Microbiology Online

Generally speaking, fluorescence microscopy is very similar to ultraviolet microscopy. Fluorescence microscopy is based on the property of some substances to produce fluorescence right after have absorbed ultraviolet energy. In microbiology, the microorganisms can be stained with a fluorescent dye, like fluorochrome, in order to produce fluorescent images through UV microscope.

The main application of fluorescence microscopy in microbiology is the technique of identification of immunological reactions, I mean, of antigen-antibody reactions. The fluorochrome couples with the antibody and makes it possible to identify single cells which react with the antibody, through the emission of fluorescence. This technique is called immunofluorescence.

In immunofluorescence, a culture of bacterial cells is incubated with an antibody, which is coupled with a fluorescent dye. This dye-coupled antibody will cover the surface of some cells, while others will remain without the coupled. With the use of ultraviolet light, just the cells covered with the dye-coupled antibody will produce fluorescence and will look shiny at the microscope.

Next article: phase-contrast microscopy

As already seen in “Principles of Microscopes and Microscopy (Part I)”, light microscopy can be divided into six sub-categories: bright field microscopy, dark field microscopy, ultraviolet microscopy, fluorescence microscopy, phase contrast microscopy and differential interference contrast (D.I.C).

The so-called ultraviolet microscopy uses ultraviolet light instead of white, common, light, or visible light, as the light source. The ultraviolet light has a wavelength of 180 to 400 nm, much smaller than the visible light, whose wavelength is from 400 to 700 nm. And the advantage is?

Let’s recap how the resolution power is calculated:


According to the above equation, the smaller the wavelength, smaller the resolution power, what means you will be able to observe even smaller objects through the microscope. That said, we can conclude that ultraviolet microscopy enables a useful magnification of about twice the magnification of bright field microscopy.

In addition to promoting a larger magnification, without prejudice of clearness, the ultraviolet microscopy also allows the observation of substances absorbed for microorganisms, which become visible as ultraviolet light reaches upon them and make them fluorescent. This is the main application of ultraviolet microscopy, I mean, histochemical.

The ultraviolet microscope differs from the conventional microscope, for since ultraviolet radiations are not visible, images need to be impressed on a photographic film, by use of an image converter tube, or by the projection on a screen, right after being captured by a phototube. In addition, ultraviolet microscopy needs special lens for the transmission of ultraviolet light and optic resources to reflect the region of interest, 230 to 350 nm.

Don’t miss the next article: fluorescence microscopy.

As we have already seen at part III of this mini-course, in light microscopy, also called bright field microscopy, the object analyzed shows darkened and the surrounding environment shows very bright. In dark field microscopy, the opposite occurs, i.e. the object shows very bright, in contrast with the dark background.

In order to fulfill its purpose, the technique of dark field microscopy makes use of a special type of condenser lens, which makes that only the light that reach an object at the slide enters the objective lens.

If the slide contains just a transparent material, like saline solution or purified water, the light will deviate and will not enter the objective lens. In this case, when looking through the ocular lens, the field will be completely dark.

However, if the slide contains any object with a different refraction index, there will be dispersion of light by reflection and refraction, and the light that reaches the object will enter the objective. In this case, when looking through the ocular lens, the object will appear illuminated against a dark field.

The condenser lens used by the dark field microscopy is called cardioid condenser and there are two types: dry and wet. The wet one requires a liquid medium between the objective and the slide, while the dry one does not. The wet one provides clearer images, in opposite of the dry one. For this reason, the most frequently used type of condenser is the wet one and the liquid used between the slide and the objective is the immersion oil.

The dark field microscopy is particularly useful for the analysis of not stained cells, components or microorganisms. An example of application is the observation of bacteria that cannot be appropriately stained by conventional means, like Borrelia burgdorferi (figure above), which infects a special kind of tick and causes the Lyme disease in humans.

Next article: ultra-violet microscopy.