Abstract

Optical section microscopy furnishes advantages over physical sectioning in visualizing the 3-D structure within a living cell. A hybrid digital-coupled fluorescence microscope for optical sectioning the specimen has been developed to measure the 3-D distribution of calcium in a single cardiac cell. The cell is initially loaded with fura-2, a fluorescent calcium-sensitive Indicator, and a stack of sectional fluorescent emission Images corresponding to different focal settings along the microscope’s axis of the specimen are obtained and then digitized for postprocessing. The experimental defocused point spread and contrast transfer functions of the overall system have been measured using the Ealing resolution target. Each sectional image of the stack is then processed digitally, using spread functions, by a VAX 11/780 to eliminate the defocused light contribution using the method described by Agard.¹ The stack of 2-D processed images is then displayed on a multiple pixel system to observe the 3-D distribution. We show results of 3-D calcium distribution in a cardiac muscle cell and its dynamic variation during metabolism achieved by our system.

© 1988 Optical Society of America