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v11.0

by z mbstaff

Introduction

This guide from the Center for Microscopy and Image Analysis shows how to start up and mount a sample on the NEW Leica Stellaris 5 confocal laser scanning microscope. 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 4 Power HyD S detectors.
  • NEW TauSense technology, which gives access to lifetime-based information, delivering additional insights to your experiments.
  • Possibility of switching between resonant and non-resonant scanner directly within the software.

Please find more detailed information about the system here.

  1. Switch ON the fluorescence lamp. Once turned on, the lamp should remain ON for a minimum of 30 min. Switch ON the "Power", "Laser", and turn the " Emission" key to "On-1" (control unit next to the microscope).
    • Switch ON the fluorescence lamp.

    • Once turned on, the lamp should remain ON for a minimum of 30 min.

    • Switch ON the "Power", "Laser", and turn the " Emission" key to "On-1" (control unit next to the microscope).

    • Switch ON the PC.

  2. Sign-in with your ZMB core credentials. Start the "LAS X" software. Make sure "machine.xlhw" is selected as "Configuration", and "DM6" as "Microscope".
    • Sign-in with your ZMB core credentials.

    • Start the "LAS X" software.

    • Make sure "machine.xlhw" is selected as "Configuration", and "DM6" as "Microscope".

    • Click "OK".

    • Click "Yes" in order to initialize the x/y stage. Please make sure nothing is placed currently on the stage.

    • x/y stage initialization is necessary to be able to use the "LAS X Navigator".

  3. Lower the stage by keeping the "LOWER Z" button pressed on the right side of the microscope. This is a mandatory step as it avoids possible collision of the stage and objective during exchange of inserts and/or samples.
    • Lower the stage by keeping the "LOWER Z" button pressed on the right side of the microscope.

    • This is a mandatory step as it avoids possible collision of the stage and objective during exchange of inserts and/or samples.

    • You can now toggle between objectives within the software (drop-down menu).

    • Select the 10x dry objective in the "Aquire" tab.

    • In order to facilitate the focusing it is recommended to start with the 10x dry objective.

  4. Slide your sample into place with the coverslip facing up . Move your sample under  the objective with the help of the external controller "Smart Move".
    • Slide your sample into place with the coverslip facing up .

    • Move your sample under the objective with the help of the external controller "Smart Move".

    • Movement in y-direction.

    • Movement in x-direction.

    • Toggle between coarse movement "XY Fast" and slow movement "XY Precise".

  5. On the touch screen at the microscope stand choose the objective tab.
    • On the touch screen at the microscope stand choose the objective tab.

    • Make sure the eyepiece is selected as the "Port: Eyepieces 100%".

  6. On the touch screen at the microscope stand choose the light path tab. Click "FLUO" and choose an appropriate "FLUO-Filtercube" : e.g. "DAP".
    • On the touch screen at the microscope stand choose the light path tab.

    • Click "FLUO" and choose an appropriate "FLUO-Filtercube" : e.g. "DAP".

    • Open the "IL -Shutter"(if activated the dot is yellow).

    • Look through the oculars and focus your sample by using:

    • or the z-wheel on the external controller ("Smart Move").

    • Moving sample upwards (towards objectives) turn z-wheels clockwise/away from you. Moving sample downwards (away from objectives) turn z-wheels counter-clockwise/towards you.

    • Toggle between "Z FINE" and "Z COARSE" directly on the "Smart Move".

  7. The storage of the focal plane is helpful in order to find the focus back if the sample or objective will be changed.
    • The storage of the focal plane is helpful in order to find the focus back if the sample or objective will be changed.

    • To save your current focus position select the "xyz tab" and the "Focusdrive Z" on the touchscreen of the microscope.

    • Click the "Upper Focus Limit" button.

    • Press "Set".

    • If done successfully you will see an upper marker line appearing.

    • Press the "Lower Limit" button in order to move down (for safe change of the objective or the sample).

  8. Lower the stage. Toggle within the software to the objective of choice. Depending on the objective different immersion media will be used. Apply directly on the sample.
    • Lower the stage. Toggle within the software to the objective of choice.

    • Depending on the objective different immersion media will be used. Apply directly on the sample.

    • Oil objectives: "Type-F" immersion liquid.

    • "Glycerin" objectives: "Type-G" immersion liquid .

    • "Water" objectives: ddH2O (always use fresh).

    • Please further consider the additional information in the next step to guaranty proper image acquisition.

    • Mount your sample again and press the "Upper Focus Limit" button.

    • Focus your sample as described previously.

  9. For optimal imaging performance on some objectives the correction collar has to be adjusted. 20x IMM (multi-immersion - Oil, Glycerin or Water) needs to be set to the corresponding  immersion media ("OIL", "GLYC" or  "0.17-W" (with cover glass) or "W-0" (without cover glass)).
    • For optimal imaging performance on some objectives the correction collar has to be adjusted.

    • 20x IMM (multi-immersion - Oil, Glycerin or Water) needs to be set to the corresponding immersion media ("OIL", "GLYC" or "0.17-W" (with cover glass) or "W-0" (without cover glass)).

    • Make sure that the cap of the spring-loaded front lens is released (working position).

    • Please, DO NOT remove the objectives for adjustment. They can be also accessed on the system.

Finish Line

Joana Raquel Delgado Martins

Member since: 04/09/2019

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