Detection of Multiple Qdots in Brain Tissue
A challenging problem is multiplexing two or more fluorophores in paraffin embedded tissue sections that contain a large amount of autofluorescence. This figure illustrates the application of spectral unmixing in brain (cerebellar region) labeled with two quantum-dot-coupled antibodies directed against glial fibrillary acid protein (GFAP, 605-nm QDot, yellow) and neurofilamin (NF, 655-nm QDot, red; data courtesy Ventana Medical Systems, Tucson, AZ). Nuclei were stained with DAPI. While DAPI normally generates a blue signal, because this dataset was acquired in the spectral range, 530 to 680 nm, only its green emission "tail" is detected here. In addition to these specific signals, a ubiquitous green-yellow autofluorescence was also detected and removed from the final image to improve contrast.
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Figure: Formalin-fixed cerebellum labeled with two quantum dots. Panel A shows an RGB image of the sample with glial fibrillary acidic protein (GFAP) immunolabeled with a 605-nm Qdot, and neurofilamin (NF) immunolabeled with a 655-nm Qdot). Nuclei were labeled with DAPI. In addition to the specific labels, tissue autofluorescence is present. The insert show the spectra that were derived from this datacube (see Materials and Methods) and used to unmix into the 4 component images (B through D), whose border colors correspond to the colored spectral graphs and to the pseudocolors used to form the composite image, F. Thus, B identifies the nuclear signal, C, the 605-nm GFAP signals, D, the 655-nm NF signals, and E, tissue autofluorescence. Because it is unmixed in the "black" channel, it is invisible in the panel F, which accounts for the greater clarity in panel F vs. the original (panel A). Sample courtesy of Ventana Medical Systems.
Panel A shows a conventional RGB image of the sample that is typical of most fluorescence-based imaging systems. The yellow and red striations of GFAP and neurofilamin (NF) protein, respectively, are visible, although not clearly, against a ubiquitous greenish autofluorescence background. Additional nuclear autofluorescent signals are also present and can be seen most prominently at the right of the image. In comparison, the Nuance system was able to overcome the hurdles surrounding autofluorescence thereby dramatically imrproving the signal-to-noise of each label. The Nuance-acquired 4 component images (B through D) identify each signal in their own respective panels, all accurately separated using the system's RCA algorithm. The composite image is shown in panel F. Panel B identifies the specific nuclear autofluorescence signal, C, the 605-nm GFAP signals, D, the 655-nm NF signals, and E, generic autofluorescence. Because the autofluorescence signal is unmixed in the "black" channel, it is invisible in panel F, which accounts for the greater clarity in panel F vs. the original (panel A).
