Following FRET through five energy transfer steps: spectroscopic photobleaching, recovery of spectra, and a sequential mechanism of FRET

Forde, Toni S; Hanley, Quentin S.

doi:10.1039/b416478d

Cited by 17 publications

(11 citation statements)

References 31 publications

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“…The T-8869 beads consist of a system of six BODIPY dyes emitting at 720, 678, 635, 592, 560 and 515 nm. Under normal conditions, these microspheres exhibit highly efficient energy transfer along a series beginning with the first donor (515 nm) to the terminal acceptor (720 nm) (Roberts et al 1998;Forde & Hanley 2005) The assumption of no competing dark processes is equivalent to assuming that the quantum yields of both the donor and acceptor are 1. If the acceptor transfers 50 per cent of its energy via FRET to a subsequent acceptor then the amount of light observed from the first acceptor will be one-half of that from the principal donor.…”

Section: Energy Transfer and Allowed Regions In Ab-spacementioning

confidence: 99%

Spectrally resolved fluorescent lifetime imaging

Hanley

2008

J. R. Soc. Interface.

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Placing an imaging spectrograph or related components capable of generating a spectrum between a microscope and the image intensifier of a conventional fluorescence lifetime imaging (FLIM) system creates a spectrally resolved FLIM (SFLIM). This arrangement provides a number of opportunities not readily available to conventional systems using bandpass filters. The examples include: simultaneous viewing of multiple fluorophores; tracking of both the donor and acceptor; and observation of a range of spectroscopic changes invisible to the conventional FLIM systems. In the frequency-domain implementation of the method, variation in the fractional contributions from different fluorophores along the wavelength dimension can behave as a surrogate for a frequency sweep or spatial variations while analysing fluorophore mixtures. This paper reviews the development of the SFLIM method, provides a theoretical and practical overview of frequency-domain SFLIM including: presentation of the data; manifestations of energy transfer; observation of multiple fluorophores; and the limits of single frequency methods.

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Section: Energy Transfer and Allowed Regions In Ab-spacementioning

confidence: 99%

Spectrally resolved fluorescent lifetime imaging

Hanley

2008

J. R. Soc. Interface.

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“…5a). In contrast to the extensively characterized T-8869 beads (17,27), the T-8883 beads do not transfer energy primarily to a single terminal acceptor. At the beginning of the experiment, evidence of three dyes was readily observed.…”

Section: Spectroscopic Lifetime Analysis Of Beads Exhibiting Energy Tmentioning

confidence: 99%

“…Spectral imaging with a prism-based spectrograph during photobleaching allows simultaneous observation of multiple fluorescent dyes (17). The advantage is that FRET relationships between the dyes can be determined, and parallel processes can be looked at in isolation.…”

Section: Imaging Spectroscopic Photobleachingmentioning

confidence: 99%

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Microspectroscopic fluorescence analysis with prism‐based imaging spectrometers: Review and current studies

2006

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Background: Fluorescence imaging spectroscopy is a powerful but under-utilized tool. This article gives perspective on the use of imaging spectroscopy, and provides two examples of imaging spectroscopy done with a prism-based system. The intent is to give insight into the power of imaging spectroscopy when used in combination with other imaging techniques. In particular, studies of intact coral photobleaching and beads designed to show energy transfer are reported. In the bead study, spectroscopic lifetime imaging was performed at each photobleaching step. Results: Spectroscopic photobleaching of the hard coral, Montastrea annularis, revealed two spectral regions. A region in the red portion of the spectrum bleached rapidly while progressively increasing fluorescence was observed over a wide portion of the spectrum. This behavior is consistent with current theories for the role of fluorescent proteins in corals.

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“…However, with suitable chromophore differentiation, functionally unidirectional transfer is exhibited in multichromophore systems, assisted by suitable chromophore disposition, and often the operation of a spectroscopic gradient. Through experimental studies involving photobleaching spectroscopy, it has recently been shown possible to trace the intricate form of such multi-step resonance energy transfer [13], allowing sequential mechanisms to be established in kinetically and spectroscopically complex systems. Whilst the principles that govern the directionality of energy flow in multichromophore systems is largely understood, other recent developments have identified new possibilities for effecting externally determined directional control.…”

Section: Introductionmentioning

confidence: 99%