Article Information

• PDF (935 kb)

PubMed

• Articles by G.E. Rice
• Articles by T.T. Bannister

Related Articles

• Temporally and Spectrally Resolved Imaging Microscopy of Lan...

  Temporally and Spectrally Resolved Imaging Microscopy of Lanthanide Chelates Biophysical Journal, Volume 74, Issue 5, 1 May 1998, Pages 2210-2222 György Vereb, Elizabeth Jares-Erijman, Paul R. Selvin and Thomas M. Jovin Abstract The combination of temporal and spectral resolution in fluorescence microscopy based on long-lived luminescent labels offers a dramatic increase in contrast and probe selectivity due to the suppression of scattered light and short-lived autofluorescence. We describe various configurations of a fluorescence microscope integrating spectral and microsecond temporal resolution with conventional digital imaging based on CCD cameras. The high-power, broad spectral distribution and microsecond time resolution provided by microsecond xenon flashlamps offers increased luminosity with recently developed fluorophores with lifetimes in the submicrosecond to microsecond range. On the detection side, a gated microchannel plate intensifier provides the required time resolution and amplification of the signal. Spectral resolution is achieved with a dual grating stigmatic spectrograph and has been applied to the analysis of luminescent markers of cytochemical specimens in situ and of very small volume elements in microchambers. The additional introduction of polarization optics enables the determination of emission polarization; this parameter reflects molecular orientation and rotational mobility and, consequently, the nature of the microenvironment. The dual spectral and temporal resolution modes of acquisition complemented by a posteriori image analysis gated on the spatial, spectral, and temporal dimensions lead to a very flexible and versatile tool. We have used a newly developed lanthanide chelate, Eu-DTPA-cs124, to demonstrate these capabilities. Such compounds are good labels for time-resolved imaging microscopy and for the estimation of molecular proximity in the microscope by fluorescence (luminescence) resonance energy transfer and of molecular rotation via fluorescence depolarization. We describe the spectral distribution, polarization states, and excited-state lifetimes of the lanthanide chelate crystals imaged in the microscope. Abstract | Full Text | PDF (797 kb)

• Late Events in the Photocycle of Bacteriorhodopsin Mutant L9...

  Late Events in the Photocycle of Bacteriorhodopsin Mutant L93A Biophysical Journal, Volume 84, Issue 6, 1 June 2003, Pages 3848-3856 R. Tóth-Boconádi, L. Keszthelyi and W. Stoeckenius Abstract In the photocycle of bacteriorhodopsin (bR) from mutant L93A, the O-intermediate accumulates and the cycling time is increased ∼200 times. Nevertheless, under continuous illumination, the protein pumps protons at near wild-type rates. We excited the mutant L93A in purple membrane with single or triple laser flashes and quasicontinuous illumination, (i.e., light for a few seconds) and recorded proton release and uptake, electric signals, and absorbance changes. We found long-living, correlated, kinetic components in all three measurements, which—with exception of the absorbance changes—had not been seen in earlier investigations. At room temperature, the O-intermediate decays to bR in two transitions with rate constants of 350 and 1800ms. Proton uptake from the cytoplasmic surface continues with similar kinetics until the bR state is reestablished. An analysis of the data from quasicontinuous illumination and multiple flash excitation led to the conclusion that acceleration of the photocycle in continuous light is due to excitation of the N-component in the fast N↔O equilibrium, which is established at the beginning of the severe cycle slowdown. This conclusion was confirmed by an action spectrum. Abstract | Full Text | PDF (179 kb)

• Multiple Phosphorylation of Rhodopsin and the In Vivo Chemis...

  Multiple Phosphorylation of Rhodopsin and the In Vivo Chemistry Underlying Rod Photoreceptor Dark Adaptation Neuron, Volume 31, Issue 1, 19 July 2001, Pages 87-101 Matthew J. Kennedy, Kimberly A. Lee, Gregory A. Niemi, Kimberley B. Craven, Gregory G. Garwin, John C. Saari and James B. Hurley Summary Dark adaptation requires timely deactivation of phototransduction and efficient regeneration of visual pigment. No previous study has directly compared the kinetics of dark adaptation with rates of the various chemical reactions that influence it. To accomplish this, we developed a novel rapid-quench/mass spectrometry-based method to establish the initial kinetics and site specificity of light-stimulated rhodopsin phosphorylation in mouse retinas. We also measured phosphorylation and dephosphorylation, regeneration of rhodopsin, and reduction of all- retinal all under identical in vivo conditions. Dark adaptation was monitored by electroretinography. We found that rhodopsin is multiply phosphorylated and then dephosphorylated in an ordered fashion following exposure to light. Initially during dark adaptation, transduction activity wanes as multiple phosphates accumulate. Thereafter, full recovery of photosensitivity coincides with regeneration and dephosphorylation of rhodopsin. Summary | Full Text | PDF (432 kb)

• …more

Abstract

Slow fluorescence transients in Chlamydomonas reinhardi arise after transitions from high light intensities to low light or dark conditions. Characteristics of the newly described transient phenomena include: (a) A slow biphasic decrease in fluorescence yield occurs in the dark, followed by an even slower, hour long, increase in fluorescence. (b) A similar, but faster, fluorescence yield decrease and subsequent increase also occurs during low intensity illumination periods separating high light intervals, or after transitions from high intensity to low intensity light. (c) Short (several seconds) flashes of light given during a dark period have no effect on the dark fluorescence decay, regardless of the flash frequency. Such flash regimes accurately monitor the dark decline of the M2 level by tracing the parallel decay of flash-generated P2 (Kautsky) peaks. However, flashes during a low light illumination period do influence the decay kinetics. Frequent flashes allow decay similar to that occurring in dark, but less frequent flashes inhibit the decrease in fluorescence yield.