Photosynthesis Research

, 102:157 | Cite as

Wide-field photon counting fluorescence lifetime imaging microscopy: application to photosynthesizing systems

  • Zdeněk Petrášek
  • Hann-Jörg Eckert
  • Klaus Kemnitz
Review

Abstract

Fluorescence lifetime imaging microscopy (FLIM) is a technique that visualizes the excited state kinetics of fluorescence molecules with the spatial resolution of a fluorescence microscope. We present a scanningless implementation of FLIM based on a time- and space-correlated single photon counting (TSCSPC) method employing a position-sensitive quadrant anode detector and wide-field illumination. The standard time-correlated photon counting approach leads to picosecond temporal resolution, making it possible to resolve complex fluorescence decays. This allows parallel acquisition of time-resolved images of biological samples under minimally invasive low-excitation conditions (<10mW/cm2). In this way unwanted photochemical reactions induced by high excitation intensities and distorting the decay kinetics are avoided. Comparably low excitation intensities are practically impossible to achieve with a conventional laser scanning microscope, where focusing of the excitation beam into a tight spot is required. Therefore, wide-field FLIM permits to study Photosystem II (PS II) in a way so far not possible with a laser scanning microscope. The potential of the wide-field TSCSPC method is demonstrated by presenting FLIM measurements of the fluorescence dynamics of photosynthetic systems in living cells of the chlorophyll d-containing cyanobacterium Acaryochloris marina.

Keywords

Fluorescence lifetime imaging FLIM Time-correlated single photon counting QA detector Photosynthesis Chlorophyll Acaryochloris marina 

Abbreviations

FLIM

Fluorescence lifetime imaging microscopy

FRET

Foerster resonance energy transfer

TCSPC

Time-correlated single photon counting

TSCSPC

Time- and space-correlated single photon counting

PMT

Photomultiplier tube

MCP

Multichannel plate

QA

Quadrant anode

DL

Delay line

IRF

Instrument response function

EET

Excitation energy transfer

PS I , PS II

Photosystem I and photosystem II

Chl

Chlorophyll

PBP

Phycobiliprotein

S–S

Singlet–singlet

DCMU

3-(3,4-Dichlorophenyl)-1,1-dimethyl urea

PC

Phycocyanin

APC

Allophycocyanin

QA

Primary plastoquinone acceptor of PS II.

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Copyright information

© Springer Science+Business Media B.V. 2009

Authors and Affiliations

  • Zdeněk Petrášek
    • 1
  • Hann-Jörg Eckert
    • 2
  • Klaus Kemnitz
    • 3
  1. 1.Biophysics group, Biotechnologisches ZentrumTechnische Universität DresdenDresdenGermany
  2. 2.Max-Volmer-Laboratory for Biophysical ChemistryTechnische Universität BerlinBerlinGermany
  3. 3.Europhoton GmbH, BerlinBerlinGermany

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