Time-resolved fluorescence microspectroscopy of visible fluorescent proteins in isolated form and in their natural cellular environment

Condensed Matter lunchtime seminar

Time-resolved fluorescence microspectroscopy of visible fluorescent proteins in isolated form and in their natural cellular environment

  • Event time: 1:00pm
  • Event date: 4th May 2009
  • Speaker: Ton Visser (Wageningen University)
  • Location: Room 2511,

Event details

Microspectroscopy can be defined as spectroscopy performed on microscopic objects. One type of microspectroscopy is polarized time-resolved fluorescence spectroscopy, which combines (sub-)microscopic spatial resolution with (sub-)nanosecond time resolution. In the Microspectroscopy Centre of Wageningen University we have developed during several decades detailed expertise in the time-correlated single photon counting (TCSPC) technique for measurements of time-resolved fluorescence. We have applied this technique to a wide range of biochemical and biophysical topics. More recently we have made an extension to multi-dimensional TCSPC recording fluorescence lifetime images of cellular objects. The method works in combination with a two-photon excitation confocal laser scanning microscope.

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria together with its differently colored mutants and new fluorescent proteins from coral species are abundantly applied as genetically encoded, brightly fluorescent markers in cell biology. Pairs of fluorescent proteins such as between the cyan fluorescent protein (CFP, donor) and the yellow fluorescent protein (YFP, acceptor) are widely used as Förster resonance energy transfer (FRET) pairs, are fused to the proteins of interest and genetically encoded in cells. FRET is a very powerful method for obtaining distance information between proteins. FRET can be measured in cells by the technique of fluorescence lifetime imaging microscopy (FLIM). Here we focus attention on two applications of single-photon timing spectroscopy: purified visible fluorescent proteins (VFPs) in cuvette-type of experiments and VFPs expressed in their natural habitat of a living cell providing spatially resolved fluorescence lifetimes. Both experiments provide complementary information. For quantitative interpretation of FRET in cellular systems we need details of the molecular fluorescence that can be obtained from experiments with isolated VFPs. For FRET experiments in cells we have developed an efficient protocol for global analysis of FLIM images. We illustrate both approaches by selected examples of VFPs and FRET pairs of VFPs.

1. J.W. Borst, S.P. Laptenok, A.H. Westphal, R. Kuhnemuth, H. Hornen, N.V. Visser, S. Kalinin, J. Aker, A. van Hoek, C.A.M. Seidel, A.J.W.G. Visser (2008) Biophys. J. 95, 5399-5411.
2. S.P. Laptenok, K.M. Mullen, J.W. Borst, I.H.M. van Stokkum, V.V. Apanasovich, A.J.W.G. Visser (2007) J. Stat. Software 18, issue 8.
3. S. P. Laptenok, J. W. Bors, K. M. Mullen, I. H. M. van Stokkum, A. J. W. G. Visser, H van Amerongen, in preparation

This is a weekly series of informal talks given primarily by members of the soft condensed matter and statistical mechanics groups, but is also open to members of other groups and external visitors. The aim of the series is to promote discussion and learning of various topics at a level suitable to the broad background of the group. Everyone is welcome to attend..

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