Cover glass thickness and resolution

The effect is highest with high-numeric aperture aperture (high magnification) objectives, and barely noticeable when using objectives of a low numeric aperture.

Types of cover glasses

Cover glasses come in all sorts of different sizes. I already wrote a post about cover glass size: Microscope Slides and Cover Glasses. In this post, we’ll now have a look at the importance of cover glass thicknesses. The table gives a summary of available thicknesses:

 

Number Thickness (mm)
#0 0.08 – 0.13
#1 0.13 – 0.16
#1.5 0.16 – 0.19
#2 0.19 – 0.25
#3 0.25 – 0.35
#4 0.43 – 0.64

Why cover glass thickness is important

Most microscope objectives have the optimum cover glass thickness engraved into them. For most objectives this is 0.17mm. Read the following post for more information on the engravings: About the numbers on the Objective. The correct cover glass thickness is important to achieve the best resolution with a given objective. But do not go out to buy the more expensive 0.17mm cover glasses, get the thinner and cheaper ones (will be explained below).

Generally speaking, the higher the numeric aperture of the objective, the more serious the loss in resolution if the wrong cover glass thickness is used. For some high-aperture objectives, a cover glass thickness of only a few micrometers can significantly reduce resolution. Therefore, some more advanced objectives possess a correction collar. This is an adjustment ring which can be turned to adjust the objective to the actual cover glass thickness which is in use.

Importance of the mounting medium

The optimum cover glass thickness of many objectives is 0.17mm. Now, why is it that the most commonly available cover glasses are of category 1 (0.13-0.16mm), which is thinner than the calculated optimum? The answer is a bit more complex: The thickness of the cover glass is not the only parameter which is important. The specimen is embedded in mounting medium. The thickness of this medium must be added to the thickness of the cover glass. A specimen which is located deep in the medium will have a larger “effective” cover glass thickness than a specimen which is located right beneath the cover glass. A calculated (ideal) cover glass thickness 0.17mm is therefore a good compromise, even if the “real” cover glass is thinner. And yes, the refractive index of the mounting medium also plays a role.

How to determine the thickness of a cover glass

Cheap cover glasses which are used for uncritical routine observations will show a statistical spread of different thicknesses. There are also assorted cover glasses available that show a much more narrow spread of thicknesses. Some people buy cheap cover glasses (with a larger spread) and then manually measure their thickness using a caliper to sort them. Is it worth the effort? When using low-magnification objectives with a low numeric aperture, the difference in cover glass thickness may not even be noticeable and the more expensive pre-selected cover glasses may only be necessary for specific applications where a high resolution is necessary and the objectives do not possess a correction collar. One should not forget that the thickness and refractive index of the mounting medium also has an impact on the resolution, and mounting medium thickness may be much more difficult to standardize.

7 thoughts on “Cover glass thickness and resolution”

  1. Hi, Oliver, I have questions which you might not be able to answer…but I always wondered about!

    Many older (circa early to mid 1950’s microscopes) microscopes have an adjustment knob for the body-tube itself of the microscope.

    And of course as you here noted, specific microscopes have ‘a correction collar’ right on the objective barrel.

    My questions…how did these oldtime microscopists adjust either ‘adjustment knob or barrel’? Did they do it by quality of a certain stardard image they ‘expected to see’ with that setup? I can not imagine how you can ‘gap-guage’ a delicate glass coverslip with a metal shop caliper micrometer. I do have old ( circa late 1800’s-early 1900’s booklets which came with such vintage scopes…I should dig these vintage manuals out and read! It just seems to me that the optical index of the mounting sandwich a specimen is within is variable to a degree far greater than the variation in coverslip thickness ( yet the cover slip thickness as you say, it varies too)…and admist all this ‘variable media’…you add the awkwardness of ‘hand held micrometer calipers’ to read (without variation!?) the thickness of a delicate glass coverslip…it seems I miss an aspect of the skilled vintage microscopist technique for adjusting both the microscope-tube compensator-knob, and that correction-collar adjustment on the objectives which have such a critical correction device.

    And yes, these questions of mine are practicle for me…as I have both vintage microscopes with that body-tube length adjustment knob, the vintage manuals which talk about adjustment for coverslip thickness, and always wondering when : size 1/2 coverslips are needed, I wonder when those very thin coverslips are needed?

    Again, no need to reply…perhaps I can tidy up my use of images of my vintage stand, it’s vintage manual describing it’s adjustments, and offer this wonderment as a thread in your forums? I can’t wait for your online magazine to come out soon, Oliver!

    charlie guevara, fingerlakes/US

  2. At last! A layman oriented explanation on this subject. Thank you. One question though, at what “higher magnification” does the cover glass thickness start to become critical? 40X, 60X, 90X? This would be useful to know as I am beginning the infancy stage of slide making in my budding carrer as a world renowned microscopist.
    Regards
    Joseph Wilhelm

  3. Joseph,
    I needed some time to think about your question, and I had to do a bit of research. I’m afraid that the answer is not as straight forward. Cover slip thickness is not a concern when the numerical aperture of the objective is 0.4 or less and it is of high importance for objectives with an NA of 0.65 or more. The following link gives more information (especially Table 1 is quite interesting):

    http://www.microscopyu.com/articles/formulas/formulascoverslipcorrection.html

  4. Joseph,

    I’m curious about the affect on very high NA oil coupled objectives (100x 1.3NA) of not only the thickness changing, but also the index. For example will a fused silica coverslip with index 1.48 affect aberrations enough to worry about the index difference (index oil is 1.52 index)?

    I notice that our oil coupled objectives are still marked for .17mm coverslip thickness, but does thickness matter if the index isn’t changing from oil to the glass?

  5. Hello Mr. Kim,

    So, if I were to use a slide with a depression in it for viewing pond water, the importance of the thickness of the cover glass wouldn’t matter much, even with 100x oil objective? I’ll fill the depression overflowingly, then put the cover glass so that excess water leaves, while not having any air bubbles trapped inside. This should negate the air refractive index, but then again, the refractive index of the water is different (I’m pretty sure on that one). This is all conjecture, as I don’t have the microscope and slides just yet. So in the end, the best way to view pond water is not with a well, but only a flat slide with only the thin layer of water under the cover glass. Sorry, I’m inexperienced, and also thinking “aloud”.

    Take care,
    Huy

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