A conventional epifluorescence microscope exhibits a partial confocal effect when a small illumination aperture is used. This effect causes a reduction in the out of focus light present when viewing a 3D object. Inserting smaller apertures than the usual iris diaphragm causes a large confocal effect; an illumination spot at the sample of, for example, 2 microns across results in the nearly complete absence of out of focus light originating more than 2 microns out of focus. We propose to develop instruments based on this effect combined with computational methods called image restoration or deconvolution that reverse the blurring of the optics and remove any remaining out of focus light. We will notify two conventional epifluorescence microscopes, one which will scan the sample across the illumination spot and another that will scan the illumination spot across the sample. Only the illumination path is changed by replacing the usual iris diaphragm by a smaller aperture. The light collection path is entirely unchanged, so high quantum efficacy CCD cameras can still be used; thus the light collection loses of conventional confocal microscopes are avoided. These instruments will have significant advantages for imaging of fluorescently labeled biological samples. They will provide high resolution 3D images in thick samples where there is too much out of focus light for seeing fine detail in conventional microscopes. In combination with image restoration these instruments will provide greater resolution, contrast, and accuracy of quantitative fluorescence measurements in living fluorescently labelled samples that is possible with either conventional side field or conventional confocal microscopes. This new instrument will be tested and applied to studies directed at understanding the control of vascular function.
| Fogarty, K E; Kidd, J F; Turner, A et al. (2000) Microtubules regulate local Ca2+ spiking in secretory epithelial cells. J Biol Chem 275:22487-94 |
| Fogarty, K E; Kidd, J F; Tuft, R A et al. (2000) A bimodal pattern of InsP(3)-evoked elementary Ca(2+) signals in pancreatic acinar cells. Biophys J 78:2298-306 |
| Dictenberg, J B; Zimmerman, W; Sparks, C A et al. (1998) Pericentrin and gamma-tubulin form a protein complex and are organized into a novel lattice at the centrosome. J Cell Biol 141:163-74 |
| Lifshitz, L M (1998) Determining data independence on a digitized membrane in three dimensions. IEEE Trans Med Imaging 17:299-303 |
| Rizzuto, R; Carrington, W; Tuft, R A (1998) Digital imaging microscopy of living cells. Trends Cell Biol 8:288-92 |
| Rizzuto, R; Pinton, P; Carrington, W et al. (1998) Close contacts with the endoplasmic reticulum as determinants of mitochondrial Ca2+ responses. Science 280:1763-6 |
