Zeiss LSM 510 confocal manual - Duke University Light Microscopy ...

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2 of 6 Focus carefully - the objective moves up and down and the stage is fixed so be careful not to hit your sample with the objective (not good for either party). Click "RL Illumination Off" on the touch pad or the shutter off button to turn off fluorescence (if you just use BF leave it on so the system automatically turns that on each time you go to ocular) Taking confocal images Click the "Acquisition" tab so you see all the settings for confocal imaging. If the "Show manual tools" box is unchecked some of the less common adjustments are hidden (the Show all check marks on each blue bar gives a similar option). Setup If you follow the smart setup path . . .
3 of 6 Manual setup Or you can manually change the excitation lasers, emission bands, dichroics etc in the Imaging Setup and Light Path tabs. The MBS (Main Beam Splitter = dichroic) needs to match the lasers used in the visible and 405 light paths) One track for simultaneous imaging of the channels, more than one track for sequential scanning For multi-track: Frame switching should be used if different dichroics, pinholes or overlapping emission bands are required. Line switching is more convenient and possible when only electronic things change between tracks (eg AOTF control of excitation, which detector is active) Ch1 and Ch2 are standard alkali PMTs, ChS1 is an array of 32 high-sensitivity GaAsP PMTs. This can be used as up to 8 channels in Channel mode (but they have to have the same gain at any time, but different digital gain is allowed). If you have created a configuration you can save this for easy reloading in the future Please ask if you need some help to design an optimal setup for your particular fluorophores and experiment. Scanning and optimizing your image Pressing Auto-Exposure is a good starting point to get an image on the screen to optimize. Auto-exposure changes the detector gain values so something is visible.
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Zeiss LSM 510 confocal manual - Duke University Light Microscopy ...

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