Light microscopy is the most largely used microscopy technique in microbiology labs. In light (optic) microscopy, the objects studied look darkened and the adjacent area looks very well illuminated. The magnification power of an optic microscope is up to 2000 times. The main difficulty in light microscopy is to obtain a high magnification power along with clearness and image definition.
In light microscopy, the resolution power represents the size of the smaller object that can be seen on the microscope with total clearness. A good microscope shall combine magnification power with resolution power; otherwise an image could be amplified 2000 times, but it would show up distorted and without definition.
The lower the resolution power, smaller the size of the object that can be observed on the microscope. That said, a light microscope with a resolution power of 0.5 µm is able to magnify, with clearness, objects of 0.5 µm or higher.
In order to calculate the resolution power of an optic microscope, we need to know:
- The light wavelength;
- The objective lenses numeric aperture;
- The condenser lens numeric aperture.
In light microscopy, the Numeric Aperture (NA) is calculated from the following equation:
θ = half angle of the cone light that enters the objective or condenser lenses. Please observe the picture below.
In the modern microscopes, the NA value of the objective and condenser lenses is indicated at the microscope user’s guide.
At last, the resolution power is calculated from the following equation:
At microbiology labs, the light microscopy technique is usually performed using immersion oil. The immersion oil has a refraction index higher than the air, what increases NA and decreases resolution power; in practical terms, it allows the observation of smaller objects, like cells and bacteria, for example.
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