Course Info

This course introduces participants to the combined use of flow cytometry and fluorescence microscopy for quantitative Förster Resonance Energy Transfer (FRET) in cell biology and biomedicine. It integrates theory with hands-on work on both conventional and spectral flow cytometers, as well as complementary imaging platforms.

Using BD LSRFortessa and Cytek Aurora systems, participants will compare conventional and spectral flow-cytometric strategies for FRET, examine their strengths and weaknesses, and learn how to recognize and avoid common artifacts. The practical sessions will cover sample preparation, selection of acquisition settings, and data processing workflows tailored to FRET assays.

The microscopy component will focus on quantitative imaging of the same samples analyzed by flow cytometry. Participants will acquire and interpret both FLIM-based and intensity-based FRET readouts:

• Confocal FLIM (time-domain): Lifetimes will be recorded on a laser-scanning system, with emphasis on instrument configuration, fitting models, and the pitfalls of lifetime heterogeneity in live-cell experiments.
• Wide-field TIRF intensity FRET: Participants will perform donor and acceptor imaging under TIRF illumination, handle correction factors, and work through issues such as bleed-through and uneven illumination.

By comparing FLIM and intensity-based approaches side-by-side, participants will see where each method succeeds, where it fails, and how the two can corroborate or contradict each other when applied to realistic biological samples.

After completing the course, participants will understand when and how flow cytometry and fluorescence microscopy can be combined in FRET studies, how to select fluorophore pairs and appropriate controls, and how to interpret the resulting data with a critical eye.

The course is intended for PhD students and early-career researchers in the life sciences who already have basic experience with fluorescence methods and want to implement or strengthen FRET-based assays.

May, 2026

BIOCEV, Vestec

The course is organized by the Imaging Methods Core Facility.

The course is supported by the National Infrastructure for Biological and Medical Imaging (Czech-BioImaging, Ministry of Education, Youth and Sports – Large Research Infrastructure, LM2023050).