Fluorescence was monitored with a custom-designed two-photon microscope (Fig. 1b ). The embedded zebrafish were placed under a water immersion 40x/0.8 numerical aperture objective (OBJ1; LUMPlanFl40XW/IR2, Olympus) that focused the excitation light from a MaiTai HP laser (Newport). Multiple horizontal planes through the cerebellum were imaged at a 2 Hz scan rate (512 × 512 pixels, ~3 planes per fish). Emitted light was collected through the 40x objective, and reflected by two dichroic mirrors (DM1, 720dcxruv; DM2, 565dcxr; Chroma Technology) into separate red (F2; ET605\70m-2p, Chroma) and green (F3; ET525\50m-2p; Chroma) channels. Fluorescence signals were detected and amplified by photomultiplier tubes (PMTs, R3896; Hamamatsu), while scattered light from the focal plane was detected by a substage photodetector (PD, PDA36A, Thorlabs). Signals from the PMTs and the PD were acquired by ScanImage93 to generate fluorescence and contrast images of specimen in the focal plane.
Lumplanfl40xw ir2
Manufactured by Olympus
The LUMPlanFl40XW/IR2 is a water immersion objective lens designed for microscopy applications. It features a 40X magnification and is optimized for infrared wavelengths. The lens is suitable for a variety of imaging techniques.
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Lab products found in correlation
2 protocols using lumplanfl40xw ir2
1
Two-Photon Imaging of Zebrafish Cerebellum
2
Zebrafish Calcium Imaging with Two-Photon Microscopy
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A custom-built two photon microscope was used for imaging calcium activity and fluorescence in the embedded zebrafish. A MaiTai HP laser (Newport) was used to focus a 915 nm light beam through a water immersion 40x/0.8 NA objective (OBJ1; LUMPlanFl40XW/IR2, Olympus) onto the fish. Multiple separate horizontal planes of the fish were acquired sequentially in the dark at 2 Hz (256×256 pixels, 550x550 µm). The emitted light from the zebrafish larva was collected by the objective and directed towards two dichroic mirrors (DM1, 720dcxruv; DM2, 565dcxr; Chroma Technology) which reflected this light into two separate channels of green (F3; ET525\50m-2p; Chroma) and red (F2; ET605\70m-2p, Chroma) to be then amplified by the photomultiplier tubes (PMTs, R3896; Hamamatsu). A substage photodetector (PD, PDA36A, Thorlabs) detected light scattered from the focal plane and together with signal from the PMTs was processed by MATLAB based software ScanImage (53) to generate fluorescence images.
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