IGC

AdvancedImagingUIC

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WARNING

This is to remind you the basic rules of fluorescence lamps and gas lasers:

  • Never turn OFF the fluorescent lamp if it's been on for less than 30 min.
  • Never turn OFF the lasers if they've been on for less than an hour (1 Hour).
  • Never turn on the lamp if it's been off for less than 30 min (check to see if it's still warm).
  • Never turn on the lasers if they've been off for less than hour (1 hour).
  • Never turn off the lamp or lasers if someone else is using the microscope within the next 4 hours.
  • The Mercury lamp should always be first-on and last-off.
  • Not respecting these instructions may lead to instrument malfunction and complete loss of function! Both lasers and lamps are extremely expensive.

Zeiss Stereo Lumar | Reservations

  • Stereo Lumar

  • The Zeiss' Stereo Lumar is a flurescence capable and fully automated stereoscope with an increased focusing range, which allows imaging of whole small animals (eg Zebrafish e c elegans), as well as cell cultures in petri dishes. Magnifications range is equivalent to ~1-12x, with variable zoom.


    Location     : Bartolomeu Dias wing, UIC Room3

    Microscope: Zeiss SteREO Lumar.V12

    Year Installed: 2011

    Optics: Fluorescence and off-axis illumination

    Camera : Hamamatsu Orca-ER monochrome (1.3MPx)

    Operating conditions : Room Temperature

    Options : None

Suggestion for description in "Materials and Methods":

Images were acquired on Zeiss STereo LUMAR stereoscope, equiped with a Hamamatsu Orca-ER CCD camera and GFP fluorescence filterset, controlled with the MicroManager v1.14 software.

Suggestion for "Acknowledgments":

We would like to thank the technical support of IGC's Advanced Imaging Facility (AIF-UIC), which is supported by the national Portuguese funding ref# PPBI-POCI-01-0145-FEDER-022122, co-financed by Lisboa Regional Operational Programme (Lisboa 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER) and Fundação para a Ciência e a Tecnologia (FCT; Portugal).

Maintenance log:

  • 12-12-2014 - Log-in system implemented at the Stereoscope with fluorescence control through Micromanager.
  • 10-12-2014 - Computer imaged back up made.

Installed Filtersets

Name
Excitation
Emission
CFP
436/20
480/40
YFP
500/20
535/30
GFP
470/40
525/50
TxRed
560/40
630/75

Spectra, lines and filters

Taking color pictures

The camera is monochrome but it is possible to obtain a color images in brightfield mode, using the same filters that are used for fluorescence. ACquire a stack of 3 pictures using the TxRed, GFP and CFP filters (with brighfield illumination and NOT fluorescence illumination!), and from this stack create an RGB image (Red, Green and Blue). Load the 3 pictures as a stack in ImageJ, assign the red color to the TxRed picture, Green color to the GFP picture, blue color to the CFP picture and auto-adjust the histogram for each channel. Finally, set the display to composite or convert the image to RGB and you now have a color picture of your sample!

Pixel size

Because this stereoscope has a continuous zoom it is important that you write down the magnification at which the images were acquired (as displayed on the microscope controler). TO calibrate the images in ImageJ or any other software you need to insert the correct pixel size by using the calculator below.

Magnification: as displayed in controller LCD.

Calculated pixel size: 0 µmeter per 1 pixel

Setup

Turn on procedures

  1. Turn on the computer (if not on already) and log-in using your Agendo username and passowrd. Remember to book on the reservations system, even for very short 1 minute sessions.
  2. Turn on the mercury lamp power source.

    3. Fluorescent lamp house

  3. Turn the Transmitted light source if needed.

    4. Transmitted light source.

  4. Turn on the stereoscope control box.

    5. Stereoscope control box

  5. Turn on the camera controller.

    6. Camera control box

  6. Start the Micro-Manager software, available as a Desktop shortcut.

    7. Micro-Manager shortcut

  7. In case everything is on but you get an error at MicroManager boot, unplug and plug back in the cable in the blue box on top of the fluorescent lamp.

    8. Lamp shutter control box

  8. Set up your experiments on the stereoscope.
  9. Press "Live" button in MicroManager to open the fluorescence shutter.

Turn off procedures

  1. Check if there is anyone after you.

  2. Close all programs and Log-off from Windows.

    • If you skip this step, the next user will not be able to confirm his entry and he will be using the stereoscope with your name.
  3. Turn off the camera controller.
  4. Turn off the stereoscope control box.
  5. If you used the mercury light source and there is no one using the stereoscope for the next 2 hours or you’re sure that the next user won’t use this light source, then turn it off.
  6. Clean the area.

Acquisition

  1. Make sure the stereoscope view is set to the CCD camera.

  2. On Micro-Manager press "Live" to get a live feed of your sample.

    live

  3. Readjust the focus on the stereoscope joystick until the image appears optimally sharp, and the exposure time on the software until you are satisfied with your image.
  4. Press "Snap" or "Snap to Album" to capture the image.
  5. On Micro-Manager save the image to your folder on D:\users.

Extra Info