Imaging biochemistry inside cells

Wouters, Fred S.; Verveer, Peter J.; Bastiaens, Philippe I. H.

doi:10.1016/s0962-8924(01)01982-1

Cited by 428 publications

(301 citation statements)

References 57 publications

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“…For example, when the transfer efficiency is 50%, then the short lifetime must be reduced from 1.14 to 0.57 ns and the longer one from 3.5 to 1.75 ns, yielding an average fluorescence lifetime of 1.34 ns as compared to 2.67 ns in the absence of FRET (see Table I). In order to eliminate the environment-sensitive lifetime of ECFP in FLIM experiments reporting on FRET, acceptor photobleaching should then be applied yielding a reference donor fluorescence lifetime [23]. Again, because of its monoexponential fluorescence decay, EYFP would be a much better donor in FLIM experiments reporting on FRET.…”

Section: Discussionmentioning

confidence: 99%

Effects of Refractive Index and Viscosity on Fluorescence and Anisotropy Decays of Enhanced Cyan and Yellow Fluorescent Proteins

et al. 2005

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The fluorescence lifetime strongly depends on the immediate environment of the fluorophore. Timeresolved fluorescence measurements of the enhanced forms of ECFP and EYFP in water-glycerol mixtures were performed to quantify the effects of the refractive index and viscosity on the fluorescence lifetimes of these proteins. The experimental data show for ECFP and EYFP two fluorescence lifetime components: one short lifetime of about 1 ns and a longer lifetime of about 3.7 ns of ECFP and for EYFP 3.4. The fluorescence of ECFP is very heterogeneous, which can be explained by the presence of two populations: a conformation (67% present) where the fluorophore is less quenched than in the other conformation (33% present). The fluorescence decay of EYFP is much more homogeneous and the amplitude of the short fluorescence lifetime is about 5%. The fluorescence anisotropy decays show that the rotational correlation time of both proteins scales with increasing viscosity of the solvent similarly as shown earlier for GFP. The rotational correlation times are identical for ECFP and EYFP, which can be expected since both proteins have the same shape and size. The only difference observed is the slightly lower initial anisotropy for ECFP as compared to the one of EYFP.

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Section: Discussionmentioning

confidence: 99%

Effects of Refractive Index and Viscosity on Fluorescence and Anisotropy Decays of Enhanced Cyan and Yellow Fluorescent Proteins

et al. 2005

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“…[89,90] Moreover, fluorescent proteins and advanced imaging techniques have revolutionized the ability to follow proteins in live cells in real time. [91][92][93] Although these approaches provide insight into the machinations of multiprotein complexes, the molecular details that underlie inter-receptor processes often are obscure. This low resolution view is largely due to an old problem: the details revealed by an experiment are limited by the resolution of the investigative methods.…”

Section: Challenges To Studying Receptor Functionmentioning

confidence: 99%

Synthetic Multivalent Ligands as Probes of Signal Transduction

2006

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Cell surface receptors acquire information from the extracellular environment and coordinate intracellular responses. Evidence from biochemical and structural studies indicates that many receptors do not operate as individual entities, but rather as part of higher-order complexes (e.g. dimers and oligomers). Coupling the functions of multiple receptors may endow signaling pathways with the sensitivity and malleability required to govern cellular responses. Moreover, multireceptor signaling complexes may provide a means of spatially segregating otherwise degenerate signaling cascades. Despite the proposed importance of receptor-receptor processes in cellular signaling, questions concerning the mechanisms, extent, and consequences of receptor co-localization and interreceptor communication remain unanswered.Chemical synthesis can provide a variety of compounds with which to address the role of receptor assembly in signal transduction. The focus of this review is one such approach -the use of synthetic multivalent ligands to characterize receptor function. Multivalent ligands can be generated that possess a variety of sizes, shapes, valencies, orientations, and densities of binding elements. Their unique architectures imbue multivalent ligands with the ability to access binding modes not available to monovalent compounds. Multivalent ligands, therefore, are capable of illuminating aspects of inter-receptor processes that are not readily probed using conventional approaches. We suggest that, as focus shifts from investigations of the function of individual proteins and toward the analysis of multi-receptor signaling complexes, multivalent ligands will become even more valuable tools with which to ask sophisticated mechanistic questions. Further, multivalent ligands may provide new opportunities for manipulating receptor systems for the deconvolution of pathways, diagnosis, and, ultimately, the treatment of disease.

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“…This paper aims to describe our approach to timedomain FLIM and to review our recent work, as presented at the ESP 2003 conference [3] in Vienna, with particular emphasis on its application to biological tissue. It is not intended as a general discussion of FLIM -for this we could suggest the papers by Cubeddu et al [4], Wouters et al [5] and Tadrous [6]. For a discussion of FLIM applied to FRET, we suggest the paper by Bastiaens and Squire [7].…”

Section: Introductionmentioning

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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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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Imaging biochemistry inside cells

Cited by 428 publications

References 57 publications

Effects of Refractive Index and Viscosity on Fluorescence and Anisotropy Decays of Enhanced Cyan and Yellow Fluorescent Proteins

Effects of Refractive Index and Viscosity on Fluorescence and Anisotropy Decays of Enhanced Cyan and Yellow Fluorescent Proteins

Synthetic Multivalent Ligands as Probes of Signal Transduction

Time-domain fluorescence lifetime imaging applied to biological tissue

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