Fluorescence lifetime imaging with picosecond resolution for biomedical applications

Dowling, K.; Dayel, Mark J.; Lever, M. J.; French, Paul M. W.; Hares, J. D.; Dymoke‐Bradshaw, A. K. L.

doi:10.1364/ol.23.000810

Cited by 145 publications

(98 citation statements)

References 8 publications

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“…Figure 1 shows a schematic of wide-field time domain FLIM, in which time-gated "snapshots" of the fluorescence emission are acquired at various nanosecond delays after the excitation -with ~ 100 ps shutter speeds, usually achieved using high speed gated optical image intensifiers (GOI's), e.g. [19]. This series of time-gated fluorescence intensity images can then be analysed to extract the fluorescence decay time for each pixel of the image.…”

Section: Wide-field Flimmentioning

confidence: 99%

“…Our work towards the development of clinical FLIM instrumentation has mainly utilised frequency-doubled mode-locked Cr:LiSAF and Ti:Sapphire lasers to provide excitation wavelengths above 400 nm, for which FLIM has been shown to provide intrinsic label-free contrast in bulk tissue, e.g. [19]. Figure 4 shows wide-field time domain FLIM maps of unstained rat tissue obtained using picosecond excitation pulses at 410 nm [18].…”

Section: Endogenous Flim For Intrinsic Tissue Contrastmentioning

confidence: 99%

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Time-domain fluorescence lifetime imaging applied to biological tissue

Elson

Requejo-Isidro

Munro

et al. 2004

Photochem Photobiol Sci

177

135

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Fluorescence lifetime imaging (FLIM) is a functional imaging methodology that can provide information, not only concerning the localisation of specific fluorophores, but also about the local fluorophore environment. It may be implemented in scanning confocal or multi-photon microscopes, or in wide-field microscopes and endoscopes. When applied to tissue autofluorescence, it reveals intrinsic excellent contrast between different types and states of tissue. This article aims to review our recent progress in developing time-domain FLIM technology for microscopy and endoscopy and applying it to biological tissue.

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Section: Wide-field Flimmentioning

confidence: 99%

Section: Endogenous Flim For Intrinsic Tissue Contrastmentioning

confidence: 99%

Time-domain fluorescence lifetime imaging applied to biological tissue

Elson

Requejo-Isidro

Munro

et al. 2004

Photochem Photobiol Sci

177

135

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“…1). Short lifetimes are close to the coordinates [1,0], whereas long lifetimes approach the origin ([0, 0]). If multiple lifetime components are present in the sample, then we obtain this inequality:…”

Section: Polar Representationmentioning

confidence: 98%

Three‐dimensional polar representation for multispectral fluorescence lifetime imaging microscopy

et al. 2009

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Multispectral fluorescence lifetime imaging microscopy is a promising and powerful technique for discriminating multiply labeled samples and for detecting molecular interactions inside thick, heterogeneous, and light-scattering milieu such as tissue. The fast and correct analysis of the spectral and lifetime images constitutes a major challenge, which requires a high level of expertise. We present here a new approach that considerably simplifies this analysis avoiding complex fitting algorithm strategies and permitting a fast and visual graphical representation of the fluorescence lifetimes. By transforming the experimental data from time domain to frequency domain for each spectral channel, we calculate the multispectral polar representation and demonstrate its interest on multiply fluorescent labeled sample. We further apply it on Förster resonance energy transfer (FRET) experiments and demonstrate that FRET measurements with a high level of precision can be performed. With addition of emission wavelength as third dimension in the polar representation, autofluorescence emitted by the sample is thus clearly identified. Analysis artifacts induced by the sample or by fitting algorithm choice become then totally inexistent. ' 2009 International Society for Advancement of Cytometry Key terms multispectral FLIM; SLIM; biological tissue; molecular interactions; FRET; phasor IN addition to intensity and wavelength, lifetime is a supplementary source of contrast in fluorescence microscopy, which greatly increases access to the biological information in living cells. Fluorescence Lifetime Imaging Microscopy (FLIM) has indeed been successfully applied to differentiate spectrally undistinguishable molecular species (1) and to map local modifications in labeled samples in terms of ion concentration (2), pH (3), and oxygen (4). FLIM has also been widely used to explore conformational change of proteins (5) or to separate interacting and non-interacting molecular fractions in Förster Resonance Energy Transfer (FRET) experiments (6,7).However, because of the low number of collected photons per pixel and the variability of the molecular environment inside a living cell, FLIM is usually limited to two lifetime components in the same pixel. This restriction makes it difficult to extract biologically relevant information from autofluorescent, heterogeneous, lightscattering samples such as biological tissue. Because of optical properties of tissue, a dedicated temporally and spectrally resolved system (called SLIM for Spectral and Lifetime Imaging Microscopy) is necessary for example to be able to explore molecular interactions on a nanometric scale (with FRET experiments).Temporal and spectral measurements can be performed using two approaches, either frequency domain (8,9) or time domain (10-12) measurements. In both cases, the determination of lifetimes and contributions of each molecular species are mainly achieved by fitting the collected data at each pixel using equations with multiple

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“…single-and multi-gate imaging by means of either slow but highly efficient point-detectors 12,43,45 or by means of fast but low-efficiency image intensifiers 11,41,49 , are alternative TD FLIM techniques compatible with two-photon microscopy. Thereby, one or more time-gates are synchronised with the pulse train of a high-repetition rate pulsed laser, e.g.…”

Section: Time-gated Flimmentioning

confidence: 99%

“…interactions between proteins in living cells 48,[82][83][84][85][86] , photophysics of complexes involved in plant photosynthesis 87 or the redox metabolism 38,50,[88][89][90] . In the last years, FLIM gained on interest in the biomedicine, as well 27,49,78,[91][92][93][94][95][96] . For instance, a FLIM device for the clinical diagnosis of malign skin tissue is commercially available (DermaInspect, JenLab, Jena, Germany) 27 .…”

Section: Bioscientific Applicationsmentioning

confidence: 99%

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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Fluorescence lifetime imaging with picosecond resolution for biomedical applications

Cited by 145 publications

References 8 publications

Time-domain fluorescence lifetime imaging applied to biological tissue

Time-domain fluorescence lifetime imaging applied to biological tissue

Three‐dimensional polar representation for multispectral fluorescence lifetime imaging microscopy

Fluorescence lifetime imaging in biosciences: technologies and applications

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