A fluorescence microscope (which uses incident ultraviolet light rather than light from the visible spectrum) is used to detect, localize and quantitate the amount of fluorescence in a specimen, and uses this to indicate the level of the labeled agent we are looking for.

UV Warning for Fluorescence Microscopy

Carl Zeiss WIDEFIELD Fluorescence Microscope

Take care using oil on the microscope

Layout of the fluorescence miscroscope

  • Turn on the computer (1), lamp power source (2), lamp (3) (ignite the lamp by holding down the ignition button (4) until the orange light stays lit), microscope (5), and camera (6).

  • Open up the “SPOT Advanced” program on the computer desktop.

Observing specimens and taking photographs using a fluorescence microscope is not quite the same as using a standard light microscope. Fluorophores are excited by being irradiated with a narrow range of wavelengths (as opposed to the large range of wavelengths in visible light). The narrow range of excitation wavelengths is achieved by passing the light through a filter. This means that usually, only one fluorophore can be visualized at any time. Therefore, if a specimen has been treated with a range of flurophores, these must be observed and photographed separately.

The light emitted by the fluorophores is often of a low intensity. Colour cameras are usually not sensitive enough to detect this light, so the camera takes images in grey scale. These images must be given a false colour to differentiate each fluorophore.

Therefore, the general procedure when taking photographs using a fluorescence microscope is :

  • Select a field

  • Select a filter

  • Take a photograph

  • Select the next filter without changing the field

  • Take a photograph

  • Select the next filter without changing the field

  • Take a photograph

  • Give each of the images taken a false colour based on the flurophore

  • Merge the three colourised images together to create a three colour image

Taking the images

For the fluorescence microscope in the SPARQ-ed laboratory, use the following procedure :

  • Place the slide on the microscope stage and position so that the specimen is directly over the lamp.

  • Make sure that the light path selector A is set at position “A” (100% to eyepiece)

  • Set the sliding filter selector B to “UV” (this is the setting for DAPI)

  • Focus on a field with good coverage of DAPI stained nuclei (they should appear pale blue)

  • Set the light path selector to position “C” (100% to camera at side)

  • In SPOT Advanced, select “Camera”, “Live Image”

Turning on Live Image


  • An image of the field should appear in the live image window. Adjust focus if necessary. If the image is too dim, increase the exposure time (expressed in milliseconds – 1 second = 1000ms) via the “Controls” button.

Taking the "blue" image and adjusting the exposure


  • When you are happy with the image, click “Snap”. The image will appear on the screen.

  • It is a good idea to retain your raw images – sometimes a strong overlapping signal may drown out a weaker one. Click “File”, “Save As” and select a unique filename that contains the word “Blue” to indicate that this is the blue DAPI channel. Save the file as a .jpeg and reduce this image down if required.

  • Change the filter slider to the green “B2” filter. Turn the light path selector back to “A” and make sure that the cells are in focus DO NOT CHANGE THE FIELD OF VIEW.

  • Change the light path selector to “C” and click on “Camera”, “Live Image”. Adjust the exposure if necessary as before

  • When you are happy with the image, click “Snap”. The image will appear on the screen.

Taking the "Green" image


  • Click “File”, “Save As” and select a unique filename that contains the word “Green” to indicate that this is the green channel. Save the file as a .jpeg and reduce this image down if required.

  • Change the filter slider to the red “↕” filter. Turn the light path selector back to “A” and make sure that the cells are in focus DO NOT CHANGE THE FIELD OF VIEW.

  • Change the light path selector to “C” and click on “Camera”, “Live Image”. Adjust the exposure if necessary as before.

Taking the "Red" image


  • When you are happy with the image, click “Snap”. The image will appear on the screen.

  • Click “File”, “Save As” and select a unique filename that contains the word “Red” to indicate that this is the red channel. Save the file as a .jpeg and reduce this image down if required.

Colourising and merging the images

  • Call up the “Blue” image

  • Select “Edit”, “Select Palette”, “Blue”, “OK”. The image should take on a blue tone

Colourising the "Blue" image


  • Call up the “Green” image

  • Select “Edit”, “Select Palette”, “Green”, “OK”. The image should take on a green tone

Colourising the "Green" Image


  • Call up the “Red” image

  • Select “Edit”, “Select Palette”, “Red”, “OK”. The image should take on a red tone

 

Colourising the "Red" Image


  • Select “Edit”, “Merge Images”.

  • Ensure that all files are included (check all boxes).

Merge image dialog box


  • Click “OK”

  • The merged, three-colour image should be presented on the screen – make sure that you save it under a new filename.

Merged Image

Shutdown procedure

  • Clean oil off objectives using lens tissue. For heavy contamination, use cotton bud immersed in 100% EtOH and gently clean objective, followed by Lens tissue wipe.

  • Ensure Mercury lamp has been on for 15 minutes before switching it off.

  • Turn off Computer and wait for full shut down

  • Turn off all switches

  • Cover microscope (leave hot lamp at the back uncovered)

  • Turn off wall switches