Fluorescence Lifetime Imaging by Multi-Detector TCSPC

Becker, Wolfgang; Bergmann, Axel; Biscotti, Giovanni; Biskup, Christoph

doi:10.1364/bio.2004.wd1

Cited by 22 publications

(36 citation statements)

References 2 publications

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“…Thus, in TCSPC experiments with high repetition-rate lasers afterpulses from many excitation periods pile up and form a virtually continuous counting background. The afterpulsing background can easily be one or two orders of magnitude higher than the thermal background (Becker, 2010;Becker et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

FLIM and FCS detection in laser‐scanning microscopes: Increased efficiency by GaAsP hybrid detectors

Becker¹,

Su²,

Holub

et al. 2010

Microscopy Res & Technique

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Photon counting detectors currently used in fluorescence lifetime microscopy have a number of deficiencies that result in less-than-ideal signal-to-noise ratio of the lifetimes obtained: either the quantum efficiency is unsatisfactory or the active area is too small, and afterpulsing or tails in the temporal response contribute to overall timing inaccuracy. We have therefore developed a new FLIM detector based on a GaAsP hybrid photomultiplier. Compared with conventional PMTs and SPADs, GaAsP hybrid detectors have a number of advantages: The detection quantum efficiency reaches or surpasses the efficiency of fast SPADs, and the active area is on the order of 5 mm², compared with 2.5 10⁻³ mm² for a SPAD. The TCSPC response is clean, without the bumps and the diffusion tails typical for PMTs and SPADs. Most important, the hybrid detector is intrinsically free of afterpulsing. FLIM results are therefore free of signal-dependent background, and FCS curves are free of the known afterpulsing peak. We demonstrate the performance of the new detector for multiphoton NDD FLIM and for FCS.

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Section: Introductionmentioning

confidence: 99%

FLIM and FCS detection in laser‐scanning microscopes: Increased efficiency by GaAsP hybrid detectors

Becker¹,

Su²,

Holub

et al. 2010

Microscopy Res & Technique

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“…These signals are fed into the SPC-730/SPC-830 TCSPC imaging module, where they are used to built up a 4-dimensional histogram of the photon distribution over time, wavelength, and spatial coordinates The imaging process is synchronized with the scanning process via the frame sync, line sync, and pixel sync signal of the LSM (Becker et al, 2002(Becker et al, , 2004Bird et al, 2004). With a special fiber adapter the TCSPC setup can also be used in conjunction with two-photon microscopy, thereby exploiting the advantages of two-photon excitation and maximizing fluorescence collection efficiency (see the accompanying paper by Becker et al, 2007 for more details).…”

Section: Fig 2 Experimental Setup: (A)mentioning

confidence: 99%

Multi‐dimensional fluorescence lifetime and FRET measurements

Biskup

Zimmer

Kelbauskas

et al. 2007

Microscopy Res & Technique

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When and where proteins associate with each other in living cells are key questions in many biological research projects. One way to address these questions is to measure the extent of Förster resonance energy transfer (FRET) between proteins that have been labeled with appropriate donor and acceptor fluorophores. When both proteins interact, donor and acceptor fluorophores are brought into close vicinity so that the donor can transmit a part of its excitation energy to the acceptor. As a result, both the intensity and the lifetime of the donor fluorescence decrease, whereas the intensity of the acceptor emission increases. This offers different approaches to determine FRET efficiency: One is to detect changes in the intensity of donor and acceptor emission, the other is to measure changes in the lifetime of the donor molecule. One important advantage of the fluorescence lifetime approach is that it allows to distinguish between free and associated donor molecules. However, like intensity measurements it lacks an intrinsic control ensuring that changes in the measured parameters are only due to FRET and not other quenching processes. Here, we show how this limitation can be overcome by spectrally resolved fluorescence lifetime measurements in the time domain. One technique is based on a streak camera system, the other technique is based on a time-correlated-single-photon-counting approach. Both approaches allow biologists to record both donor and acceptor fluorescence emitted by the sample in a single measurement.

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“…11,12 A recent compromise has been the use of large fiber bundles for this purpose. [12][13][14] This approach maintains some of the aforementioned advantages of fiber coupling, but the large bundle face can allow for the relaying of significant scattered light, and bundles further suffer loss of light from the inherent light rejection in the interstitial regions of the fiber packing. Finally, the bundle suffers from a significant amount of temporal broadening and skewing of photon arrival times as a function of coupling angle [numerical aperture (NA)].…”

Section: Introductionmentioning

confidence: 99%

Forward-collected simultaneous fluorescence lifetime imaging and coherent anti-Stokes Raman scattering microscopy

et al. 2011

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Abstract. We demonstrate the simultaneous collection and separation of femtosecond-laser-based forwardcollected coherent anti-Stokes Raman scattering (F-CARS) and two-photon-excitation-induced fluorescence lifetime images (FLIM) using time-correlated single photon counting (TCSPC). We achieve this in a nondescanned geometry using a single multimode fiber without significant loss of light, field of view, and most importantly, TCSPC timing fidelity. In addition to showing the ability to separate CARS images from FLIM images using time gating, we also demonstrate composite multimodal epicollected FLIM imaging with fiber-collected F-CARS imaging in live cells. C 2011 Society of Photo-Optical Instrumentation Engineers (SPIE).

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Fluorescence Lifetime Imaging by Multi-Detector TCSPC

Cited by 22 publications

References 2 publications

FLIM and FCS detection in laser‐scanning microscopes: Increased efficiency by GaAsP hybrid detectors

FLIM and FCS detection in laser‐scanning microscopes: Increased efficiency by GaAsP hybrid detectors

Multi‐dimensional fluorescence lifetime and FRET measurements

Forward-collected simultaneous fluorescence lifetime imaging and coherent anti-Stokes Raman scattering microscopy

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