FLIM
Fluorescence lifetime imaging microscopy (FLIM) is a fluorescence-based imaging technique that not only measures intensity differences on a pixel basis, but also detects the decay dynamics of excited fluorophores.
This allows differentiation between fluorophores that overlap in their excitation and emission spectra, but exhibit differences in fluorescence decay dynamics. Additionally, some fluorophores show specific changes in fluorescence lifetimes dependent on their microenvironment. Thus, FLIM is ideally suited to measure changes in chloride concentration, local pH, and other factors. Moreover, Förster Resonance Energy Transfer (FRET) sensors change their lifetime dependent on activity, which allows additional analyses
Tau-STED
Stimulated emission depletion (STED) microscopy is a superresolution microscopy technique that overcomes the resolution barrier of conventional light microscopy. Using a donut-shaped depletion laser the effective focal excitation spot is reduced beyond Abbe’s diffraction limit. Under optimal conditions, resolution is improved from 200 nm to less than 40 nm. STED microscopy is suited for both fixed specimen and living samples. Data is acquired and analyzed in real time. A multitude of established dyes are suited for this technique. However, superior results are expected by using specialized dyes that show improved photostability.
The Leica tau-STED technology is based on the combination of fluorescence intensity and fluorescence lifetime information. The STED depletion laser reduces the lifetimes of fluorophores depending on their distance from the focus point. This further improves the signal-to-noise ratio.
Equipment
The 3DSR Core Facility operates an inverted Leica SP8 Tau-STED3X.
A White Light Laser (WLL) allows to excite fluorophores at tunable wavelengths in a range of 470 to 670 nm. FLIM experiments can be performed using WLL light.
This system also incorporates an argon laser (458 nm, 476 nm, 488 nm, 496 nm, 514 nm) and an UV-laser (405 nm DMOD).
For STED and tauSTED superesolution imaging, two different depletion lasers are available: 592 nm and 775 nm. TauSTED experiments can only be executed utilizing WLL excitation (470 to 670 nm).
The microscope has a transparent enclosure for long-term live-cell imaging experiments that is temperature-controlled at both 22.5°C [MS1] and 37°C and provides humidified air with 5% CO2. The facility also allows experiments at biosafety level 1 (S1).
