Leica TCS SP8 gSTED-FLIM-FCS overview
- Pico-second pulsed white light laser (WLL) provides fully tuneable excitation from 470nm to 670nm in 1nm intervals.
- Excitation, emission or excitation-emission scanning to provide full spectral characteristics of sample (can be combined with lifetime measurements, see below).
- AOTF enables fast switching between up to 8 excitations lines from WLL.
- Galvo Z drive for fast Z acquisition.
- Resonant scanner; up to 8000 Hz scan speed
- Two gated hybrid detectors provide high sensitivity (~ twice as sensitive as PMTs) and minimise unwanted laser reflections to increase S/N.
- Gated 3D STED provides ~50nm xy resolution and ~150nm z resolution.
- Two depletion lines; 592nm and 660nm enables use of wide variety of fluorophores (Green to Red)
Fluorescence Lifetime Imaging Microscopy (FLIM):
- Monitoring the lifetime of the fluorophore rather than the intensity provides information on the local microenvironment. Potential applications are:
- Förster resonance energy transfer (FRET), both intermolecular and intramolecular.
- Measuring analytes such as calcium, chloride or potassium ions or signalling molecules like IP3, PIP2 and caspase among others.
- Measurement of the physical environment of the fluorophore, for example local temperature, viscosity etc.
- Inherently ratiometric technique and therefore less susceptible to scattering and requires far fewer calibration measurements to account for cross talk, spectral bleedthrough etc.
- Pulse picker on WLL allows selection of repetition rate (80,40,20 or 10 MHz)
Fluorescence correlation spectroscopy (FCS):
- Analysis of the fluorescence fluctuations measured within stationary laser volume allows the user to measure complex dynamic single molecule processes such as diffusion, kinetics and binding events.
- FCS may be complemented by STED to reduce focal volume in 3D facilitating the measurement of FCS data at much higher concentration of fluorophores than with diffraction limited confocal laser spot.