Thionin staining and imaging technique

• US 5,168,066 A
• Filed: 11/25/1991
• Issued: 12/01/1992
• Est. Priority Date: 03/07/1990
• Status: Expired due to Term

• Current Assignee: Cytyc Corporation (Hologic Incorporated)
• Original Assignee: Cytyc Corporation (Hologic Incorporated)
• Inventors: Oud, Petrus S., Lapen, Daniel C., Zahniser, David J.
• Primary Examiner(s): Housel, James C.
• Assistant Examiner(s): Snay, Jeffrey R.
• Application Number:
  US07/797,312
• Time in Patent Office: 372 Days
• Field of Search: 436/17, 436/63, 436/64, 436/166, 436/72, 436/174, 436/800, 436/813, 424/3, 424/7.1, 382/1, 382/6, 356/36, 356/51, 356/39
• US Class Current: 436/63
• CPC Class Codes: G01N 1/30 Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
• CPC Class Codes: Y10T 436/25 including sample preparation

Abstract

Cellular analysis in accordance with the invention involves staining cellular material with a thionin stain to distinguish the nuclear portion from the cytoplasm, counterstaining, illuminating the thionin-stained cellular material with energy in the infrared spectrum, and imaging the stained cellular material. The analysis can be automated or manual.

Claims

1. A method of staining cellular material for contrasting nuclear material from cytoplasmic material, for producing stained cellular material suitable for automated machine analysis, and for imaging the stained cellular material, said method comprising the successive steps of

staining the cellular material with an organic thionin stain having a predetermined pH in the range selected from about pH 4 to about pH 9 such that said thionin stain binds distinguishably more to said nuclear material than to said cytoplasmic material and attains a selected infrared absorbance when so bound with said nuclear material, said stain including a thionin salt and a pH buffer solution,

counterstaining the cellular material with a second stain having an infrared absorbance by cytoplasmic material which is distinguishably less than said infrared absorbance of nuclear material with said thionin stain,

illuminating the stained cellular material with energy in the infrared spectrum, and

imaging the stained cellular material in response to output infrared energy from the stained cellular material due to said infrared illumination.

2. The method of claim 1 wherein said imaging step includes detecting said output energy with an opto-electric transducer.

3. The method of claim 1 wherein said counterstaining step involves staining with a counterstain compound to produce stained cellular material wherein the cytoplasmic material is relatively transparent to said infrared illumination.

4. The method of claim 1 wherein the pH is acidic.

5. The method of claim 1 wherein said imaging step comprises digitizing said output energy.

6. The method of claim 5 further comprising the step of pathologically testing said digitized image information.

7. The method of claim 6 wherein said testing step comprises classifying said cellular material.

8. The method of claim 6 wherein said testing comprises a cytological analysis.

9. The method of claim 6 wherein said testing comprises a histological analysis.

10. The method of claim 1 wherein said staining step includes preparing a thionin stain consisting essentially of a thionin salt, an organic solvent, and a pH buffer.

11. The method of claim 10 wherein said organic solvent is selected from the group consisting of low molecular weight alcohols and DMSO.

12. The method of claim 11 wherein said organic solvent is selected from the group consisting of methanol, ethanol, isopropyl alcohol and DMSO.

13. A method of staining cellular material to contrast nuclear material from cytoplasmic material, thereby producing stained cellular material suitable for automated machine analysis and for imaging the stained cellular material, said method comprising the steps of

preparing a thionin stain consisting essentially of a thionin salt, an organic solvent, and a pH buffer, said thionin stain having a predetermined pH in the range selected from about pH 4 to about pH 9 such that said thionin stain binds distinguishably more to said nuclear material than to said cytoplasmic material and attains a selected infrared absorbance when so bound with said nuclear material,

staining the cellular material with said thionin stain for staining the nuclear material to have at least selected opacity to infrared energy and the cytoplasmic material to have a lesser opacity to infrared energy,

preparing a Papanicolaou-type counterstain consisting essentially of a first staining solution, a second staining solution, and alcoholic rinses,

wherein said first staining solution consists essentially of an orange dye, an alcoholic solvent, and pH buffer, and

wherein said second staining solution consists essentially of a green dye, a red dye, a pH buffer, and an alcoholic solvent,

counterstaining the cellular material, subsequent to said thionin-staining step, with said Papanicolaou-type counterstain,

illuminating the stained cellular material, subsequent to said counterstaining step, with energy in the infrared spectrum, and

imaging the output radiant infrared energy from said stained cellular material in response to said infrared illumination.

14. A method of staining cellular material to contrast the nuclear material from the cytoplasmic material, thereby producing stained cellular material suitable for automated machine analysis and for imaging the stained cellular material, said method comprising the steps of

preparing a thionin stain consisting essentially of a thionin salt, an organic solvent, and a pH buffer, said thionin stain having a predetermined pH in the range selected from about pH 4 to about pH 9 such that said thionin stain binds distinguishably more to said nuclear material than to said cytoplasmic material and attains a selected infrared absorbance when so bound with said nuclear material,

staining the cellular material with said thionin stain for staining the nuclear material to have at least selected opacity to infrared energy and the cytoplasmic material to have a lesser opacity to infrared energy,

preparing an eosin-type counterstain consisting essentially of an Eosin Y dye, an organic solvent, and a pH buffer,

counterstaining the cellular material, subsequent to said thionin-staining step, with said eosin-type counterstain,

illuminating the stained cellular material, subsequent to said counterstaining step, with energy in the infrared spectrum, and

imaging the output radiant infrared energy from said stained cellular material in response to said infrared illumination.

15. A method of staining cellular material for contrasting nuclear material from cytoplasmic material, for producing stained cellular material suitable for automated machine analysis and for imaging the stained cellular material, said method comprising the successive steps of

staining the cellular material with an organic thionin stain, having a predetermined pH in the range selected from about pH 4 to about ph 9 such that said thionin stain binds distinguishably more to said nuclear material than to said cytoplasmic material and attains a selected infrared absorbance when so bound with said nuclear material, said stain including a thionin salt and a buffer solution,

counterstaining the cellular material with a second stain having a near-infrared absorbance by cytoplasmic material which is distinguishably less than said near-infrared absorbance of nuclear material with said thionin stain,

illuminating the stained cellular material with energy in the near-infrared spectrum, and

imaging the stained cellular material in response to output energy from the stained cellular material due to said near-infrared illumination.