Separating out different color wavelengths to better understand target distribution

The nuance multispectral imaging system from PerkinElmer is pretty cool. Basically it's just a camera with some spectral filters that sits on top of your microscope, and some smart software that can make sense of the captured image. The system lets you isolate the color signal of specific stains, filter out the noise that comes from autofluorescence, figure out which cells have taken up which stain, and impartially calculate the strength of the staining response. Best of all it can use some wavelength addition and subtraction calculations to highlight areas that have taken up more than one stain. 

In a recent study conducted in our CLIA certified lab, we used this system to show co-localization of a particular protein and the mRNA transcript from which that protein was translated. The 4 images below are of the same region of a human prostate section. IHC was used to stain a smooth muscle cytoskeletal protein called "smoothelin". ISH was then used to stain the same material so that we can see the mRNA transcripts from which the protein was translated. 

The first image shows the presence of antibody stained protein as brown, the blue stain is the mRNA. Note the localization of the mRNA signal to the stroma (long arrow) and the lack of signal in the epithelia (short arrow). In the first image (brightfield microscopy), it's sort of hard to see if the brown and the blue stains are really tightly co-localized.

In the next pair of images, fluorescence microscopy is used to separately show the smoothelin protein (green) and the mRNA transcript (blue).

Finally, in the last image, the nuance system helps us to see BOTH types of staining simultaneously, with the dual stained regions shown as yellow. This makes the co-expression much easier to see. For more information on the Nuance system here.