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 Microscope
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)


Deconvolution Algorithm
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


Results
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.


Resolution
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.