FLIM - Picoquant Fluorescent Lifetime Imaging Microscope

System Description

FLIM measures the total fluorescence lifetime of fluorophores (as in, how long on average a fluorophore stays in its excited state following excitation from a short excitation light pulse). This can give you a wealth of information on the fluorophore’s local chemical and physical environment (“local” meaning nanometer scale!). It can also be used to study translational and rotational behaviour of the fluorophores and their relative orientations as well as orientational dynamics (thereby allowing an estimation of the respective properties of the attached molecules). It also allows highly accurate FRET imaging, eliminating the crosstalk and/or bleaching problems inherent to standard ratiometric FRET imaging, and it provides an elegant tool for (time-resolved) anisotropy measurements

FLIM approach allows high accuracy FRET imaging, eliminating the crosstalk and/or bleaching problems inherent to standard ratiometric FRET imaging.

This microscope, although officially belonging to CSF, can be used by IMP-IMBA-GMI users as any other system of the BioOptics facility, without any extra costs.

Microscope frame


Olympus iX71 inverted microscope frame

Excitation

Laser: 440 nm, 510 nm

Objective lenses (Olympus)

PL 20 x PlanAchromat Objektiv, NA = 0.4

PL 40 x PlanAchromat Objektiv, NA = 0.65

UPLSAPO 60 x Ultra-Planapochromat, NA = 1.2 (water immersion)

PLAPON 60 x  Plan Apo, NA = 1.42 (oil immersion)

Filters & Beam Splitters

These vary according to the experimental demands. Any common (and many uncommon) filter / beamsplitter combinations can be installed. 

Detector

Hybrid Photomultiplier Detection Assembly with < 50 ps time resolution 

Analysis Software

SymPhoTime 64 (more details on the Picoquant webpage)

External Links

More information on TCSPC theory and data analysis available on a Picoquant Wikipage

Further information also available on the CSF Homepage

Reservation & Training

If you wish to use (or learn more about) the FLIM system, please send an email to microscopy(at)imp.ac.at in order to arrange the training.