、成像系统以及成像方法
CN110160958B (zh) * 2019-05-23 2024-04-30 佛山科学技术学院 一种光学相干层析成像装置、成像系统以及成像方法
WO2021067726A1 (fr) * 2019-10-03 2021-04-08 The Regents Of The University Of California Imagerie en fond clair imitant la fluorescence
US11808703B2 (en) 2019-10-03 2023-11-07 The Regents Of The University Of California Fluorescence imitating brightfield imaging
CN112698503A (zh) * 2020-08-11 2021-04-23 江苏省海洋水产研究所 一种显微染色鉴别肝肠胞虫的方法及其专用装置
WO2023061068A1 (fr) * 2021-10-12 2023-04-20 The Hong Kong University Of Science And Technology Tomographie par sectionnement à excitation par ultraviolets rapide translationnelle assistée par apprentissage profond
CN114279991A (zh) * 2021-12-30 2022-04-05 宜宾五粮液股份有限公司 白酒品牌鉴定的方法
CN114279991B (zh) * 2021-12-30 2023-05-12 宜宾五粮液股份有限公司 白酒品牌鉴定的方法
CN115746840A (zh) * 2022-11-28 2023-03-07 陕西科技大学 一种黄色碳量子点石油醚荧光探针溶液、制备方法及应用
CN115746840B (zh) * 2022-11-28 2024-02-02 陕西科技大学 一种黄色碳量子点石油醚荧光探针溶液、制备方法及应用
CN117070213A (zh) * 2023-08-21 2023-11-17 扬州大学 一种基于荧光碳点的金胺o响应型比率型荧光探针及可视化试纸条及其用途

Similar Documents

Publication Publication Date Title
US9964489B2 (en) System and method for controlling depth of imaging in tissues using fluorescence microscopy under ultraviolet excitation following staining with fluorescing agents
AU2015318251B2 (en) System and method for controlling depth of imaging in tissues using fluorescence microscopy under ultraviolet excitation following staining with fluorescing agents
WO2018175565A1 (fr) Système et procédé pour commander la profondeur d'imagerie dans les tissus à l'aide de la microscopie de fluorescence sous excitation aux ultraviolets après coloration avec des agents fluorescents
Fereidouni et al. Microscopy with ultraviolet surface excitation for rapid slide-free histology
EP3414553B1 (fr) Procédé et appareil d'imagerie d'échantillons de tissu non sectionnés
JP5540102B2 (ja) 高められた病理学的決定のための複数モダリティコントラストおよび明視野コンテキスト表現、および組織内の複数検体検出
TWI753448B (zh) 分析組織標本的方法
EP3164689B1 (fr) Nouveaux procédés de traitement de tissus pour imagerie profonde desdits tissus
JP6074427B2 (ja) 蛍光画像を用いて明視野画像を生成するためのシステム及び方法
Yoshitake et al. Direct comparison between confocal and multiphoton microscopy for rapid histopathological evaluation of unfixed human breast tissue
US11808703B2 (en) Fluorescence imitating brightfield imaging
US11940359B2 (en) Preparation of tissue sections using fluorescence-based detection
US20170108414A1 (en) High-resolution three-dimensional imaging of mammalian hearts
EP3785008A1 (fr) Imagerie tridimensionnelle de tissu de grade histologique à l'aide d'un microscope à excitation de surface ultraviolette
JP7231345B2 (ja) 蛍光に基づく検知を使用する組織切片の調製
JP2024505077A (ja) 蛍光ベースの検出を使用した包埋組織試料の分析
Dey Fluorescence microscope, confocal microscope and other advanced microscopes: basic principles and applications in pathology
US12298210B2 (en) Preparation of tissue sections using fluorescence-based detection
Yoshitake Nonlinear Microscopy System and Protocol for Rapid Evaluation of Freshly Excised Human Tissue
Wu et al. High-throughput, nondestructive, and low-cost histological imaging with deep-learning-assisted UV microscopy
Paul Development and Evaluation of Whole Slide Hyperspectral Confocal Fluorescence and Brightfield Macroscopy

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18770740

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18770740

Country of ref document: EP

Kind code of ref document: A1

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载