Different regions of a cell perform
different tasks, and this subcellular specialization is only possible
because of the stringent control a cell maintains over where proteins
are located; the cell's molecular architecture. Using a combination of
fluorescence microscopy and immunocytochemical techniques we have begun
to map the molecular architecture of smooth and cardiac muscle cells,
with particular emphasis on those proteins which regulate the
intracellular calcium concentration.
Acquisition of 3D Images
A series of 2D images are acquired of the
specimen at 0.25 μm intervals. All aspects of image capture and
microscope function are controlled through a custom written Windows
interface on the PC. Images are then transferred to a Silicon Graphics
or Linux Workstation for analysis.
Point Spread Function of a Wide-Field
Images of a sub-resolution bead labeled
with fluorescein. When the bead is at the plane of focus of the
objective it appears as a small point (centre image). When the bead is
out of the plane of focus of the objective it is still visible, but its
emitted fluorescence is spread over a large area. The greater the
distance the objective is from the plane of focus of the bead, the
greater the area over which the light is distributed. This effect
distorts microscope images since objects which are out of focus are
always visible, but will appear to be blurred the farther we are from
their plane of focus. This blur will be superimposed on objects which
are currently in focus and the result is a washed out image of poor
contrast. (28 kb)(1.23 mb)
Several laboratories have developed
algorithms which place the out of focus energy back into its point of
origin, a process called deconvolution. We use the deconvolution
algorithm developed by Dr. Walter Carrington of the Biomedical Imaging
Group at the University of Massachusetts Medical School. Although other
imaging options are available, 3D data can be obtained by confocal and
multi-photon microscopes, deconvolution of a wide field image provides
the best results for discretely organized objects from which the
emitted fluorescence intensity is low.1
These are images of the distribution of the
ryanodine receptor in a ventricular cardiomyocyte isolated from a rat
ventricle. The images on the left were acquired from the microscope.
The images on the right have been deconvolved.
One measure of resolution, and of the
effectiveness of the deconvolution algorithm, is the full width at half
maximum amplitude of a deconvolved point source object. We have
examined the FWHM amplitude through the centre of a sub-resolution bead
labeled with fluorescein in both XY and Z for 4 different imaging
situations; wide field microscopy, confocal microscopy (a Zeiss LSM 410
scanning microscope), wide field microscopy coupled with deconvolution,
and confocal microscopy coupled with deconvolution.
1. Carrington, W.A., Fogarty, K.E. and Fay,
F.S. In: The Handbook of Biological Confocal Microscopy, J.
Pauley (ed.), Plenum Press,1989.