The Fluorescence Microscope
To be able to see very small specimens or minute gradations in the specimen itself, some microscopists might employ staining or looking at the sample through darkfield or phase contrast microscopy. On the other hand, to provide a good contrast between the part of the specimen being examined and the background in general, fluorescence microscopy might be used not only for the best possible contrast but also for a high resolution image of the sample being viewed under magnification.
Fluorescence microscopy works when the light used in the microscope for illumination excites or irradiates the specimen being observed under the microscope lens. This specimen in turn emits a radiation which wavelength is shorter than the light of excitation. This kind of property in matter is called luminescence and is the basic principle in the fluorescence microscopy procedure.
There are two forms of luminescence: one is wherein the secondary luminescence goes on even after the light of excitation has been taken away, which is called phosphorescence. Another form is when the secondary luminescence stops along with the light of excitation, which is called fluorescence.
With a fluorescence microscope, a light with short wavelength like UV or blue light is used to illuminate the sample so that some component of the sample would react by way of emitting a light called fluorochrome that can be seen by the observer through the microscope eyepiece. Fluorochromes can be seen as brilliantly lit parts against a very dark background. Some substances naturally contain fluorochromes and fluoresce when illuminated under UV or blue light through the fluorescence microscope. This naturally occurring fluorescence is called autofluorescence or primary fluorescence.
On the other hand, it is perfectly possible for the microscopist to apply fluorochrome onto the specimen so that the parts that need to be observed under magnification would fluoresce and be seen through the ocular of the fluorescence microscope, making for an image where the marked parts of the specimen seem to be glowing brightly against an almost black background.
Applying dyes with fluorochromes give the specimen applied or secondary fluorescence. Since this kind of fluorescence is easier to control than autofluoresence and is more reliable, application of fluorescent dyes like fluorescein iso-thyocyanate (FITC) onto the sample before looking under the optical microscope has become the golden standard for such procedures. Other dyes with fluorochromes are acridine orange, thioflavine T, auramine and rhodamine.
Another important factor in fluorescence microscopy is the kind of light being used for illumination. The wavelength of the light of excitation for each fluorochromes differ enough that the microscopist needs to take into account not only the amount of contrast they need to see under the microscope but also whether to use transmitted (coming from below the specimen) or incident (coming from above) light. One problem with working with transmitted light is that some of the illumination passing through the specimen might get into the way of the received image seen through the ocular. Since working with a fluorescence microscope requires the microscopist to perform their examinations in a dim to dark room, any kind of accidental light could result to a lower resolution image than would be the case if the observation had been done under the best possible conditions. This is why fluorescence microscopy done under transmitted light illumination is done best with an immersion-type darkfield condenser, which will result to a maximum contrast between the fluorescent parts of the specimen and the background.
On the other hand, working with incident light to illuminate the sample being viewed under the microscope would result to a higher level of fluorescence. Epi-fluorescence, as the incident light illumination is called, produces very bright images with a higher contrast than that from transmitted illumination.
Since it is capable of providing a high contrast image (against a dark to black background) as well as a higher resolution compared to other types of microscopes, investing in a fluorescence microscope is a perfect choice for microscopists who need to look at specific parts of biological specimens to be stained with fluorescence dyes.
