The relaxation of fluorescence from diffraction-limited sources of photoactivatable green fluorescent protein (PAGFP) or sinks of photobleached enhanced GFP (EGFP) created by multiphoton photo-conversion was measured in solutions of varied viscosity (eta), and in live, spherical Chinese hamster ovary (CHO) cells. Fluorescence relaxation was monitored with the probing laser fixed, or rapidly scanning along a line bisected by the photoconversion site. Novel solutions to several problems that hamper the study of PAGFP diffusion after multiphoton photoconversion are presented. A theoretical model of 3D diffusion in a sphere from a source in the shape of the measured multiphoton point-spread function was applied to the fluorescence data to estimate the apparent diffusion coefficient, D(ap). The model incorporates two novel features that make it of broad utility. First, the model includes the no-flux boundary condition imposed by cell plasma membranes, allowing assessment of potential impact of this boundary on estimates of D(ap). Second, the model uses an inhomogeneous source term that, for the first time, allows analysis of diffusion from sources produced by multiphoton photoconversion pulses of varying duration. For diffusion in aqueous solution, indistinguishable linear relationships between D(ap) and eta(-1) were obtained for the two proteins: for PAGFP, D(aq)= 89 +/- 2.4 microm2 s(-1) (mean +/- 95% confidence interval), and for EGFP D(aq)= 91 +/- 1.8 microm2 s(-1). In CHO cells, the application of the model yielded D(ap)= 20 +/- 3 microm2 s(-1) (PAGFP) and 19 +/- 2 microm2 s(-1) (EGFP). Furthermore, the model quantitatively predicted the decline in baseline fluorescence that accompanied repeated photobleaching cycles in CHO cells expressing EGFP, supporting the hypothesis of fluorophore depletion as an alternative to the oft invoked 'bound fraction' explanation of the deviation of the terminal fluorescence recovery from its pre-bleach baseline level. Nonetheless for their identical diffusive properties, advantages of PAGFP over EGFP were found, including an intrinsically higher signal/noise ratio with 488-nm excitation, and the requirement for approximately 1/200th the cumulative light energy to produce data of comparable signal/noise.
"The titanium-sapphire laser (Chameleon Ultra II; Coherent) was tuned to 800 nm for excitation of the Alexa Fluor 568 fluorochrome. Custom MATLAB (MathWorks) scripts (Calvert et al., 2007; Nikonov et al., 2008) were used for segmenting complete cones or oriented " slabs " from the 3D Z-stacks, and for extraction of voxel intensity values (in photon counts) from the 3D image stacks. "
"Lateral diffusion of Rho-PAGFP in disc membranes is directionally heterogeneous Initially, we examined Rho-PAGFP diffusion using the mFRAPa approach in the " side-on " imaging orientation (Calvert et al., 2007, 2010) (Fig. 3). We chose to use mFRAPa with a Rho-PAGFP fusion protein because the signal after photoactivation is 100-fold higher than on the nonphotoconverted background, which allows tracking of activated molecules over longer distances and times than can be achieved with traditional fluorescence recovery after photobleach (FRAPb) of EGFP (Calvert et al., 2007). Rho-PAGFP in rod outer segments of live retinal slices was photoactivated with a brief exposure from the Ti–sapphire laser applied at the radial center of outer segments. "
[Show abstract][Hide abstract] ABSTRACT: G protein-coupled receptor (GPCR) cascades rely on membrane protein diffusion for signaling and are generally found in spatially constrained subcellular microcompartments. How the geometry of these microcompartments impacts cascade activities, however, is not understood, primarily because of the inability of current live cell-imaging technologies to resolve these small structures. Here, we examine the dynamics of the GPCR rhodopsin within discrete signaling microcompartments of live photoreceptors using a novel high resolution approach. Rhodopsin fused to green fluorescent protein variants, either enhanced green fluorescent protein (EGFP) or the photoactivatable PAGFP (Rho-E/PAGFP), was expressed transgenically in Xenopus laevis rod photoreceptors, and the geometries of light signaling microcompartments formed by lamellar disc membranes and their incisure clefts were resolved by confocal imaging. Multiphoton fluorescence relaxation after photoconversion experiments were then performed with a Ti-sapphire laser focused to the diffraction limit, which produced small sub-cubic micrometer volumes of photoconverted molecules within the discrete microcompartments. A model of molecular diffusion was developed that allows the geometry of the particular compartment being examined to be specified. This was used to interpret the experimental results. Using this unique approach, we showed that rhodopsin mobility across the disc surface was highly heterogeneous. The overall relaxation of Rho-PAGFP fluorescence photoactivated within a microcompartment was biphasic, with a fast phase lasting several seconds and a slow phase of variable duration that required up to several minutes to reach equilibrium. Local Rho-EGFP diffusion within defined compartments was monotonic, however, with an effective lateral diffusion coefficient D(lat) = 0.130 ± 0.012 µm(2)s(-1). Comparison of rhodopsin-PAGFP relaxation time courses with model predictions revealed that microcompartment geometry alone may explain both fast local rhodopsin diffusion and its slow equilibration across the greater disc membrane. Our approach has for the first time allowed direct examination of GPCR dynamics within a live cell signaling microcompartment and a quantitative assessment of the impact of compartment geometry on GPCR activity.
The Journal of General Physiology 08/2012; 140(3):249-66. DOI:10.1085/jgp.201210818 · 4.79 Impact Factor
"The reaction product is insoluble in water and alcohol and after standard preparation can be used to achieve differentiated subcellular localization of molecules with a high spatial resolution in the electron microscope (Fig. 1). Photoconversion on the other hand characterizes the light induced structural change of fluorophores, i.e. light can activate or convert fluorescent proteins to states that are excitable and/or emit in spectral bands distinct from those characterizing the native state (Calvert et al., 2007). Examples are the fluorescence enhancement of GFP-tagged proteins Fig. 1. "
[Show abstract][Hide abstract] ABSTRACT: The combination of the capabilities of light microscopical techniques with the power of resolution of electron microscopy along with technical advances has led to a gradual decline of the gap between classical light and electron microscopy. Among the correlative techniques using the synergistic opportunities, photooxidation methods have been established as valuable tools for visualizing cell structures at both light and electron microscopic level. Fluorescent dyes are used to oxidize the substrate diaminobenzidine, which in its oxidized state forms fine granular precipitates. Stained with osmium, the diaminobenzidine precipitates are well discernible in the electron microscope, thus labelling and defining the cellular structures, which at light microscopy level are recorded by fluorescent probes. The underlying photooxidation reaction is based on the excitation of free oxygen radicals that form upon illumination of fluorochromes; this is a central step in the procedure, which mainly influences the success of the method. This article summarizes basic steps of the technology and progresses, shows efforts and elaborated pathways, and focuses on methodical solutions as to the applicability of different fluorochromes, as well as conditions for fine structural localizations of the reaction products.
Journal of Microscopy 09/2009; 235(3):322-35. DOI:10.1111/j.1365-2818.2009.03220.x · 2.33 Impact Factor
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