System and method for controlling depth of imaging in tissues using fluorescence microscopy under ultraviolet excitation following staining with fluorescing agents

Patent metadata

Field	Value
Publication number	US-2017191937-A1
Application number	US-201715466195-A
Country	US
Kind code	A1
Filing date	Mar 22, 2017
Priority date	Sep 16, 2014
Publication date	Jul 6, 2017
Grant date	—

Abstract

A method is disclosed for analyzing a thin tissue sample and adapted to be supported on a slide. The tissue sample may be placed on a slide and exposed to one or more different exogenous fluorophores excitable in a range of about 300 nm-200 nm, and having a useful emission band from about 350 nm-900 nm, and including one or more fluorescent dyes or fluorescently labeled molecular probes that accumulate in tissue or cellular components. The fluorophores may be excited with a first wavelength of UV light between about 200 nm-290 nm. An optical system collects emissions from the fluorophores at a second wavelength, different from the first wavelength, which are generated in response to the first wavelength of UV light, to produce an image for analysis.

First claim

What is claimed is:

1 . A method for imaging with enhanced contrast thin, flat tissue specimens with a micron-scale thickness and being supported on at least one of a glass surface or a UV-transparent surface, the method comprising:
immobilizing a sectioned tissue sample; exposing the sectioned tissue sample to one or more different exogenous fluorophores excitable in a range of about 300 nm to about 200 nm and having a useful emission band from about 350 nm to about 900 nm, and including one or more fluorescent dyes or fluorescently labeled molecular probes that accumulate in tissue or cellular components; 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; 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, 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.

2 . The method of claim 1 , wherein the second wavelength of excitation light has a wavelength from about 350 nm to about 950 nm.

3 . The method of claim 1 , wherein, prior to staining, the tissue sample is prepared through a process including fixation, dehydration, and infiltration with a cutting support compound, and sectioning a tissue specimen with a microtome to provide the tissue sample, the tissue sample having a uniform thickness, and mounting the tissue sample on a support, and rehydration of the tissue sample.

4 . The method of claim 1 , wherein, prior to staining, the tissue sample is prepared through a process including cryo-sectioning, the cryo-sectioning including freezing a tissue specimen and supporting it in freezing compound within a cryotome, thin-sectioning using a cutting device to create the tissue sample, and mounting the tissue sample onto the glass slide.

5 . The method of claim 4 , further comprising post-sectioning processing of the tissue sample, prior to staining.

6 . The method of claim 5 , wherein the post-sectioning processing involves exposure to solutions containing at least one constituent from the list, formaldehyde, paraformaldehyde, glutaraldehyde, ethanol, methanol, acetic acid, acetone.

7 . The method of claim 1 , further comprising, prior to staining, sectioning a tissue specimen to obtain the tissue sample by using sectioning instrument to provide the tissue sample with a uniform thickness.

8 . The method of claim 1 , further comprising preparing a smear or other thin distribution of non-solid samples containing at least one of cells, tissue elements, foreign material, parasites, either prior to or after staining.

9 . The method of claim 1 , wherein a surface of the tissue sample not in contact with the glass slide is uncovered.

10 . The method of claim 1 , wherein a surface of the tissue sample not in contact with the glass slide is covered by a UV transparent material through which excitation is able to occur.

11 . The method of claim 1 , wherein a surface of the tissue sample not in contact with the glass slide is covered by a coverslip.

12 . The method of claim 11 , wherein the coverslip is comprised of at least one of: quartz, fused silica, sapphire or UV-transparent plastic, such as cyclic olefin copolymers, TPX® polymethylpentene or the like.

13 . The method of claim 11 , wherein the coverslip is comprised of a UV-transmissive liquid coverslip material that dries or hardens into an optically clear and flat surface.

14 . The method of claim 1 , wherein the image produced for analysis comprises more than two colors, and;
further converting the image from a more than two color image to a two color, pseudo-H&E stained image.

15 . The method of claim 1 , in which the more-than-two color image is converted into a different color space by remapping into an H&E-like overall appearance supplemented by additional colors for specific contrast.

16 . The method of claim 1 , further comprising using multiple excitation sources and excitation spectroscopy to enhance detection and separability of the emissions from the exogenous fluorophores.

17 . The method of claim 1 , wherein the one or more fluorescent dyes or fluorescently labeled molecular probes are selected to enhance contrast of tissue or cell components viewed under a microscope, and preferentially bind to of at least one of:
subcellular organelles; lipids; extracellular tissue constituents including at least one of connective tissue including collagen and extracellular matrix; cyst contents; foreign bodies; infectious agents; pigments; exogenous marking dyes for orientation; in a case of molecular probes:
proteins;
post-translational modifications;
DNA or RNA sequences including genes, chromosomal regions or DNA constituents;
RNA transcripts, coding and non-coding; and
lipid rafts.

18 . The method of claim 1 , wherein the one or more dyes or fluorescently labelled molecular probes comprise at least one of the following:
dyes conjugated to molecular probes including at least one of antibodies, peptides, affibodies, DNA and RNA-targeting reagents.

19 . The method of claim 1 , in which the one or more different exogenous fluorophores include histological or histochemical fluorescent dyes and include at least one of: Eosin dye family, toluidine blue O, methylene blue, DAPI, Acridine Orange, DRAQ 5, Hoechst 33342 and 33528, calcein-AM, propidium iodide, Nile Blue, Nile Red, Oil Red O, Congo Red, Fast Green FCF, DiI, DiO, DiD, TOTO® dye, YO-PRO® dye, Neutral Red, Nuclear Fast Red, Pyronin Y, acid fuchsin, astrazon-family dyes, MitoTracker dye, mitochondrial dyes, LysoTracker dye, lysosome dye, safranine dyes, thioflavine dyes, fluorescent phalloidins, plasma membrane stains, calcofluor white or fluorescent compounds that bind to infectious agents.

20 . A method for imaging with enhanced contrast a tissue specimen having a micron-scale thickness, the method comprising:
obtaining a specimen as at least one of:
a microtome sectioned, support-matrix infiltrated specimen;
a cryotome-sectioned frozen tissue specimen;
a vibratome-sectioned fresh or fixed tissue specimen;
a cytology specimen;
a tissue culture preparation; or
a blood sample;
supporting the specimen on at least one of a glass support or a UV-transparent support; immobilizing the specimen and exposing the specimen to one or more different exogenous fluorophores excitable in a range of about 300 nm to about 200 nm and having a useful emission band from about 350 nm to about 900 nm, and including one or more fluorescent dyes or fluorescently labeled molecular probes that accumulate in tissue or cellular components; 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; and 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, 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.

21 . The method of claim 20 , further comprising prior to exciting with an ultraviolet (UV) light source the one or more different exogenous fluorophores, washing off unbound portion

Assignees

• L Livermore Nat Security Llc

Inventors

• Levenson Richard
• Demos Stavros

Classifications

• G01N21/6458 Primary

  Fluorescence microscopy (fluorescence microscopes per se G02B21/0076 and G02B21/16) · CPC title

• G01N33/52

  Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper {and including single- and multilayer analytical elements (immunological elements G01N33/54386; involving labelled immunochemicals G01N33/58; for haemoglobin or occult blood G01N33/72)} · CPC title

• G01N21/6428

  Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53) · CPC title

• G01N21/6445

  Measuring fluorescence polarisation · CPC title

• A61B5/0071

  by measuring fluorescence emission · CPC title