Fast data acquisition: Conventional point illumination scan rates (~1 frame/sec) are inadequate to temporally resolve many biological events, such as intracellular ion activity and many enzymatic reactions. Focusing the excitation beam to a line (rather than a point) allows video rate scan speeds (30 frames/sec) and thus permits imaging of biomolecular events at the tens of millisecond time scale. Our lab has constructed a real-time MPE fluorescence microscope, based on a Bio-Rad line scanning confocal microscope (diagram to right). The image at the lower right shows a measured spontaneous calcium transient in a Fluo-3 loaded neonatal rat myotube. The image was acquired by holding the focused line stationary. Note that the temporal resolution is 70 microseconds.

We are currently characterizing and optimizing the MPE line scanner. At the present level of engineering, the instrumentation suffers from two primary problems: (1) The supplied cylindrical lens introduces axial aberrations which cause a twofold degradation of the axial resolution, a fault that can be remedied by a redesign of the lens. (2) Image signal-to-noise is currently limited by the noise associated with the image intensifier. We plan to decrease the detector noise by utilizing a cooled CCD. To increase the readout time to video rates we will use multiple fractions of the CCD face for sequential images. Real-time image acquisition from a point scanning microscope. Using a 1 MHz, 12 bit A/D data streaming system, image data can be saved directly to an AV hard disk. At imaging rates of up to 7 frames/sec (256 x 256 pixels), the total number of frames stored depends only on the size of the hard disk.

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Last update: August 13, 2003