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

Becker, Wolfgang; Su, Bertram; Bergmann, Axel

doi:10.1117/12.908663

Cited by 7 publications

(10 citation statements)

References 3 publications

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“…Improvements can be expected from techniques that use repetitive stimulation of the sample and triggered accumulation of a time series. An example for TCSPC is fluorescence lifetime‐transient scanning (Becker et al ., 2012), but similar principles can be applied to other FLIM techniques as well. Another, yet data‐intensive, way to record a fast time series of FLIM data, and to apply correlation analysis to the temporal data of the series.…”

Section: Discussionmentioning

confidence: 99%

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Fluorescence lifetime imaging – techniques and applications

Becker¹

2012

Journal of Microscopy

731

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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Section: Discussionmentioning

confidence: 99%

“…Fast dynamic processes can be recorded by using the time after a stimulation of the sample as an additional recording coordinate. A technique called ‘fluorescence lifetime‐transient scanning’ uses line scanning to record lifetime changes down to a resolution of 1 ms (Becker et al ., 2012).…”

Section: Time‐domain Flim By Tcspcmentioning

confidence: 99%

Fluorescence lifetime imaging – techniques and applications

Becker¹

2012

Journal of Microscopy

731

553

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“…The minimum fluorescence lifetime detectable with this setup is~300 ps. FLIM data were analyzed with a binning of one using the SPCImage V4.6 software (Becker & Hickl), which uses an iterative reconvolution method to recover the lifetimes from the fluorescence decays (50). The goodness of the fit was evaluated from the c 2 values, which ranged from 0.9 to 1.2, and from the plot of the residuals.…”

Section: Two-photon Flimmentioning

confidence: 99%

“…In contrast to solution measurements, FLIM does not allow recording of more than a few thousand photons per pixel. This results in poor statistics, which precludes analysis of decays with more than one or two lifetime components (50). By monitoring the emission centered at 580 nm, where the contribution of lifetimes of %2.2 ns are negligible (Fig.…”

Section: Guvs Labeled By F2n12smentioning

confidence: 99%

Fluorescence Lifetime Imaging of Membrane Lipid Order with a Ratiometric Fluorescent Probe

Kilin

Glushonkov

Herdly

et al. 2015

Biophysical Journal

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To monitor the lateral segregation of lipids into liquid-ordered (Lo) and -disordered (Ld) phases in lipid membranes, environment-sensitive dyes that partition in both phases but stain them differently have been developed. Of particular interest is the dual-color F2N12S probe, which can discriminate the two phases through the ratio of its two emission bands. These bands are associated with the normal (N(∗)) and tautomer (T(∗)) excited-state species that result from an excited-state intramolecular proton transfer. In this work, we investigated the potency of the time-resolved fluorescence parameters of F2N12S to discriminate lipid phases in model and cell membranes. Both the long and mean lifetime values of the T(∗) form of F2N12S were found to differ by twofold between Ld and Lo phases as a result of the restriction in the relative motions of the two aromatic moieties of F2N12S imposed by the highly packed Lo phase. This differed from the changes in the ratio of the two emission bands between the two phases, which mainly resulted from the decreased hydration of the N(∗) form in the Lo phase. Importantly, the strong difference in lifetimes between the two phases was preserved when cholesterol was added to the Ld phase. The two phases could be imaged with high contrast by fluorescence lifetime imaging microscopy (FLIM) on giant unilamellar vesicles. FLIM images of F2N12S-labeled live HeLa cells confirmed that the plasma membrane was mainly in the Lo-like phase. Furthermore, the two phases were found to be homogeneously distributed all over the plasma membrane, indicating that they are highly mixed at the spatiotemporal resolution of the FLIM setup. Finally, FLIM could also be used to sensitively monitor the change in lipid phase upon cholesterol depletion and apoptosis.

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“…67,68 A way to resolve even faster dynamic lifetime e®ects is to combine the TCSPC recording with line scanning, which is also called°uorescence lifetimetransient scanning (FLITS). 18,39,69 The fastest line times for galvanometer scanners are in the range of 1 ms, consequently transient e®ects can be resolved down to about 1 ms. The application to Ca 2þ imaging in live neurons has been shown.…”

Section: Temporal Mosaic Recording With Triggered Accumulationmentioning

confidence: 99%

Fast fluorescence lifetime imaging techniques: A review on challenge and development

Liu

Lin

Becker

et al. 2019

J. Innov. Opt. Health Sci.

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Fluorescence lifetime imaging microscopy (FLIM) is increasingly used in biomedicine, material science, chemistry, and other related research fields, because of its advantages of high specificity and sensitivity in monitoring cellular microenvironments, studying interaction between proteins, metabolic state, screening drugs and analyzing their efficacy, characterizing novel materials, and diagnosing early cancers. Understandably, there is a large interest in obtaining FLIM data within an acquisition time as short as possible. Consequently, there is currently a technology that advances towards faster and faster FLIM recording. However, the maximum speed of a recording technique is only part of the problem. The acquisition time of a FLIM image is a complex function of many factors. These include the photon rate that can be obtained from the sample, the amount of information a technique extracts from the decay functions, the efficiency at which it determines fluorescence decay parameters from the recorded photons, the demands for the accuracy of these parameters, the number of pixels, and the lateral and axial resolutions that are obtained in biological materials. Starting from a discussion of the parameters which determine the acquisition time, this review will describe existing and emerging FLIM techniques and data analysis algorithms, and analyze their performance and recording speed in biological and biomedical applications.

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

Cited by 7 publications

References 3 publications

Fluorescence lifetime imaging – techniques and applications

Fluorescence lifetime imaging – techniques and applications

Fluorescence Lifetime Imaging of Membrane Lipid Order with a Ratiometric Fluorescent Probe

Fast fluorescence lifetime imaging techniques: A review on challenge and development

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