{"id":192,"date":"2019-07-05T15:35:06","date_gmt":"2019-07-05T19:35:06","guid":{"rendered":"https:\/\/sites.bu.edu\/biomicroscopy\/?page_id=192"},"modified":"2019-07-15T09:43:36","modified_gmt":"2019-07-15T13:43:36","slug":"autoconfocal","status":"publish","type":"page","link":"https:\/\/sites.bu.edu\/biomicroscopy\/research\/autoconfocal\/","title":{"rendered":"Autoconfocal microscopy"},"content":{"rendered":"

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ACM is a nonlinear-optical microscopy that uses a femtosecond-pulsed infrared laser beam to trans-illuminate a tissue sample. As opposed to other nonlinear-optical techniques such as two-photon excited fluorescence (TPEF) microscopy, the laser beam does not generate signal within the sample. Instead, the laser beam traverses the sample and is then re-focused onto a nonlinear crystal. The detected signal is the SHG produced by the crystal. Because this signal is nonlinear, the crystal acts as a \u201cvirtual pinhole\u201d and an ACM exhibits 3D resolution and background rejection similar to a transmission-confocal microscope. An ACM requires no sample labeling, is technically simple, and can be readily combined with existing nonlinear imaging modalities such as TPEF microscopy.<\/p>\n

We have shown that the equivalent effect of a \u201cvirtual pinhole\u201d can be achieved by thermionic emission in a standard PMT photocathode. Because of the nonlinear dependence of thermionic emission on absorbed laser power, a PMT is found to produce a virtual pinhole effect that rejects unfocused light at least as strongly as a physical pinhole. This virtual pinhole effect can be exploited in a scanning transmission confocal microscope equipped with a cw laser source. Because the area of the PMT photocathode is large, signal de-scanning is not required and thermionic detection acts as a self-aligned pinhole.<\/p>\n

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Simultaneous graded-field ACM (gray) and TPEF (green) images of a rat hippocampus slice (400 \u03bcm thickness) stained with sulforhodamine 101, which preferentially labels astrocytes.<\/figcaption><\/figure>\n