How fast can TCSPC FLIM be made?

Katsoulidou, Vicky; Bergmann, Axel; Becker, Wolfgang

doi:10.1117/12.735550

Cited by 21 publications

(10 citation statements)

References 10 publications

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“…Typical examples are Z stack FLIM (Becker, 2010) and time-series FLIM (Katsoulidou et al, 2007). Figure 10 shows a Z stack of two-photon autofluorescence FLIM images of pig skin.…”

Section: Flim Applicationsmentioning

confidence: 98%

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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“…Typical examples are Z stack FLIM (Becker, 2010) and time-series FLIM (Katsoulidou et al, 2007). Figure 10 shows a Z stack of two-photon autofluorescence FLIM images of pig skin.…”

Section: Flim Applicationsmentioning

confidence: 98%

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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“…This is not quite correct, as has been shown by Becker et al . (2004b, 2009) and Katsoulidou et al . (2007).…”

Section: Time‐domain Flim By Tcspcmentioning

confidence: 99%

Fluorescence lifetime imaging – techniques and applications

Becker¹

2012

Journal of Microscopy

734

553

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SummaryFluorescence lifetime imaging (FLIM) uses the fact that the fluorescence lifetime of a fluorophore depends on its molecular environment but not on its concentration. Molecular effects in a sample can therefore be investigated independently of the variable, and usually unknown concentration of the fluorophore. There is a variety of technical solutions of lifetime imaging in microscopy. The technical part of this paper focuses on time-domain FLIM by multidimensional time-correlated single photon counting, time-domain FLIM by gated image intensifiers, frequency-domain FLIM by gainmodulated image intensifiers, and frequency-domain FLIM by gain-modulated photomultipliers. The application part describes the most frequent FLIM applications: Measurement of molecular environment parameters, protein-interaction measurements by Förster resonance energy transfer (FRET), and measurements of the metabolic state of cells and tissue via their autofluorescence. Measurements of local environment parameters are based on lifetime changes induced by fluorescence quenching or conformation changes of the fluorophores. The advantage over intensity-based measurements is that no special ratiometric fluorophores are needed. Therefore, a much wider selection of fluorescence markers can be used, and a wider range of cell parameters is accessible. FLIM-FRET measures the change in the decay function of the FRET donor on interaction with an acceptor. FLIM-based FRET measurement does not have to cope with problems like donor bleedthrough or directly excited acceptor fluorescence. This relaxes the requirements to the absorption and emission spectra of the donors and acceptors used. Moreover, FLIM-FRET measurements are able to distinguish interacting and noninteracting fractions of the donor, and thus obtain independent information about distances and interacting and noninteracting protein fractions. This is information not accessible by steady-state FRET techniques. Autofluorescence FLIM exploits changes in the decay parameters of endogenous fluorophores with the Correspondence to:

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“…Recording the non-photochemical transient is relatively easy: It can even be recorded by normal time-series FLIM [2,5]. To record the transients by FLITS we used a bh DCS-120 confocal scanning FLIM system [3].…”

Section: Non-photochemical Transientmentioning

confidence: 99%

Spatially resolved recording of transient fluorescence-lifetime effects by line-scanning TCSPC

Becker

Bergmann

2012

SPIE Proceedings

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We present a technique that records transient effects in the fluorescence lifetime of a sample with spatial resolution along a one-dimensional scan. The technique is based on scanning a sample with a high-frequency pulsed laser beam, and building up a photon distribution over the distance along the scan, the arrival times of the photons after the excitation pulses, and the time after a stimulation of the sample. The maximum resolution at which lifetime changes can be recorded is given by the line scan time. With repetitive stimulation and triggered accumulation transient lifetime effects can be resolved at a resolution of about one millisecond.

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How fast can TCSPC FLIM be made?

Cited by 21 publications

References 10 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

Fluorescence lifetime imaging – techniques and applications

Spatially resolved recording of transient fluorescence-lifetime effects by line-scanning TCSPC

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