Learn to identify when all cells in a sample have the same or very similar autofluorescence spectral characteristics, as demonstrated by a single defined signature in the spectral plot.
▶ 00:23
2
Recognize two unmixing approaches available
Understand that for homogeneous autofluorescence, you can choose to unmix either with or without autofluorescence extraction, as only a single spectral signature can be removed.
▶ 01:07
3
Access unmixing wizard and select autofluorescence
Open the unmixing wizard and select autofluorescence as a fluorescent tag to extract autofluorescence from all samples.
▶ 01:25
4
Gate on population of interest for extraction
In the unmixing wizard, set gates on the unstained sample and ensure you gate only on your population of interest (e.g., lymphocytes) to extract the autofluorescence of cells you will analyze.
▶ 01:50
5
Compare unmixed results with and without extraction
Perform unmixing with autofluorescence extraction, check the n by n matrices, then repeat unmixing without autofluorescence extraction to compare the two approaches.
▶ 02:26
6
Evaluate resolution trade-offs for each marker
Place the two unmixed results side by side and assess whether autofluorescence extraction improves resolution on some markers while potentially worsening it on others, using identically scaled plots.
▶ 03:11
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Select optimal unmixing approach for your analysis
Decide whether to use autofluorescence extraction based on which approach provides better overall marker resolution for your population of interest, considering that the same autofluorescence signature may perform differently depending on the cell population being analyzed.