Introduction
In this guide of the Center for Microscopy and Image Analysis we show how to start up and mount a sample on the NEW Leica Stellaris 5 inverse confocal laser scanning microscope located at the UZH, Zollikerstrasse 107, 8008 Zürich.
It will introduce users in the new Stellaris 5 confocal laser scanning microscope. The most relevant new features are:
- NEW Spectrally flexible white light laser (WLL) with an extended spectral output on the red and near-infrared spectrum (485 nm up to 685 nm).
- NEW 3 Power HyD S detectors.
- NEW TauSense technology, which gives access to lifetime-based information, delivering additional insights to your experiments.
Please find more detailed information about the system here.
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Switch ON the "Power", "Laser", and turn the "Emission" key to "On-1" (control unit next to the microscope).
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The LED (for fluorescence observation via the oculars) switches on automatically. No action needed.
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Switch ON the PC.
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Sign-in with your ZMB core credentials.
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Start the "LAS X" software.
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Make sure "machine.xlhw" is selected as "Configuration", and "DMI8" as "Microscope".
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Click "OK".
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Click "Yes" in order to initialize the x/y stage. Please make sure nothing is placed currently on the stage.
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Lower the objective by keeping the "LOWER Z" button pressed on the right side of the microscope.
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In the "Acquire" tab you can now toggle between objectives (drop-down menu).
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Select the 5x dry objective for easy sample navigation and focusing.
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Here correct and flat.
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Here not inserted correctly (stage not flat and shaky).
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Push the condenser arm of the microscope back.
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Insert your sample with the coverslip facing down .
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Move your sample under the objective with the help of the external controller "Smart Move".
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Movement in y-direction.
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Movement in x-direction.
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Toggle between coarse movement "XY Fast" and slow movement "XY Precise".
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On the touch screen at the microscope stand choose the light path tab.
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Click "FLUO" and choose an appropriate "FLUO-Filtercube" : e.g. "DAPI".
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Open the "IL -Shutter" (if activated the dot is yellow).
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Look through the oculars and focus your sample by using:
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The z-wheel on the external controller ("Smart Move").
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Toggle between "Z FINE" and "Z COARSE" directly on the "Smart Move".
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To save your current focus position select the "xyz tab" and the "Focusdrive Z" on the touchscreen of the microscope.
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Click the "Upper Focus Limit" button.
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Press "Set".
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If done successfully you will see an upper marker line appearing.
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Press the "Lower Limit" button in order to move down (for safe change of the objective or the sample).
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Lower the objectives, remove your sample and toggle within the software to the objective of choice.
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Oil objectives: "Type-F" immersion liquid.
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"Glycerin" objectives: "Type-G" immersion liquid.
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"Water" objectives: ddH2O (always use fresh).
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Mount your sample again and press the "Upper Focus Limit" button.
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Focus your sample as described previously.
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20x IMM (multi-immersion - Oil, Glycerin or Water) needs to be set by moving the black dot to the corresponding immersion media ("OIL", "GLYC" or "0.17-W" (with cover glass) or "W-0" (without cover glass)).
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Make sure that the cap of the spring-loaded front lens is released (working position). (Please note: Displayed objective just an example, not exactly same as installed on system) .
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You are now ready to acquire your images. Please check the corresponding follow up guides.
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