Heterogeneous Autofluorescence

Review the unstained sample using a spectral plot to identify that multiple cell types in the tissue sample produce different autofluorescence signatures rather than a single clean signature.

Open a raw n-by-n permutation worksheet to compare all fluorescent channels. Create a spectral plot of UV and YG1 channels to identify regions of heterogeneity and locate distinct cell populations with different spectral characteristics.

Gate around the identified populations based on their positions in the UV/YG1 plot to isolate different spectral signatures. Create spectral plots for each gated population alongside scatter plots to confirm they represent distinct cell types with different autofluorescence profiles.

For each distinguished spectral signature, identify the peak channel and gate on the brightest events. Export at least 300-500 events from each population as FCS files for use in unmixing, including the dim autofluorescent negative population.

Create a new autofluorescent tags group in the spectral flow software library. Add entries for each distinguished autofluorescent population with their respective detector and peak emission wavelength information.

Add the newly created autofluorescent tags to the experiment as reference controls. Import the exported FCS files for each population and assign the dim autofluorescent population as the negative control for spillover calculation.

Run the unmixing wizard through all controls, setting gates for the cell populations. Check the similarity matrix to confirm that autofluorescent signatures have similarity indices below the 0.98 threshold, indicating they are distinct spectral signatures.