Method of determining a nucleotide sequence of a nucleic acid segment from a region of interest in a biological sample

1. A method for determining a nucleotide sequence of a nucleic acid segment present in a biological sample, comprising the steps of:

a) generating one or more first fluorescent images of said sample by a protocol comprising immunofluorescence detection of at least five different target proteins in said sample by repeated cycles of i) immunofluorescence staining, ii) capture of an immunofluorescent image, and iii) bleaching of the sample, and generating a plurality of second fluorescent images of said sample by the repeated cycles of i)-iii);

b) selecting a region of interest of said sample by comparing said one or more first fluorescent images and second fluorescent images to a pattern of expression levels for the different proteins, wherein the pattern is qualitative, semiquantitative, or quantitative;

c) removing a subsample from said region of interest, and;

d) determining a nucleotide sequence of a nucleic acid segment present in said subsample,

wherein each of the plurality of second fluorescent images are generated by a protocol comprising immunofluorescence detection of at least one of the at least five target proteins in said sample and fluorescence detection of at least one morphological feature in said sample, and

wherein each of the plurality of second fluorescent images separately capture each of the at least five different target proteins.

2. The method of claim 1 , wherein in step a) said one or more first images comprise a composite image obtained by overlaying a plurality of immunofluorescent images of said sample.

3. The method of claim 1 , wherein said immunofluorescence staining comprises contacting the sample with an antibody against one of the at least five different target proteins, conjugated with a fluorophore.

4. The method of claim 3 , wherein said fluorophore is a cyanine dye comprising Cy3 or Cy5.

5. The method of claim 1 , wherein said bleaching comprises contacting said sample with a peroxide or an organic borate.

6. The method of claim 1 , wherein the sample is further stained with a fluorescent marker that provides morphological information.

7. The method of claim 6 , wherein signals from said fluorescent marker are used to provide location information for the overlaying of said immunofluorescent images.

8. The method of claim 6 , wherein said fluorescent marker is a nucleus stain comprising 4′,6-diamidino-2-phenylindole.

9. The method claim 1 , wherein said at least five different target proteins comprise at least one of EGFR, HER2, HER3, cMET, PI3K, phosphorylated AKT, S6, phospho ERK1/2, PCK26, AE1 and NaKATPase.

10. The method of claim 1 , wherein step a) further comprises fluorescence in situ hybridization staining of said sample followed by capture of a fluorescent hybridization image and overlaying of said fluorescent hybridization image with said plurality of immunofluorescent images.

11. The method of claim 10 , wherein said fluorescence in situ hybridization staining comprises contacting the sample with an oligonucleotide capable of hybridizing with a target nucleic acid region in said sample, conjugated with a fluorophore.

12. The method of claim 11 , wherein said target nucleic acid region is cMYC or CEP8.

13. The method of claim 1 , wherein said biological sample is affixed to a solid support.

14. The method of claim 13 , wherein said solid support is a microscope slide having a positively charged surface.

15. The method of claim 13 , further comprising detecting an autofluorescence intensity of the solid support, wherein said solid support has the autofluorescence intensity of less than 1% of an average immunofluorescence intensity over a region of interest in said sample.

16. The method of claim 1 , wherein in step c) said subsample is removed using laser microdissection or micromilling.

17. The method of claim 1 , wherein a circle-equivalent diameter of said subsample is less than 2 mm.

18. The method of claim 1 , wherein in step d) said nucleotide sequence is determined using ion semiconductor sequencing or Illumina dye sequencing.

19. The method of claim 1 , wherein in step d) said nucleotide sequence is determined directly from said subsample.

20. The method of claim 1 , wherein step d) is preceded by amplification of said nucleic acid segment.

21. The method of claim 1 , wherein said biological sample is a tissue section.