If epifluorescence microscopy sounds suspiciously similar to fluorescence microscopy, it’s because the former is a setup of the latter.  The optical trickery here is practical, designed to provide for a more efficient means to use illumination and process the fluorescence produced by the specimen sample.  This allows the observer to see only the fluorescence emitted by the sample and not the image produced by the illumination from the light source.

Epifluorescence microscopy is one of the most widely utilized instruments in live sciences, particularly biology, biochemistry and many branches of medicine.  It is one of the most effective means to study living cells and organisms, allowing observers to view cellular structures, physiological events and biochemical changes within cells.  It is one of the few microscopy instruments that allow effective viewing of live cell cultures, for example.  As such, it is one of the most popular microscopy techniques used.

Using the epifluorescence microscope
To know how to use the epifluorescence microscope, it is important that the user understands how the setup works.  The epifluorescence microscope works in a similar manner to the fluorescence microscope.  The only difference is that the light source of the epifluorescence microscope is directed from above the specimen and that the objective lens is used both for focusing light to the sample specimen and processing the emission (or fluorescence) it produces.  The epifluorescence microscope does not require a separate lens or condenser in order to use UV light on the specimen.

Using the dichroic mirror
The dichroic mirror is probably the most important component of the epifluorescence microscope.  Dichroic means two-color, which refers to the mirror’s ability to reflect and process two different wavelengths or colors of light.  The dichroic mirror is the part of the epifluorescence microscope that separates the path of the excitatory light and the path of the emission light.  This produces to the image the observer will be viewing through the eyepiece.

Preparing the specimen
The epifluorescence microscope is best used for viewing live specimens or cell cultures.  In order to use it efficiently, it is also important that the observer knows how to prepare the specimen properly.

First, it is important to determine whether a specimen is capable of producing fluorescence on its own or not.  Some samples, such as chlorophyll or certain rocks and minerals, are capable of fluorescing without the use of chemicals.  However, for most specimens, a chemical stain is often required in order to encourage them to emit light.

For living cells, a chemical stain called the calcein/AM dye is often used.  The dye itself is not naturally fluorescent but it does react to the specimen and encourages it to produce its own light under the conditions used with the epifluorescence microscope.  This dye will produce a green fluorescence when viewed through the eyepiece.  This staining technique is used best to observe cellular structures within a living specimen.

Using the double dye technique
To view the dead cells within the specimen, a chemical stain called the propidium iodide may be used along with the calcein/AM stain.  Dead cells within the same specimen will not react to the calcein/AM dye and therefore will not be affected.  However, they will fluoresce when stained with the propidium iodide dye, which binds to the cells’ DNA.  As a result, living cells will fluoresce green while dead cells will fluoresce red using UV light.

The double dye technique is best used when the observer wishes to compare living cell populations with dead cell populations, such as those that appear in toxicology studies.

Using lighting with the epifluorescence microscope
One of the problems that are commonly encountered when using light microscopy is that the technique itself often damages the sample.  The process known as photobleaching can often alter or even destroy the specimen before it can be processed for proper viewing by the observer.  To avoid this problem, it is often necessary to reduce lighting intensity used with the sample.  Using the epifluorescence microscope also requires the observer to work in dim lighting in order to optimize the fluorescence produced by the specimen.  Strong ambient lighting in the environment often dilutes the fluorescence of the specimen, which might mean that it will not be fully appreciated by the observer.

The epifluorescence microscope
The epifluorescence microscope is a very valuable piece of microscopy for many biology-, chemistry- or medical-related fields.  It offers a finer look at the structures of living organisms and cells, allowing the observer to take note of slight changes in quantity or characteristics.  Antibodies, DNA and enzymes, for example, are best viewed using an epifluorescence microscope.  With other types of microscopy, these substances and other chemicals are just too difficult to observe. Continue research on this page