[Histonet] Coverslipping for excellent optical quality

Rene J Buesa rjbuesa <@t> yahoo.com
Tue Jan 31 08:28:02 CST 2012

You have brought up some concerns that have been "into focus" since the mid XIX century, when the objectives manufacturers started to improve the quality of objectives.
Let me go step by step:
1- you already claim that you have mastered the Köhler's illumination whose purpose is limited to obtaining maximum illumination limited only in the area covered by each objective. This illumination is of paramount importance for observation but fundamentally for photomicrography.
2- the next problem tackled by the manufacturers (specially Zeiss and Leitz) was the development of objectives corrected in a way that allowed minimal color distortion and that is how the fluorite and apochromatic objectives appeared in the market.
Now is where you "thickness" issue came to be of concern:
3- the space between the stained tissue and the frontal lens of the objective was calculated by each manufacturer because, in early models, the focal distance of the whole assembly (objective + ocular) was specific: 160mm for Zeiss, Bausch&Lomb and Spencer (American Optical) and 170mm for Leitz.
4- besides the required optical thickness the recommendation was always to have the least amount of mounting medium and glass coverslip between the tissue and the frontal lens of the objective and two fundamental types of coverslips were developed; the so called "No1" which was/is thinner; and the "No2" which thicker. For photomicrography you would never use a "No2" coverslip.
5- to determine the thickness of the coverslips you take a given number (lets say 10) and use a mechanics micrometer to determine the thickness of the selected group and divide by 10 (or whatever amount of coverslips you used for the measurement).
6- the amount of mounting medium will vary but you have to make sure the the coverslip is "pressed down" enough as to make sure that the contact is intimate and minimal.
7- as you can see,these are too many "approximations" so the objectives manufacturers came with an optic-mechanical solution and developed the "correction collar" which is a group of middle lenses mounted on a moving thread that allows those lenses to focus the image to compensate for the thickness of the space mounting medium + coverlip to obtain a perfect compensated focus.
8- those collars were provided with "high-dry" objectives (usually of magnification 40:1 and higher with NA 0.95 and higher) and only for apochromatic objectives. Baush&Lomb, Zeiss and Leitz (among others) developed such objectives with correction collars.
9- but all of that became of secondary importance when in the late 1950's all manufacturers developed objectives with focus at infinity (the are marked as ∞) and those limitations were eliminated.
My final consideration is that for routine work you do not need neither Köhler's illumination or have to worry about the thickness of the space between the specimen and the frontal lens of the objective.
Some pathologists bemoan about the use of film to cover the sections because they think that the "uneven" thickness of the film will hamper photomicrography quality, but that is not the case.
My recommendation to you is that you should not worry that much. If you intend to take photomicrographies during your work, just use Köhler's illumination to limit the field of vision, use the condenser diaphragm to control the intensity of light, use coverslips "No1" making sure that there is no excess mounting medium. Try to use at least fluorite objectives and take your photos. They will come very good.
René J. 

--- On Tue, 1/31/12, Jonathan Cremer <Jonathan.Cremer <@t> med.kuleuven.be> wrote:

From: Jonathan Cremer <Jonathan.Cremer <@t> med.kuleuven.be>
Subject: [Histonet] Coverslipping for excellent optical quality
To: "histonet <@t> lists.utsouthwestern.edu" <histonet <@t> lists.utsouthwestern.edu>
Date: Tuesday, January 31, 2012, 5:56 AM

Dear Histonetters,

I am currently reading up on proper use and maintenance of microscopes, and inevitably stumbled across Köhler's illumination. No one ever explained this to me, although I have a 'microscopy protocol' that comes close to properly explaining the correct setup.
So now that I know how to properly use a microscope fitted with such a system, there is still the issue of the coverslip/mounting medium which are considered the 'first lens'. I understand why there is a narrow tolerance for coverslip/mounting medium thickness to achieve optimal performance, but how do you control that thickness?

Currently, when I mount a slide, I keep it wetted with xylene, put a sufficient amount of mounting medium on the coverslip and mount in the usual 'rolling' motion. I then push on the coverslip to remove trapped bubbles (if any) and be sure to spread the glue under the entire coverslip.
However, how can one be sure about the thickness of the mounting medium layer? Using more/less glue and applying more/less pressure to the coverslip must surely influence this? Especially since we're talking microns.

Does anybody have any insights or tips, or I am just nitpicking and trying to achieve an impossible result? :)


Jonathan Cremer
Laboratory Technician
KU Leuven, Belgium

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