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Laser Scanning Confocal Fluorescence, Fluorescence Recovery After Photobleaching (FRAP)

The Opterra Multipoint Scanning Confocal Microscope is an inverted laser scanning confocal microscope being developed in collaboration with LOCI for improvements in speed, software analysis, photomanipulation and fast spectral collection. The current prototype at LOCI can collect 8 spectral channels at 4 frames a second using an Amici prism incorporated into a multi-pinhole confocal to allow dynamic spectral scanning for live cell imaging in real-time.  The software-controlled aperture design uses a combination of one-dimensional pinhole arrays for maximum resolution with half of the crosstalk, as well as aperture slits for high-speed acquisition. This combination results in sharper images, higher resolution, greater depths, and faster speeds than spinning disk confocal. The short acquisition times minimize photobleaching and phototoxicity, and this cell-friendly 4D instrument is ideal for studies including protein localization and trafficking, microtubule dynamics, mitosis, and FRAP.

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Mitosis in Xenopus laevis embryo

Image shown
: Cell membrane is labeled with targeted mTagBFP, microtubules are labeled with eGFP-tubulin, and chromatin is visualized with mCherry-Histone H2B. The image was captured with 500 ms exposure time. The image was captured with filter configuration 2 (see section "System Dichroics and Emission Filters") with a 60x oil objective and excitation at 405 nm, 488 nm, and 561 nm.




Emission filters:





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