Multi‐dimensional fluorescence lifetime and FRET measurements

Biskup, Christoph; Zimmer, Thomas; Kelbauskas, Laimonas; Hoffmann, Birgit; Klöcker, Nikolaj; Becker, Wolfgang; Bergmann, Axel; Benndorf, Klaus

doi:10.1002/jemt.20431

Cited by 74 publications

(73 citation statements)

References 32 publications

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“…Considering the fact that many endogenous chromophores (quenched donors in FRET, NADH, NADPH) are characterised by fluorescence lifetimes in ps-range, the time-accuracy provided by FD FLIM is often insufficient. In contrast, TD FLIM techniques reach time-resolutions in the low ps-range or even fs-range [8][9][10][11]40,[45][46][47] .…”

Section: Time-domain Flimmentioning

confidence: 95%

“…It makes use of the fact that the detection of several photons in different detection channels in one laser period is unlikely. Therefore, the single photon pulses from all detector channels can be combined into a common photon pulse line and sent through the normal time measurement procedure of the TCSPC module 10,40,46 .…”

Section: Time-correlated Single Photon Counting (Tcspc)mentioning

confidence: 99%

“…The technique can reasonably be used for up to four individual ultrafast MCP-PMTs (e.g. R3809U by Hamamatsu) or one 16 channels or 32 channels multi-anode PMT 10,40,46 .…”

Section: Time-correlated Single Photon Counting (Tcspc)mentioning

confidence: 99%

“…Synchronously with the detection of a photon, the detector channel number n for the current photon is read into the detector channel register. If light is split into different wavelength intervals in front of the detectors, n represents the wavelength of the detected photon 10,40,46 .…”

Section: Time-correlated Single Photon Counting (Tcspc)mentioning

confidence: 99%

“…Each block can be regarded as an image containing a full fluorescence decay curve in each pixel 10,40,46 .…”

Section: Time-correlated Single Photon Counting (Tcspc)mentioning

confidence: 99%

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Fluorescence lifetime imaging in biosciences: technologies and applications

Niesner

Gericke

2008

Front. Phys. China

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The biosciences require the development of methods, which allow a non-invasive and rapid investigation of biological systems. In this frame high-end imaging techniques allow an intravital microscopy in real-time, providing information on a molecular basis. Far-field fluorescence imaging techniques are some of the most adequate methods for such investigations. However, there are great differences between the common fluorescence imaging techniques, i.e. wide-field, confocal one-photon and two-photon microscopy, respectively, as far as their applicability in diverse bioscientific research areas is concerned. In the first part of this work, we briefly compare these techniques. Standard methods used in the biosciences, i.e. steady-state techniques based on the analysis of the total fluorescence signal originating from the sample, can successfully be employed in the study of cell, tissue and organ morphology as well as in monitoring the macroscopic tissue function. However, they are mostly inadequate for the quantitative investigation of the cellular function at molecular level. The intrinsic disadvantages of steady-state techniques are counteracted by using time-resolved techniques. Among these the fluorescence lifetime imaging (FLIM) is currently the most common. Different FLIM principles as well as applications of particular relevance for the biosciences, especially for fast intravital studies are discussed in this work.

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Section: Time-domain Flimmentioning

confidence: 95%

Section: Time-correlated Single Photon Counting (Tcspc)mentioning

confidence: 99%

“…The technique can reasonably be used for up to four individual ultrafast MCP-PMTs (e.g. R3809U by Hamamatsu) or one 16 channels or 32 channels multi-anode PMT 10,40,46 .…”

Section: Time-correlated Single Photon Counting (Tcspc)mentioning

confidence: 99%

Section: Time-correlated Single Photon Counting (Tcspc)mentioning

confidence: 99%

“…Each block can be regarded as an image containing a full fluorescence decay curve in each pixel 10,40,46 .…”

Section: Time-correlated Single Photon Counting (Tcspc)mentioning

confidence: 99%

See 3 more Smart Citations

Fluorescence lifetime imaging in biosciences: technologies and applications

Niesner

Gericke

2008

Front. Phys. China

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FLIM‐FRET‐based analysis of S100A11/annexin interactions in living cells

