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

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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.

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Notes

  1. Singlet–singlet-annihilation can occur if the photon density of the exciting laser pulse is high enough to produce simultaneously more than one excited singlet states per photosynthetic domain. During their random migration through the domain to the reaction center two excitations can meet on the same chromophore. This leads to a loss of one excitation due to internal conversion and thus to a shorter fluorescence lifetime.

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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Acknowledgments

We would like to thank Dr. Artur Napiwotzki for providing the data for Fig. 2, and Monika Weß and Sabine Kussin for cultivating A. marina. We further thank Marco Vitali, Franz-Josef Schmitt, Dr. Christoph Theiss and Prof. Hans-Joachim Eichler for their support. This work was supported by Marie Curie Fellowship (Z. P.) of the European Community program “Quality of Life” under the contract number QLK2-CT-200-60076 and by the Deutsche Forschungsgemeinschaft (SFB 429, TP A1).

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Authors and Affiliations

  1. Biophysics group, Biotechnologisches Zentrum, Technische Universität Dresden, Tatzberg 47-51, 01307, Dresden, Germany

    Zdeněk Petrášek

  2. Max-Volmer-Laboratory for Biophysical Chemistry, Technische Universität Berlin, Straße des 17. Juni 135, 10623, Berlin, Germany

    Hann-Jörg Eckert

  3. Europhoton GmbH, Berlin, Mozartstraße 27, 12247, Berlin, Germany

    Klaus Kemnitz

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  1. Zdeněk Petrášek
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Correspondence to Hann-Jörg Eckert.

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Petrášek, Z., Eckert, HJ. & Kemnitz, K. Wide-field photon counting fluorescence lifetime imaging microscopy: application to photosynthesizing systems. Photosynth Res 102, 157–168 (2009). https://doi.org/10.1007/s11120-009-9444-0

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