Research - Research Groups - Biophysics Research Group - Observation Volume in Confocal Microscopy and Fluorescence Correlation Spectroscopy
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The Observation Volume in Confocal Microscopy and Fluorescence Correlation Spectroscopy Depend Strongly on Objective Underfilling and Confocal Detector Aperture. Observation profile images in log-scale pseudocolor, for a 1.2 NA objective, plotted for various values of the detector aperture rd and underfilling fraction ?. Images show the axial direction (u) horizontally and the radial direction (?) vertically away from the focus. The logarithmic color scale emphasizes the low amplitude fringes away from the focus. Closing the detector aperture (from left to right) constrains the observation volume, reducing fluorescence collection from the tails of the illumination profile which are particularly periodic and non-Gaussian for an overfilled back aperture (top row). Underfilling the back aperture (from top to bottom) elongates and smoothes the illumination profile and hence the observation volume, but the effect is not drastic until ? >1.25. Using small rd and an underfilled back aperture (? >1) results in a more nearly Gaussian observation volume. An exactly Gaussian profile is shown (bottom row left) with 20 optical unit scale bar (center) and intensity color scale (right) for reference.
An HAb2 fibroblast cell (left, lower left, and bottom) with attached bleb (just off center) labeled with the fluorescent lipids lissamine-Rhodamine B-DOPE (red channel) and perylene (blue channel) imaged by laser scanning confocal fluorescence microscopy. The phase-separated cell attached bleb shows two coexisting fluid phases and was induced at room temperature by the addition of a chemical agent. This result is surprising because normally phase separation in cell membranes is not visible to light microscopy due to the extremely small size of domains in unperturbed cell membranes. The image is 36.6 x 36.6 microns in size.
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