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How to obtain the best resolution with your microscope

Found in: Microscopy Basics, Techniques, Theory

The resolution that a microscope is capable of achieving is probably the single most important factor that determines the quality of a microscopic image. Without a sufficiently high resolution, magnification is not possible without loss of quality. 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.

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The resolution that a microscope is capable of achieving is probably the single most important factor that determines the quality of a microscopic image. Without a sufficiently high resolution, magnification is not possible without loss of quality. Read the following introductory post: Magnification and Resolution.

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
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