Coverslip Secrets

Coverslips typically come in 4 thicknesses: #0 = approximately 0.10 mm thick #1 = approximately 0.12- 0.15 mm thick #1-1/2 = approximately 0.17 mm thick #2 = approximately 0.2mm thick. For the best image, you want the right coverslip: 170 microns. However, in the case where you want additional depth penetration into your sample, you might use a thinner coverslip, and trade optical quality for increased depth. This is because each objective has a maximum working distance at which it can focus. The thinner the glass, the deeper you can ultimately focus.

For example, our 40X/1.30 Plan Neofluar can penetrate 120 microns into your sample if you use the #1.5 (170 microns thick), but you could go 180 microns with a #1 (which is 90-110 microns thick), or 230 microns using a #0 (50-60 microns thick). Image quality will degrade at all depths with these thinner glasses, but you'll be able to image deeper into the sample.

Since these estimates assume an optically clear sample with no refraction, practical distances will be somewhat less than that, although you could approach these distances by employing clearing steps (with e.g. methyl salicylate) and a mounting medium with the proper index of refraction. Also note that the thickness values are nominal, and can vary by large amounts, but the general message is still the same.

All of the foregoing assumes that you've already taken care of the most common problem we encounter: Do not allow excessive distance from your coverslip to your sample. This can not be stressed too highly. Some people worry about crushing their cells. In practice, it's usually the opposite problem; if the cells have a 100 micron layer of medium between them and the coverslip, image quality will suffer greatly, unless you pay a lot of attention to the optical quality of the medium. The ideal situation is to grow the cells on the coverslip that you will be imaging through, reducing the distance to zero, and giving us the clearest possible image. This is why we frequently recommend Mat-Tek dishes.

Various Mounting Media and Anti-fade Reagents:
(Not a complete listing. This is a collection of suggestions from many users. Please contact me with your suggestions, corrections, and additions.)

Mounting Media:

The sample's surroundings will affect the image to a great degree. For optimal quality, you would ideally like your sample to be immersed in a material with the same index of refraction as our immersion oil (1.518). If the material is able to solidify, you will avoid problems with movement of the sample (or the coverslip) relative to the slide. And for unstable fluorophores, which tend to photobleach quickly, many of the commercial mounting media contain anti-fading reagents in them.

Note that your sample's characteristics will determine whether you want an aqueous medium or an organic one.

• PermaFluor from Lipshaw:
  (aqueous, sets hard)
• FluorSave:
  (aqueous)
• ProLong B from Molecular Probes:
  (sets hard)
• Fluoroguard from Biorad
• VectaShield from Vector Labs
• PolyMount or Aqua-PolyMount from Polyscience
• MOWIOL from Calbiochem
• DPX
• 50% glycerol, 50% PBS
  (homemade, buffered to correct pH for your fluorophore)
• methyl salicylate
  (organic, dissolves plastic and nail polish, but has excellent optical properties)

Anti-fade additives:

Many people prefer to add specific anti-fade reagents, such as the following, to their mounting media (often made from scratch). Although the mechanism of the photobleaching has not been completely elucidated, it has been found that you can reduce the effect by using free-radical scavengers, minimizing the amount of oxygen in the sample, etc.

• 1,4-diazobicyclo-(2,2,2)octane (DABCO)
• n-propyl gallate
• p-pheylenediamine dihydrochloride
• ascorbic acid

Keep in mind that every fluorophore has an optimal pH range, and buffer your sample accordingly.

Axiophot Image Size

The following values apply to the Axiophot microscope front and back camera ports. Note that both cameras clip the edges of the field, so that you take a picture of only the center of the view through the oculars.

The "magnification" factor of an image is dependent on the size that you ultimately display or print the image. When looking through the microscope while using a 20X objective lens, with the standard 10X ocular lenses, what you see is a 200X magnified image.

But consider this example: If you take an image with the 20X objective lens using the MicroMax CCD, your field size is 450 micrometers. If this is printed, as is common, to take up one column in a journal, it will be about 55mm wide, which is roughly 122X magnification. But if the same image is printed on the cover, taking up the full page, it will be about 200mm, or almost 444X.

For this reason, it is often preferable to simply insert a scale bar in the image, the length of which is determined by its size as a fraction of the whole image, which is known.