Epifluorescent microscopes are one of the most commonly used types of microscopy in the medical field today.  Epifluorescence is a rather familiar microscopy technique, being another form of light fluorescence microscopy.  It is also one of the most important techniques used for research and experiment, especially those involving the study of living organisms and live cell samples.

An epifluorescent microscope uses a slightly different optical setup than that of its cousin, the fluorescent microscope.  An epifluorescent microscope uses an objective lens to perform two tasks: to focus light upon the specimen being observed and to collect light being emitted by that specimen, which is fluorescent.  As a result, the observer will only see the fluorescent light, not the ordinary image produced through direct illumination.

Breaking down the modern fluorescent microscope
Epifluorescence microscopy has evolved over the years, thanks in large part to its use in a wide variety of fields.  It is a very interesting instrument, with a simple setup capable of much more complicated tasks.

The most important components of the modern epifluorescent microscope include: the dichroic mirror, the excitation filter, the emission filter and the light source.  The light source in an epifluorescent microscope is located above (’epi’) the specimen, illuminating it from that location.

The dichroic mirror
The dichroic mirror separates the excitation light from the emission light.  It has its own set of transition wavelength value.  This is the wavelength of half the transmission.  The dichroic mirror can reflect light wavelengths below the wavelength value and transmit the wavelengths above that value.  This is why it is called the dichroic (two colors) mirror because it is capable of handling two light wavelengths.

The excitation filter
The excitation filter is one of the two filters used for the modern epifluorescent microscope.  This is placed in the path of the excitation light – the light coming from the lamp source.  In the case of the epifluorescent microscope, this light comes from above the sample.

The emission filter
The emission filter in the modern epifluorescent microscope is used to select the specific ideal wavelength of the emission light coming from the specimen.  It also filters out any excitation light that may still be present.  As a result, the observer does not see a specimen being illuminated ordinarily from above (such as the image often produced by ordinary light microscopy) but a specimen that fluoresces with its own light, usually aided by a chemical stain.

Both the excitation and emission filters are chosen to equal the characteristics of the spectral excitation and the emission of the fluorophore being utilized with the specimen.

Lighting
The modern epifluorescent microscopy uses a highly efficient light source to aid in the process of fluorescence for the specimen being observed.  Traditional fluorescent microscopes utilized transmitted light from carbon arc lamps, which were later replaced by 200-watt mercury lamps.  The modern epifluorescent microscope now uses lower wattage xenon, tungsten or mercury lamps, usually starting at 50 watts.  These lamps are the main source of ultraviolet light used to focus on the specimen and excite its molecules.

The type of light being used in epifluorescence microscopy also affects how the specimen is viewed.  Quite often, a special staining agent is used in order to enhance the image or provide the observer with a means to perform a more accurate count.  The dyes are fluorochromes, one of the types of histological stains used with certain materials and live cells.  Either UV light or blue light is used on the specimen in order to encourage it to produce long wavelengths of emission light.  This will result to a fluorescence that is seen as either red or green.

Designing the light path
The main difference between traditional fluorescent microscopes and epifluorescent microscopes is their use of light.  With the modern epifluorescent microscope, the objective lens is used to focus light on the sample.  Once the specimen fluoresces, the objective lens also collects this light.  The objective lens works with the excitation and emission filters, which separates the two light paths within the instrument.

The modern epifluorescent microscope
Epifluorescent microscopes are highly useful instruments for observing biological and physiological structures within living cells.  They are also very efficient for viewing biochemical changes, such as those that occur in live cell cultures.  The modern epifluorescent microscope is the instrument of choice for many important observations and studies involving genetics, particularly those related to stem cell research.

Epifluorescence microscopy is so widely used that many of the major brands manufacturing microscopy today are producing it.  The modern epifluorescent microscope is also the inspiration for many of the advanced instrument designs in microscopy, such as the TIRF or total internal reflection fluorescence microscope and the confocal laser scanning microscope. Article link