XB-IMG-124731
Xenbase Image ID: 124731
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Figure 3. Equilibration of PAGFP throughout the cytoplasm of a rod after
photoactivation in the IS. (A) Infrared image of the retinal slice
before experiment. The rod in which PAGFP was photoconverted is
indicated by the arrowhead. (B–D) All images were obtained with
the microscope operating in confocal mode, with the power of the 488-nm
argon ion laser line attenuated to 2 µW (at the sample). (B)
Initial fluorescence distribution of PAGFP in the central
z image of the rod, intensity scaled to more
clearly show the cell structure. The red dot in the center of the image
is an overlay of the field intensity profile of the
psf, indicating the spatial position in
x–y and the dimensions of
the multiphoton photoconversion pulse. OS, outer segment; IS, inner
segment; N, nucleus; S, synaptic region. (C) The final time series image
of the rod (25.5 min from the onset of photoconversion), where the
boundary between the IS and OS was more clearly identifiable, was used
to delineate regions (red polygons) over which fluorescence levels were
integrated in each of the time series images. See results for details of
the analysis. (D) Selected images of the fluorescence time series,
starting with an image taken just before the photoconversion exposure.
Photoconversion was effected by a 100-ms, 20-mW (at the sample) pulse
from the Ti:S laser tuned to 820 nm. The times in subsequent images are
measured from the moment of pulse initiation. The color bar to the right
codes the fluorescence intensity in photon counts and is relevant to the
images in C and D only. (E) The integrated fluorescence
(F) in the OS (Region 1 in C) and the IS (Region 2
in C), normalized to the integrated fluorescence within the respective
region just before photoconversion (F0), as
a function of time after the photoconversion pulse. The dashed line
drawn at F/F0 = 5.3
indicates the time-averaged magnitude of total cell fluorescence
increase post-photoconversion, and thus represents the value of
F/F0 expected for all
compartments upon equilibration (see Fig. 5 and Results for details).
T1/2 is the time required for the OS
compartment to reach 1/2 the
F/F0 value at
equilibrium. (F) Integrated fluorescence over the entire cell image
normalized to the integrated prepulse fluorescence
(Ftot/Ftot,0),
as a function of time from pulse onset. The total post-pulse
fluorescence remains within ∼7% of the median post-pulse value.
(A–D) Bars, 10 µm. See Video 1. Image published in: Calvert PD et al. (2010) © 2010 Calvert et al. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike license Larger Image Printer Friendly View |