The design and construction of a laser scanning microscope for quantitative imaging of fluorescently-labelled cells is proposed. Fluorescence techniques permit the sensitive detection and monitoring of a variety of important chemical and biophysical processes within living cells, including membrane potentials, pH, pCa, and mobility of selected molecules. In a laser scanning microscope, the focussed laser beam is a spatially resolved optical probe which can be used to make precise measurements of fluorescent intensity, and potentially of fluorescence lifetime and anisotropy. This microscope will be constructed in several steps. Laser scanning optics previously developed in the P.I.'s laboratory will be adapted to an inverted microscope stand. A new laser scan controller will provide video-like scans, region-of-interest and random-access (point hopping) scans. Image acquisition, processing, and control functions will be hosted by a DEC MicroVAX II computer, with a high speed Ethernet data link to larger computers for intensive processing. Laser scanning will be integrated with conventional video imaging via a sophisticated graphical interface. A precise Z-axis stage control will permit through-focus image series to be acquired for tomographic reconstruction. The hardware and software requirements for fluorescence lifetime imaging will be analyzed. The microscope will be tested both with standard fluorescent preparations and in specific cell biology investigations. The microscope will be a flexible research instrument permitting the development and evaluation of new scanning based approaches to fluorescence.
