Figure 2.

Spectral analysis of quantum dot-based in situ hybridization to resolve signal against tissue autofluorescence in routinely processed human clinical tissue. i: H&E-stained human glioma is marked by the asterisk, and the arrow points to the tumor normal brain interface. All tissue was routinely formalin fixed, paraffin embedded, and imaged at ×20 magnification. ii: Raw (real color) image of a human glioma with adjacent normal cerebral tissue, hybridized with a 5′ thiol-labeled antisense ASCL1 oligonucleotide probe/amine-coated 580-nm QD conjugate, showing poor signal resolution due to high green autofluorescence of brain tissue. iii and iv: Pseudocolorized images following spectral analysis of image in ii showing signal for ASCL1 (iii) and NKX2-2 (iv) in red and tissue autofluorescence in green, demonstrating ability of spectral imager to resolve hybridization signal against high levels of inherent tissue autofluorescence.