Microscope Cover Slips

How do you know when to use a microscope cover slip? And if you do need to use one, what thickness is appropriate? This post will answer all of these questions and hopefully help you figure out what mediums will produce the best images under different microscopes.

What type of microscope are you using?

The first question you should answer is what type of microscope are you using? Some microscopes do not require the use of a cover slip at all. Below is a list of a variety of microscopes and their use of cover slips:

• Stereo Microscopes - when using a stereo microscope you do not need to use a cover slip. The sample sits directly on the microscope stage and is not typically placed on a microscope slide at all.
• Inverted Biological Microscopes - Petri dishes are used with inverted microscopes in order to contain living samples in liquid. Cover slips are not used with a Petri dish, but the thickness of the Petri dish can be important. We will talk more about this below.
• Inverted Metallurgical Microscopes - when using an inverted metallurgical microscope the sample will be flat and may be polished. The sample is placed directly on the stage and no slide or cover slip is used.
• Upright Biological Microscopes (Compound Microscopes) - Upright biological microscopes are sometimes referred to as compound microscopes. When using an upright biological microscope both a slide and cover slip is used.
• Upright Metallurgical Microscopes - Upright metallurgical microscopes do not require the use of a slide or cover slip. Occasionally one is used if the sample is a powder and must be flattened or contained, but typically the sample is placed directly on the stage. Filter patches are also placed directly on the stage under an upright metallurgical microscope.
• Polarizing Microscopes - polarizing microscopes may be used to view thin sections of rocks, minerals, or even powdery substances. Depending on the sample, a slide and cover slip is used at times to flatten and contain the sample.
  

When viewing sections or smears (typically biological in nature), they must be fixed to a slide. The sample is preserved or prepared with a medium (stain) by placing a cover slip on top of the sample on the slide. Thin sections of plant samples can be placed on a slide with a drop of water with a cover slip on top of the plant to flatten it.

Take a look at the microscope objective lens shown at left. This objective is a 20x plan achromat objective lens with a Numerical Aperture (NA) of 0.45. The infinity symbol tells us that it is an infinity corrected lens and after this symbol the lens shows "0.17". This refers to the  cover slip thickness.

Standard transmitted light objective lenses are designed for a 0.17mm cover slip between the sample and the lens. Standard cover slips have a thickness of 0.13-0.16mm, taking into consideration the added layer of an embedding medium or water into account in the sample results in meeting the rule of 0.17mm.

The higher the Numerical Aperture of an objective lens, the higher the sensibility for deviations from the value on the objective lens. For example, a 4x/0.10 objective lens can be used with or without a cover slip and no difference will be noted. But if an objective lens with a value of 40x/0.95 were used, any variation off the cover slip requirement could result in an image that is not clear and crisp. In this situation using the appropriate cover slip thickness, as well as ensuring that samples were precisely cut in a thin section (1-5 microns thick) will result in the clearest high quality images.

Additionally, if too much of a solution is used between the cover slip and microscope slide (such as stain or liquid), it can ruin the image. When preparing slides it can help to press the cover slip into the slide with the tip of a needle (to avoid fingerprints on the slide while flattening it), as well as mopping up extra fluid with a paper towel placed at the edge of the cover slip.

Some objective lenses will be marked with a "0". This indicates that the objective lens does not require use of a cover slip at all. In some fields these objective lenses are used to save time by making slide preparation faster.

When using an inverted biological microscope, Petri dishes, flasks or well plates hold the microscopy samples. These vessels have a thickness of 1mm on the bottom. Therefore the objective lenses used with these vessels are marked with a 1.1mm indicator. The additional 0.1mm comes from the water or agar medium in the Petri dish.

Due to the improved working distance in inverted microscope objectives lenses, they are not driven to maximum resolution (NA). The inverted microscope was constructed for the improved handling freedom it provides with specimens and was not created for the evaluation of resolution limits. If you wish to use a 0.17mm objective lens on an inverted microscope, make sure to use a Petri dish with a 0.17mm glass bottom, or you could turn the glass slide upside down. Of course if you are using a slide containing liquid, turning it upside down will not be beneficial.

Next time you are preparing samples for your microscope, take a look at the objective lens and ensure you are using the proper cover slip thickness for the lens.

If you have questions regarding microscope cover slips and obtaining the best image with your objective lens, contact Microscope World and we will be happy to help.