Fluorescence lifetime imaging microscopy (FLIM) has emerged as a powerful technique for multi-color imaging, allowing researchers to distinguish fluorochromes beyond traditional spectral separation methods. By leveraging fluorescence lifetime differences, FLIM provides additional fluorochrome discrimination, enabling more complex imaging experiments with fewer spectral constraints. This approach is particularly valuable in confocal and Stimulated Emission Depletion (STED) microscopy, where spectral overlap and bleed-through can limit the number of detectable fluorophores. Phasor analysis of fluorescence lifetimes offers a rapid and photon-efficient alternative to traditional curve-fitting methods, making it well-suited for imaging experiments with low signal-to-noise ratios or fast acquisition times.
In a 2024 Methods in Microscopy publication, Pisfil et al. used Biotium’s CF® Dyes CF®594 and CF®680R to validate fluorescence lifetime separation in both confocal and STED multi-color imaging. CF®680R was effectively distinguished from far-red ATTO647N and AS635P due to its shorter fluorescence lifetime, enabling clear separation in the phasor plot. WGA-CF®594 was used to stain cell surfaces and was successfully distinguished from Alexa Fluor Plus 594 in the same red channel (Figure 1 ). Their study demonstrated that FLIM could successfully separate two fluorochromes within a single spectral channel, increasing the number of distinguishable targets while maintaining imaging quality comparable to spectral separation methods.
Fluorescence lifetime separation is a promising advancement for multi-color fluorescence microscopy and can be optimized with the incorporation of Biotium’s bright and photostable CF® Dyes. As new fluorophores with broader lifetime ranges become available, FLIM may further improve the resolution and accuracy of cellular imaging studies. This technique offers great potential for investigating intricate biological processes, from cellular signaling to disease pathogenesis, in both fixed and live-cell imaging applications.
Learn more about Biotium’s next-generation CF® Dye probes featuring exceptional brightness and signal-to-noise, now available in 40 colors from blue to near-IR. See a full list of CF Dyes validated for STED and other super-resolution applications.