Fluorescence lifetime imaging using a confocal laser scanning microscope

Buurman, E. P.; Sanders, Renata; Draaijer, A.; Gerritsen, Hans C.; Veen, J.; Houpt, P.M.; Levine, Y.K.

doi:10.1002/sca.4950140305

Cited by 185 publications

(109 citation statements)

References 11 publications

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“…However, as with many spectroscopic techniques, lifetimes measured in a conventional widefield microscope are subject to distortions arising from contributions of out-of-focus light originating above and below the plane of best focus. This realization has led to the development of optically sectioned lifetime imaging techniques implemented in single (3,4, this publication) and multiphoton configurations (1,(5)(6)(7)(8). Image reconstruction procedures applied to widefield and structured illumination data have also been used to generate sectioned fluorescence lifetime images.…”

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confidence: 99%

Fluorescence lifetime imaging in an optically sectioning programmable array microscope (PAM)

et al. 2005

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Background: The programmable array microscopes (PAMs) are a family of instruments incorporating arbitrary control of the patterns of illumination and/or detection. The PAM can be used in sectioning and nonsectioning modes, thereby constituting a useful platform for fluorescence lifetime imaging. Methods and Results: We used a PAM for acquisition of optically sectioned and widefield fluorescence lifetime images, in which contrast was increased predominantly by suppressing out-of-focus light contributions. We simu-

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confidence: 99%

Fluorescence lifetime imaging in an optically sectioning programmable array microscope (PAM)

et al. 2005

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“…Technically, this can be done by using a Time Correlated Single Photon Counting (TCSPC) system (10), or by measuring the fluorescence signal either using a time-gated detector (12), or a streak camera (13). Regardless of the technique applied, the theoretical fluorescence intensity profile I(t) is defined as:…”

Section: The Characterization Of Dynamic Interactions Between Biomolementioning

confidence: 99%

Upgrading time domain FLIM using an adaptive Monte Carlo data inflation algorithm

et al. 2011

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Fluorescence Lifetime Imaging Microscopy (FLIM) is a powerful technique to investigate the local environment of fluorophores in living cells. To correctly estimate all lifetime parameters, time domain FLIM imaging requires a high number of photons and consequently long laser exposure times. This is an issue because long exposure times are incompatible with the observation of dynamic molecular events and induce cellular stress. To minimize exposure time, we have developed an original approach that statistically inflates the number of collected photons. Our approach, called Adaptive Monte Carlo Data Inflation (AMDI), combines the well-known bootstrap technique with an adaptive Parzen kernel. We here demonstrate using both Monte Carlo simulations and live cells that our robust method accurately estimate fluorescence lifetimes with exposure time reduced up to 50 times for monoexponential decays (corresponding to a minimum of 20 photons/pixel), and 10 times for biexponential decays (corresponding to a minimum of 5,000 photons/pixel), compared to standard fitting method. Thanks to AMDI, in Förster resonance energy transfer experiments, it is possible to estimate all fitting parameters accurately without constraining any parameters. By reducing the commonly used spatial binning factor, our technique also improves the spatial resolution of FLIM images. ' 2011 International Society for Advancement of Cytometry

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“…To date these techniques have been largely restricted to intensity-based imaging, providing mainly structural rather than functional information, and as such can be prone to the artefacts associated with other intensity-based imaging modalities. Fluorescence lifetime imaging (FLIM) [8][9][10] is an increasingly utilized tool in biological research that can provide robust contrast between different fluorescent proteins/species and can also yield quantitative information concerning the local fluorophore environment, e.g. pH, temperature, refractive index [11][12][13], etc., including protein-protein interactions via Fö rster Resonance Energy Transfer (FRET) [14,15].…”

Section: Biophotonicsmentioning

confidence: 99%

Fluorescence lifetime optical projection tomography

McGinty¹,

Tahir

Laine

et al. 2008

Journal of Biophotonics

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One of the recent trends in biological research has been a progression from average measurements on cell populations (e.g. homogenous well-plate assays) to fixed and live cell imaging for sub-cellular spatial localization, study of metabolic signals and spatiotemporally resolved dynamics. There is now an increasing appreciation that in vitro cell mono-layers may exhibit non-physiological behaviour due to the artificially planar environment. This has led to the development of 3-D cell cultures BIOPHOTONICSWe describe a quantitative fluorescence projection tomography technique which measures the 3-D fluorescence lifetime distribution in optically cleared specimens up 1 cm in diameter. This is achieved by acquiring a series of wide-field time-gated images at different relative time delays with respect to a train of excitation pulses, at a number of projection angles. For each time delay, the 3-D time-gated intensity distribution is reconstructed using a filtered back projection algorithm and the fluorescence lifetime subsequently determined for each reconstructed horizontal plane by iterative fitting to a monoexponential decay. Due to its inherently ratiometric nature, fluorescence lifetime is robust against intensity based artefacts as well as producing a quantitative measure of the fluorescence signal. We present a 3-D fluorescence lifetime reconstruction of a mouse embryo labelled with an alexa-488 conjugated antibody targeted to the neurofilament, which clearly differentiates between the extrinsic label and the autofluorescence, particularly from the heart and dorsal aorta.

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Fluorescence lifetime imaging using a confocal laser scanning microscope

Cited by 185 publications

References 11 publications

Fluorescence lifetime imaging in an optically sectioning programmable array microscope (PAM)

Fluorescence lifetime imaging in an optically sectioning programmable array microscope (PAM)

Upgrading time domain FLIM using an adaptive Monte Carlo data inflation algorithm

Fluorescence lifetime optical projection tomography

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