Five-Color Fluorescence Microscopy

With increased fluorophore options and the abundance of directly conjugated antibodies, it is possible to use fluorescence microscopy to look at 5 different markers on a single sample. For larger microscopy panels, it is especially critical to know your microscope and be sure it has the appropriate lasers and filters to capture the emission and excitation spectra of each of these distinct fluorophores.

We stained frozen C57BL/6 mouse spleen tissue using antibodies against CD4 and CD8a to detect T cells, B220 to stain B cells, and CD169 and F4/80 to detect tissue-resident macrophages. For this staining, we took advantage of antibodies directly-conjugated to bright photostable fluorophores including the Brilliant Violet™ and Alexa Fluor® dyes.



Microscope Excitation and Emission Filters

By optimizing your filter set-up to allow for the detection of Brilliant Violet 421™ and Brilliant Violet 510™, you can expand your microscopy panels. See how we optimized our set-up below.