The Problem with Coverslip Mountant.
Have you ever noticed that a freshly stained and coverslipped slide is slightly hazy under the microscope?
Have you noticed that after a week or so the same slide image has become clearer?
Many microscopists are aware of this sharpening of the slide image over time, often noted when slides are retrieved for review or by pathologists when presenting cases at multidisciplinary team/tumour board meetings. The increase in resolution over time is due to the gradual drying of the coverslip mountant. The mixture of polymer and solvent dries by evaporation of the solvent which can only occur very slowly as it is trapped beneath the coverslip. Because the mountant at the edges of the coverslip dries first a peripheral seal is formed further retarding drying of the central area beneath the coverslip. Dried coverslip mountant is manufactured to achieve as close a refractive index to the microscope slide and coverslip glass (usually borosilicate crown glass – refractive index of ~1.53) as possible but, when wet due to the solvent content, the refractive index of the mountant is significantly less than that of crown glass (e.g. xylene, a commonly used solvent in mountant, has a refractive index of ~1.49). Not only is there a refractive index mismatch but, importantly, the refractive dispersion (the variation of refractive index with wavelength) is different for wet mountant, dry mountant and coverslip glass. For any given refractive index, liquids generally have a much higher dispersion than solids. The dispersion of a substance is measured by its Abbe value V – the lower the number, the greater the dispersion. Xylene (a typical mountant solvent) has an Abbe number V ~ 30, whereas most mountants when dry have an Abbe number V ~ 45 to 55. Borosilicate crown glass coverslips typically have an Abbe number V ~ 55 to 60. The lower the value of the Abbe number V, the higher the chromatic aberration due to dispersion.
Apart from the optical qualities of the various materials in the microscope’s light path there is the issue of precise distance variation caused by the presence of coverslip mountant. For compound light microscopy the microscope manufacturer has designed the objective lens to be corrected for spherical aberration at its defined optical tube length in order to generate a perfectly focused primary image. For high magnification, high numerical aperture objective lenses (and especially so for “dry”, i.e. non-immersion oil, lenses) the thickness of the glass coverslip is critical. High numerical aperture objective lenses will have the ideal coverslip thickness engraved on their barrel. This is usually “0.17” which means 0.17mm (coverslip thickness #1.5). If a thicker or thinner coverslip is used the result is increased spherical aberration. The presence of coverslip mountant causes an effective increase in the overall coverslip thickness, shifting the primary image away from its ideal plane of focus – i.e. adding to spherical aberration. Apart from the physical thickness of the coverslip plus mountant it is important to also consider the virtual thickness – a function of the coverslip and mountant refractive indices including any mismatch therein (as mentioned above). Furthermore, over time (hours to days) both the physical and virtual coverslip + mountant thicknesses change as the coverslip mountant solvent slowly evaporates (real distance, refractive indices and Abbe values all change). This causes a shift in the position of the primary image with time. The major microscope manufacturers’ solution to deal with the problem of variable and changing effective coverslip thickness is to manufacture high numerical aperture objective lenses with a correction collar. Adjusting the correction collar alters the optical tube length of the objective lens to re-align the primary image and reduce spherical aberration. Using the correction collar, however, can be tricky because after each tentative adjustment the image has to be re-focussed.
The CALACLEAR microscope slide avoids the optical problems of coverslip mountant entirely by eliminating its presence between the tissue section/cytological preparation and the microscope objective lens (see diagram in “About our product”). Because the sample is applied directly to the undersurface of the coverslip that forms the window, the virtual and effective coverslip thickness is fixed, with no variation over time.
At all time intervals following slide preparation, the calaCLEAR microscope slide delivers higher image resolution than does a conventional glass slide but, for the aforementioned reasons, the difference is most striking when comparing freshly prepared calaCLEAR and conventional microscope slides - i.e. precisely the time when microscope slides are usually examined.
A series of excellent articles on the light microscope by Jeremy Sanderson.
A step-by-step guide to getting the most from your microscope; includes bright field microscopy and light microscopy variants.
The major microscope manufacturers all have useful educational resource pages on their websites.