Resolution and Magnification in Microscopy
In the simplest case, a microscope consists of one lens close to the specimen
(objective) and one lens close to the eye (eyepiece). The magnification of a microscope is the product of the factors of both lenses. A 40x objective and a 10x eyepiece, for example, provide a 400x magnification.
The light wave defines the limit
However, it is not only the magnification but also the resolution that indicates the performance capacity of a microscope. Resolution is the ability to render
two closely adjacent dots separately. According to the Rayleigh criterion, the minimum distance between two dots able to be separately imaged corresponds
to approximately one-half the wavelength of the light.
- Fig. 3: Only the interaction of complex lens systems allows optimum image quality.
Therefore, with blue light, the resolution limit is approximately d = 0.2 μm; with red light, around d = 0.35 μm. UV objectives attain a resolution just under 0.2 μm. With the naked eye, we are not able to differentiate structures smaller than 0.2 millimetres.
The value n × sin α corresponds to the numerical aperture (NA), the measure of the light gathering capacity and the resolution of an objective. Because the aperture angle cannot exceed 90° and the refractive index is never less than 1 (nair = 1), NA is always below 1 for air. When immersion oil is used (n > 1), the numerical aperture increases (to up to approx. 1.45) and, along with it, the resolution.
- Fig. 1: The numerical aperture of the objective determines the detail resolution and brightness of the image
- Fig. 2: Wavelengths of the light
- Fig. 4: Images taken with different numerical apertures.
- Fig. 5: Different magnifications, same resolution – no additional details are visible.
Immersion oil increases resolution
To make the microscopic resolution detectable to the eye, the image appears in the eyepiece with corresponding magnification. The resolution and magnification
are always directly interdependent. An objective with low magnification has a low numerical aperture and thus a low resolution. For a highmagnification objective, the numerical aperture is also high, typically 0.8 for a 40x dry objective. However,
because the numerical aperture cannot be increased beyond a certain point, the usable magnification range is also limited in classic light microscopes. The “useful” magnification is between 500 × NA and 1,000 × NA.
Even more magnification
Everything beyond the “useful” magnification is called “empty” magnification. Though structures appear larger, no additional details are resolved. For high-resolution analysis of microstructures, empty magnification is not desirable. Nevertheless, empty magnification can sometimes be quite useful for making details more easily visible for the human eye. Examples of this are video microscopy and also digital microscopy, where greatly enlarged images
are displayed on a monitor, for example.