There are a variety of different factors that determine the achievable resolution. Some of these factors can not be actively influenced by the microscopist, others can. Some of the factors play a larger role, others a smaller one. In the following post, I want to summarize some of these factors.

Objective-related factors

• Correction of lens errors: In contrast to achromatic objectives, apochromatic objectives focus more colors of the spectrum to one point. This results in a sharper image.
• The numerical aperture of the objective: This value is printed on the objective. The higher the value, the higher the resolution. The numerical aperture is a dimension less value which represents the cone of light that can be caught by the objective.

Lighting system

• General color of light: The shorter the wavelength, the higher the resolution. If your microscope uses halogen or tungsten lamps (instead of LEDs), then the color of the light will shift towards the red end of the spectrum with increasing age. This will reduce the resolution. The color of the light also changes with its intensity. If you turn up the light to maximum intensity, then the color of the light will be more towards the blue end of the spectrum (shorter wavelength and higher resolution). LEDs do not change their color with age or brightness.
• Light spectrum (color range): The color range may also impact on resolution. In the case of monochromatic light, chromatic aberration does not play a role and the light can be focused on one point.

Specimen-related factors

• The correct thickness of the cover glass: The correct cover glass thickness is extremely important for high numerical-aperture objectives. For other objectives, the effect may not be noticeable.
• The correct refractive index of the cover glass: This is something that you do not have to worry about, this is the task of the cover glass manufacturer.
• The correct refractive index of the mounting medium: This one should be as close to the refractive index of glass as possible.
• Thickness of the mounting medium: the thinner the better.
• The presence of immersion oil: Objectives that carry the label “OIL” need the correct immersion oil for best resolution.

Adjustments of the microscope

• The correct condenser diaphragm setting: This setting must match the numerical aperture of the microscope in use.
• The correct setting of the correction collar: Some objectives have a correction collar (a turnable ring) to adjust to the cover glass thickness. Most objectives do not have one, however.

Maintenance-related factors

• The cleanness of the optical parts: Dust and dirt generally decrease image quality and are a big annoyance, especially if one uses dark-field microscopy.

Stability of the photomicrographic system

• Moving objects: Moving cells naturally cause a blurring when long exposure times are used. This decreases resolution of the moving object.
• Stability: A shaky photographic system generally decreases resolution of the image.

The checlkist: how to obtain the best image quality

• Use new light bulbs and turn up the light. This will reduce the wavelength of the light. Alternatively, use a blue filter.
• Use cover glasses of the correct thickness and make sure that the mounting medium has a refractive index which is close to the refractive index of glass.
• Adjust the condenser aperture diaphragm to the numerical aperture of the objective
• If you use oil immersion, make sure that the oil has the correct refractive index
• Use fresh light bulbs (low in red light, high in blue light)
• Keep the microscope free of dust
• Make sure that the objectives, eye pieces are clean

Kiwi Fruit: stacked with the software Combine ZP.

Materials: microscopic slides, cover glass, a soft kiwi, tissue paper.

Method:

1. Take a small piece of the soft part of a kiwi (not the seed and not the white center) and place it between the slide and the cover glass.
2. Carefully tap against the cover glass with a hard object, such as a pen. This is to test if the kiwi is actually compressible (or if it is not ripe enough). A hard kiwi may result in a broken cover glass.
3. Place a small piece of tissue paper on top of the cover glass and carefully and gently press down on the cover glass using your fingers (provided that the fruit is soft enough). Excess kiwi juice will be absorbed by the tissue paper. Be careful: do not move the cover glass horizontally. A thin, green kiwi film should have formed between slide and cover glass.
4. Observe under the microscope as normal.

Note: The image on the right shows some (unidentified) structures found in a kiwi fruit. The final picture is a stack of 12 images, processed with the program Combine ZP. Stacking of the images increases its depth of field. Without stacking, some of the green bubbles would not be in focus.

Impression of a leaf epidermis on white wood glue, oblique illumination. The color levels of the left image were adjusted to use the maximum contrast range. The right image shows the original color.

• Enhancing contrast: Photo editing software (such as Adobe Photoshop or GIMP) contain functions that enhance the contrast of an image. Find the menu point “Auto Levels” or simply “Levels”. This tool will make the darkest part of the image black (even if it was not black before) and the brightest part white. The resulting image will have the same information content, of course, but it may be easier to see the different structures. The photomicrograph will also not have its original color distribution anymore. This may be desired if the original picture has a red color tint due to the lamp of the microscope.
• Sharpening: Photomicrographs can be sharpened. This process results in aesthetically more pleasing images (if not overdone) but it too will not increase the information content of the image. The software enhances the contrast of the edges that it finds. An over-sharpening of photomicrographs results in so-called artifacts. The background noise (random color fluctuations) of the image is increased as well and structures that are not relevant may become more pronounced.
• Increasing depth of field: It is in the nature of compound microscopes to possess a limited depth of field. This can be an advantage, because it allows the observer to “slice-through” the different layer of a sample. By turning the fine-focus knob, it is possible to observe the different depths of a sample. When making photomicrographs, this may be a disadvantage, however. There are software packages available (see the links page) which are able to combine several photomicrographs (each on taken with a different part of the specimen in focus) into one final image. This process is called image stacking. The quality of the final photomicrograph depends both on the number of different images processed and if the focus of the images was sufficiently close together. See a stack of six separate photomicrographs of a Kiwi fruit.