We propose to develop total internal reflectance fluorescence (TIRF) microscopy in an imaging modality with a sharper focal plane (approximately 60 nm) and sub-millisecond time resolution (2000 frames/sec) for the study of rapid signaling near the cell membrane. To achieve this we shall use a novel optical design and operate in a mode where the response time of the fluorescent probe (e.g. Fluo- 3) is governed primarily by its diffusion out of the evanescent field (<<1 ms), not by its association time (k/off/-1 approximately- 10 ms). Preliminary experiments performed with a prototype system (approximately 100 nm focal plane, 734 frames/sec) have demonstrated proof-of-principle and resolved subsarcolammel Ca 2v signals in voltage-clamped rat atrial cardiomyocytes in terms of apparent single-channel Ca 2+ fluxes and intermittently active Ca 2+ release-components of Ca 2+ sparks.
The specific aims are: -1-To sharpen the focal plane (100->60 nm) by using denser optical materials (n: 151->1.77) and purchase and use a faster, more sensitive CCD camera (734-> 2000 frames/sec). -2- To evaluate the performance of this system with respect to speed, sensitivity and noise while varying the penetration (200-60 nm) and using diffusible and non-diffusible fluorescent probes. -3-To ascertain if the subsarcolemmal Ca2+-signals in atrial cardiomyocytes can be resolved in terms of single channel Ca 2+ fluxes produced by DHP- and/or ryanodine-receptors. -4-To evaluate, in a model system of cultured adrenal chromaffin cells, if protons are co-released in sufficient numbers to modulate synaptic transmission by transient acidification of synaptic clefts. While the two listed applications are relevant to our ongoing research, we believe that implementation of the TIRF technique with sufficient sensitivity and speed to match electrical recordings of single-channel currents will be of considerable general usefulness in the study of the structures and regulatory processes that are associated with cell membranes and their immediate vicinity.
