Methods for generating an image of a biological sample
A method for generating an image of a region of interest in a biological sample comprising the steps of: generating a first image including the region of interest of the biological sample having undergone a first protocol but not a second protocol; and generating a second image including the region of interest of the biological sample after having undergone a second protocol; wherein the region of interest is smaller than said sample. Also provided is a method of analyzing a biological sample, comprising providing an image of the biological sample according to the method for generating an image of a region of interest in a biological sample, and analyzing the biological sample from the image. Further provided are system and kit that comprise the means for executing the novel methods.
1. A method of generating a composite image of a region of interest in a human tissue sample comprising the steps of:
1) generating a first image including said region of interest of said sample having undergone a first protocol but not a second protocol, wherein the first protocol comprises: (i) binding a human target protein-specific monoclonal antibody labeled with a fluorophore to the human target protein in the sample, wherein said human target protein is selected from the group consisting of EGFR, Her2, ALK, galactosyl transferase II, neuron specific enolase, proton ATPase-2, acid phosphatase, Ki67, cyclin E, p53 and cMet; (ii) detecting by immunofluorescence the bound fluorophore to generate the first image, and (iii) staining the sample with a fluorescent marker that provides morphological information, and;
2) digesting said sample with a protease after generating the first image but before said sample has undergone the second protocol;
3) generating a second image including said region of interest of said sample after having undergone the second protocol, wherein the second protocol comprises: (i) hybridizing a nucleic acid probe labeled with a fluorophore to a target nucleic acid in the sample, wherein the target nucleic acid encodes a selective portion of the human target protein; and (ii) detecting by immunofluorescence the hybridized probe fluorophore to generate the second image wherein the nucleic acid probe is a DNA or RNA of from 4 to 50 nucleotides; and
4) generating a composite image that includes at least the region of interest from each of the first and the second images by registering fluorescent signals from the first image with fluorescent signals from the second image and aligning and overlaying the first and second images based on the morphological information.
2. The method of claim 1 , wherein the region of interest is selected by comparing the first image of the tissue sample to pre-determined criterion.
3. The method of claim 1 , wherein the first protocol further includes immunofluorescence detection of at least one additional protein wherein the additional protein provides the morphological information.
4. The method of claim 1 , wherein said hybridizing reaction is selected from the group consisting of FISH, IQ-FISH, in-situ PCR, rolling circle amplification and primed in situ labeling.
5. The method of claim 1 , wherein the second protocol comprises staining the sample with a fluorescent marker that provides morphological information.
6. The method of claim 5 , wherein said fluorescent marker stains the nucleus of a cell.
7. The method of claim 1 , wherein at least one of the first or the second protocol comprises detecting autofluorescence of the tissue sample.
8. The method of claim 1 , wherein the first and second images are fluorescent images.
9. The method of claim 8 , wherein generating the first image and/or generating the second image comprises generating a brightfield type image.
10. A method of analyzing a human tissue sample, comprising providing an image of the sample according to claim 1 , and analyzing protein expression and nucleic acids of the sample from the image.
11. The method of claim 1 , wherein said second image covers a smaller area of said sample compared to an area of said sample covered by said first image.