Intracellular free calcium has been recognized as a regulator of many cellular processes and plays a key role in mediating actions of many drugs. To elucidate subcellular spatial calcium changes throughout the cell in three dimensions (3-D), optical sectioning microscopy was applied using digital imaging coupled fluorescence microscopy. The cell was loaded with a fluorescent indicator, fura-2, and a stack of sectional fluorescent images were acquired, digitized and finally stored on-line for post image analysis. Each sectional image was then deconvolved, to remove contaminating light signals from adjacent planes, using the Nearest Neighboring Deconvolution Algorithm (NNDA) and the overall imaging system's empirical Point Spread Function (PSF) that is measured with a 0.25 micrometers fluorescent bead. Using this technique, we measured that the addition of growth factors caused a 2 - 3 fold increase (1) in nuclear calcium compared to cytosolic calcium in blood cells and (2) in both nuclear and cytosolic calcium in liver cells. Such spatial information, which is important in understanding subcellular processes, would not be possible to measure with other methods.
Cardiac cells exhibit rhythmic contractions when electrically paced and maintain striation patterns throughout the contraction-relaxation cycle similar to one-dimensional diffraction grating that changes in spatial frequency. The current study demonstrates the application of an on-line hybrid optical-digital processor with liquid crystal television (LCTV) acting as a spatial light modulator(SLM) to monitor the dynamics of contraction of single cardiac cells in real-time . The processor which is interfaced with a phase contrast microscope performs the Fourier transformation of the cell''s striated image optically and records the Fourier spectra digitally using a charge coupled device (CCD) camera. A series of digitized images of the Fourier spectra each of duration of 16 . 67 msec (non-interlaced frames) is captured during one contraction cycle (''4000 msec). The sarcomere length at each time point and contraction velocities are calculated. The overall system is initially calibrated using a high-resolution Ealing target. Experimental results demonstrate the improvement of contrast by about 300 using the LCTV and the optical-digital processor can faithfully track sarcomere mechanics. 1 .