Melle,

Hoffmann,

Wiesenburg

et al. 2024

FEBS Open Bio

Self Cite

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Proteins achieve their biological functions in cells by cooperation in protein complexes. In this study, we employed fluorescence lifetime imaging microscopy (FLIM)‐based Förster resonance energy transfer (FRET) measurements to investigate protein complexes comprising S100A11 and different members of the annexin (ANX) family, such as ANXA1, ANXA2, ANXA4, ANXA5, and AnxA6, in living cells. Using an S100A11 mutant without the capacity for Ca2+ binding, we found that Ca2+ binding of S100A11 is important for distinct S100A11/ANXA2 complex formation; however, ANXA1‐containing complexes were unaffected by this mutant. An increase in the intracellular calcium concentration induced calcium ionophores, which strengthened the ANXA2/S100A11 interaction. Furthermore, we were able to show that S100A11 also interacts with ANXA4 in living cells. The FLIM‐FRET approach used here can serve as a tool to analyze interactions between S100A11 and distinct annexins under physiological conditions in living cells.

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Multiphoton Fluorescence Light Microscopy

Palikaras

Tavernarakis

2012

Encyclopedia of Life Sciences

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Multiphoton fluorescence microscopy is a powerful imaging technique that depends on complex quantum mechanical interactions between photons and matter for fluorophore excitation. In conventional fluorescence microscopy, a fluorescent molecule is pumped to an excited state by absorbing a single photon. The molecule subsequently falls back to its ground state by emitting a less energetic photon. This is a linear process of absorbing and emitting energy in the form of single photons. By contrast, multiphoton microscopy is based on nonlinear interactions between light and matter, whereby multiple photons are absorbed to bring single fluorophore molecules to an excited state. Two‐photon fluorescence microscopy is the most commonly used multiphoton imaging technique. In two‐photon microscopy, the fluorescent molecule absorbs two photons simultaneously in a single event, and their combined energies provoke the electronic transition of the molecule to the excited state. Advantages of two‐photon fluorescence, compared to typical single‐photon epifluorescence microscopy, include reduced autofluorescence, deeper tissue penetration, inherent confocality and three‐dimensional (3D) imaging, as well as, minimised photobleaching and photodamage. Thus, two‐photon microscopy facilitates optical sectioning of thick biological specimens in vivo , which would not be possible with conventional imaging techniques. Recent advances in fluorescence microscopy have expanded the application spectrum and usability of multiphoton imaging, which has become an important and versatile tool in modern biomedical research. Key Concepts: Multiphoton microscopy is based on nonlinear interactions between light and matter. Two‐photon excitation transpires only at the objective lens focal volume, where photon density is high enough to generate sufficient absorption events. Multiphoton microscopy requires the use of ultra‐fast femtosecond, pulsed laser light sources to achieve appropriate fluorophore excitation conditions at the focal point. Because of sharply focused excitation in two‐photon microscopy, photodamage is reduced and viability of the biological specimen is increased. Multiphoton microscopy uses photons with near infra‐red wavelengths that are poorly absorbed and less scattered by biological material, allowing deeper light penetration into specimens. As a result of using photons of longer wavelength for excitation, two‐photon fluorescence microscopy is limited to slightly lower resolution, compared with single‐photon confocal microscopy. In two‐photon excitation, photodamage and thermal damage are considerably reduced and become significant only at the focal point. Photoconvertible fluorophores have been developed for multiphoton microscopy applications that allow localised photochemical reactions in a biological sample. Conventional fluorophores exhibit distinct two‐photon absorption spectra, which are not directly related to their single‐photon excitation properties. Two‐photon microscopy is suitable for in vivo imaging and monitoring of neuronal function in freely moving and behaving animals.

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Multi‐dimensional fluorescence lifetime and FRET measurements

Cited by 74 publications

References 32 publications

Fluorescence lifetime imaging in biosciences: technologies and applications

Fluorescence lifetime imaging in biosciences: technologies and applications

FLIM‐FRET‐based analysis of S100A11/annexin interactions in living cells

Multiphoton Fluorescence Light Microscopy

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