Jan. 03, 2012
Applications

Time Correlated Single Photon Counting

Choose Your Excitation Wavelength

  • Fig. 2: Fluorescence lifetime imaging of both a negative (mb-eGFP) and positive (mb-eGFP-mCherry) FRET reference using either the WLL or the Ti:Sa laser. Acquisitions were performed using a 40x, NA 1.25 oil immersion objective. Scale bar: 40 µm.Fig. 2: Fluorescence lifetime imaging of both a negative (mb-eGFP) and positive (mb-eGFP-mCherry) FRET reference using either the WLL or the Ti:Sa laser. Acquisitions were performed using a 40x, NA 1.25 oil immersion objective. Scale bar: 40 µm.

Fluorescence Lifetime Imaging Microscopy (FLIM) is widely used to quantify protein-protein interaction by measuring the FRET (Förster Resonance Energy Transfer) occurring between two fluorophores spaced by a few nanometers. FLIM is also used in a large array of applications ranging from tissue imaging to fluorophore environment probing. Although time correlated single photon counting (TCSPC) is the method of choice for fluorescence lifetime quantification, it requires dedicated instrumentation including i) a pulsed laser source, ii) a photon counting card, and iii) a fast detector. Such technical requirements render TCSPC acquisitions difficult to perform and/or narrow down the choices of usable fluorophores.

The Leica TCS SP5 X (Leica Microsystems) overcomes these limitations since it combines a pulsed white light laser (WLL) with an ultrafast photo-multiplier (PMT), allowing tunable spectral detection. This system is highly versatile and user-friendly for FLIM experiments. In this paper, we illustrate its potential in two biological applications: interaction studies and autofluorescence multispectral imaging.

Authors
Corentin Spriet, Ph.D.

Interdisciplinary Research Institute
Science and Technology University of Lille, France

Anja Schué (corresponding author)
Communications & Corporate Identity
Leica Microsystems GmbH
Wetzlar, Germany

 

 

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