# US 2013/0157261 A1 · Compositions and Methods for Quantitative Histology, Calibration of Images in Fluorescence Microscopy, and ddTUNEL Analyses

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- Number: US 2013/0157261 A1
- Document ID: US20130157261A1
- Status: Published application
- Kind: A1
- Application number: 13/487,058
- Filed: 2012-06-01
- Published: 2013-06-20
- Earliest claimed: 2011-06-01
- Assignees: The Methodist Hospital Research Institute
- Inventors: Martyn Alun Sharpe; David S. Baskin
- Web page: https://patentgrep.com/patent/US20130157261A1

## CPC

- G01N 21/6428: {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)}
- C12Q 1/68: involving nucleic acids
- G01N 21/6458: {Fluorescence microscopy (fluorescence microscopes per se G02B21/0076 and G02B21/16)}
- G01N 33/53: Immunoassay; Biospecific binding assay; Materials therefor
- G01N 33/567: utilising isolate of tissue or organ as binding agent
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

Disclosed are compositions and methods for quantitation and calibration of images in fluorescence microscopy. Also provided are tissue phantoms that contain known amount(s) of fluorophore standard(s), as well as components and diagnostic kits containing the same for use in various histological analyses. In certain embodiments, three distinct nucleic-acid based assays provide improvements over conventional TUNEL methods to facilitate precise quantitation of a variety of nucleic acids obtained from a biological sample.

## Claims

### Claim 1

A tissue phantom comprising gelatin that is operably linked to at least a first detection moiety.

### Claim 2 (depends on 1)

The tissue phantom of claim 1, wherein the gelatin is porcine skin gelatin.

### Claim 3 (depends on 1)

The tissue phantom of claim 1, wherein the gelatin is covalently-linked to the first detection moiety using one or more amine-reactive crosslinking agents attached to a first calibration standard.

### Claim 4 (depends on 3)

The tissue phantom of claim 3, wherein the first calibration standard comprises a chromophoric dye, a first fluorophoric dye, a first oligonucleotide, a first protein, a first peptide, a first enzyme, a first antibody, or any combination thereof.

### Claim 5 (depends on 1)

The tissue phantom of claim 1, wherein the amine-reactive crosslinking agent is suberic acid bis(N-hydrosuccinimide ester, or a derivative or analog thereof.

### Claim 6 (depends on 1)

The tissue phantom of claim 1, wherein the concentration of gelatin is about 7.5% to about 20%.

### Claim 7 (depends on 4)

The tissue phantom of claim 4, wherein the first peptide, the first protein, the first enzyme, or the first antibody may be directly crosslinked to the gelatin by paraformaldehyde fixation.

### Claim 8 (depends on 1)

The tissue phantom of claim 1, wherein the first calibration standard is adapted and configured for use in UV, visible, fluorescence, or epifluorescence microscopy.

### Claim 9 (depends on 1)

The tissue phantom of claim 1, adapted and configured for use on a microscope slide or in one or more wells of a multi-well assay plate.

### Claim 10 (depends on 9)

The tissue phantom of claim 9, wherein the microscope slide comprises a population of distinct tissue phantoms each of which comprises a different, known quantity of the first calibration standard.

### Claim 11 (depends on 1)

The tissue phantom of claim 1, further comprising a second distinct detection moiety, or a second distinct calibration standard.

### Claim 12 (depends on 9)

The tissue phantom of claim 9, wherein the presence of the population of distinct tissue phantoms comprising differing, but known quantities, of the first calibration standard permits quantitation of one or more selected compounds of interest within a specimen, a sample, a tissue, a cell, or any combination.

### Claim 13 (depends on 4)

The tissue phantom of claim 4, wherein the first calibration standard comprises 6-FITC.

### Claim 14 (depends on 4)

The tissue phantom of claim 4, wherein the first calibration standard comprises a Cy3-labeled protein, a Cy3-labeled antibody or antigen binding fragment, or any combination thereof.

### Claim 15 (depends on 1)

An article of manufacture comprising the tissue phantom of claim 1.

### Claim 16 (depends on 16)

The article of manufacture of claim 16, comprising a plurality of distinct tissue phantoms, each of which includes a distinct, known amount of the first calibration standard, adapted and configured to facilitate the generation of a standard curve to quantitate one or more selected compounds of interest within a specimen, a sample, a tissue, a cell, or any combination thereof.

### Claim 17 (depends on 15)

The article of manufacture of claim 15, defined as a cuvette, a cell culture plate, a microscope slide, a microtiter dish, or a multi-well assay plate.

### Claim 18

A method of modifying a terminal deoxynucleotidyl transferase nick end labeling assay, comprising substituting a 3′ dideoxy UTP substrate for a dUTP substrate under conditions effective to permit quantitation of the —OH 3′ ends present in an assayed biological sample suspected of containing a population of nucleic acids.

### Claim 19 (depends on 18)

The method of claim 18, wherein the presence of the ddUPT permits the stoichiometric addition of a single label at each original —OH 3′ end present in the sample.

### Claim 20

A method of quantitating —PO4 3′ ends in a nucleic acid molecule, comprising using calf intestinal alkaline phosphatase to convert the —PO4 3′ ends to —OH 3′ ends, and then assaying the converted —OH 3′ ends using a ddTUNEL assay.

### Claim 21 (depends on 20)

The method of claim 20, further comprising oxidizing or acetylating at least a first nucleobase of the polynucleotide molecule, comprising contacting the sample with an effective amount of formamidopyrimidine DNA glycosylase (Fpg).

### Claim 22 (depends on 21)

The method of claim 21, comprising the further step of treating the resulting oxo-species by borohydride reduction or by derivatization with 2,4-dinitrophenyl hydrazine (DNP-H).

### Claim 23

A method of visualizing reactive oxygen species damage within a biological cell, wherein one or more 2,4-dinitrophenyl hydrazine-derivatized oxo-species are localized in the cell by detecting the presence of a labeled anti-DNP antibody.

### Claim 24 (depends on 1)

A method of interrogating cell death within one or more cells present in a biological sample, comprising performing one or more ddTUNEL, CIAP-ddTUNEL, Fpg-ddTUNEL assays using a signal-calibrated tissue phantom in accordance with claim 1, under conditions effective to monitor the level of apoptosis in one or more such cells.

### Claim 25 (depends on 24)

The method of claim 24, wherein the step of monitoring includes epifluorescence microscopy.

### Claim 26 (depends on 25)

The method of claim 25, wherein the epifluorescence microscopy is adapted and configured with one or more optical filter blocks that include: a) a DAPI channel, b) an FITC channel, c) a Texas Red channel, or any combination thereof.

### Claim 27 (depends on 25)

The method of claim 25, wherein a) the DAPI channel is adapted and configured for the detection of a biological probe that comprises 4′,6-diamidino-2-phenylindole; b) the FITC channel is adapted and configured for the detection of a biological probe that comprises an amine-reactive fluorescein derivative; or c) the Texas Red channel is adapted and configured for the detection of a biological probe that comprises sulforhodamine 101 acid chloride or a derivative or analog thereof.

### Claim 28 (depends on 27)

The method of claim 27, wherein the amine-reactive fluorescein derivative is fluorescein isothiocyanate, Alexa Fluor 488, or DyLight488, or any combination thereof.