Application Note – Microbiology N.6 – The correct use of a fluorescence microscope


The reported notes are available on the web

In fluorescence microscopy, fluorophores are used to reflect an image of a given sample or specimen. A fluorescence microscope is generally made up of a specialized light source, either Mercury or Xenon, excitation and emission filters, and a dichroic mirror. The following steps will instruct you how to use a fluorescence microscope properly and safely.

Step 1: Remove the protective cover of your fluorescence microscope. Make sure it is set at low power before plugging and switching it on. Turn on the mercury lamp as well. You will have to wait approximately fifteen minutes before the microscope can provide full brightness. Switch on the motorized focus box.

Step 2: Place your prepared slide properly on the stage. Secure the slide with the stage clips. Peek through the eyepiece and slowly make the necessary adjustments to bring your sample into focus. The coarse knobs are there to lift or lower the stage while the fine knobs are there to provide a sharper and clearer image of your specimen.

If you are switching to a different objective, do so by holding the collar of your fluoresce microscope’s nosepiece. Never exert pressure on the objective lenses themselves because this could cause them to lose their alignment.

Change of filters is best performed while the microscope is set at low power. If you are going to adjust the condenser for Kohler illumination, refrain from adjusting the stage knobs.

Step 3: If you are interested in taking pictures of the sample, you can attach a camera eyepiece to the microscope. Images will be stored on your camera’s built-in memory or on a connected storage device.

Step 4: If you are finished using the microscope, you can only switch off the microscope if you have consumed at least thirty minutes. If so, remove the slide from the microscope then switch off the focus control box. Switch off the mercury lamp. You can turn on the fluorescence microscope again after half an hour.

Step 5: Unplug the fluorescence microscope and return it to its original place and under its protective cover.

Other Tips When Using a Fluorescence Microscope

- Mercury lamps release extremely potent and visible UV radiation so avoid looking at them directly. Do not disassemble the lamp housing. Do not look into the fluorescence microscope’s eyepieces when you are changing filters. Certain filters can reflect the UV rays directly to your eyes.
- Always record how many hours you’ve used the mercury lamp of a fluorescence microscope. Going beyond its expected lifespan can put your fluorescence microscope at risk of exploding.
- Switching a mercury lamp on and off frequently can reduce its lifespan. Leave it on if you expect someone else to use the fluorescence microscope in two hours’ time.
- If you are going to use oil immersion objectives for your fluorescence microscope, be careful when you are using low power objectives. Oil from your slide could contaminate your objectives when you swing the microscope’s nosepiece the wrong way. After using them, you can clean the lenses with lens paper by dabbing off the oil. Do not wipe harshly.

Using Epifluorescent Illumination with Your Fluorescence Microscope

Step 1: Place the appropriate filter. If you are going to use an epi-polarization filter, make sure to switch off the mercury lamp first. You can only look in the eyepiece after replacing the filter.

You can use bright field or other techniques – reflected or transmitted – to focus on your sample. Switch on the Mercury lamp.

Place in the correct filter cube for your microscope’s fluorochrome. Check if the analyzer slider has been detached; if not, then signal intensity would be reduced.

Step 2: If the Nomarski prism is still inserted in the space on top of your microscope’s objective, remove it. Its presence only reduces the quality of images captured by your fluorescence microscope.

Step 3: Take out your microscope’s shutter slider and place it in the desired filter position.

Step 4: Push the control lever in to open up the condenser aperture of your fluorescence microscope. Another push would open the field aperture of your condenser.

Step 5: You can now start observing your specimen. Adjust what’s necessary. If you’re done, push the fluorescence shutter in. Forgetting to do so places your sample at risk of overexposure and consequently, photobleaching. In photobleaching, fluorophores lose their ability to “glow” when they are illuminated.

You now know how to use a fluorescence microscope. Make sure to have a copy of this article posted near your work table to avoid forgetting any of the important safety tips included here.