Fluorescence-lifetime imaging microscopy (FLIM) not only for biologists - practical aspects with hands-on experience

Syllabus

http://web.natur.cuni.cz/IMCF/FNOB/programme.html

Annotation

Fluorescence microscopy became an indispensable tool for biologists to uncover the secrets of live. The more we want to know, the more details we need. Fluorescence Lifetime Imaging Microscopy (FLIM) offers a set of tools that allow to utilize extra information offered by fluorescence probes and sensors, which is otherwise difficult to access. Typical examples, which will be demonstrated during the course, are metabolic activity by NAD(P)H imaging, protein interactions by FLIM-FRET and lipid membrane micro-environment sensing by Laurdan imaging.

The course focuses on explaining the principles of nano-second time-resolved fluorescence detection, on demonstration of different hardware realizations, on trying various applications of FLIM in biological imaging and on testing of several ways of FLIM data analysis.

The aim is to uncover the richness of information hidden in multi-parametric fluorescence imaging and to inspire the participants to use the easily obtainable extra contrast in their imaging applications. An important part is to make the FLIM data analysis understandable.

The course will cover the most widespread FLIM realization – acquisition in time domain based on raster scanning combined with pulsed lasers and time-correlated single photon counting (TCSPC). Participants will hands-on three different systems, covering pulsed diode lasers, white-light laser source as well as two-photon excitation, SPAD, HyD and GaAsP NDD detectors and three different TCSPC systems and software.

A focus will be given on common pitfalls, instrumental artifacts and ways of avoiding them.

The demonstrated applications will include FLIM-FRET, environmental sensing, metabolic NAD(P)H imaging and FLIM data analysis.