Epifluorescence is one of the most important and fascinating techniques in microscopy.  Without the epifluorescence technique, it would be impossible to study many of the physiological and biochemical processes involved in the research of live specimens.  Epifluorescence is one of the types of fluorescence microscopy, a very essential tool for many medical and biochemical researches and related experiments.

Epifluorescent microscopes are some of the most commonly used for many fields of study, primarily medicine and medicine-related areas.  It is considered as a highly efficient type of microscopy, even more so than transmitted light fluorescent microscopy.

How does epifluorescence work in microscopy?
To understand how the principle of epifluorescence works in microscopy, it’s important that fluorescence is studied first.  Fluorescence refers to the capability of certain organisms to absorb light and emit it back.  This occurs when atoms and molecules within the organism are manipulated by light, allowing them to bounce it back at a certain wavelength.

When an organism is hit by light, it absorbs this light and atoms within it become excited.  Excitation results to electrons within the atoms to jump to another level of energy.  After a short while, these electrons return to their previous ground state.  The interval between the absorption of light by the organism and the act of emission is called the fluorescence lifetime.  This interval tends to be very short, lasting only approximately 1/100,000th of a second (some last even less), although there are also fluorescence lifetimes that extend for hours at a time.  Fluorescence is the term used to describe a short fluorescent lifetime while phosphorescence is used to refer to a longer interval.

Fluorescence occurs as a result of the photon the electron releases when it returns to its original state.  This photon produces a short light pulse.  This is the light that an observer sees when the specimen is being viewed using a microscope.

Epifluorescence microscopy
‘Epi’ is a prefix that means ‘above’, referring to the source of the light that is used to illuminate and allow electrons to emit light.  In its most basic design, the epifluorescent microscope is a light microscope.  However, it also has additional features that allow it to work differently.

Epifluorescence microscopy utilizes light to excite the atoms of the specimen but light coming from the source is first passed through the instrument’s objective lens before it strikes the specimen.  In other forms of light microscopy, light is transmitted to pass through the specimen.  With epifluorescent microscopy, transmitted light is filtered out, allowing it to produce highly detailed and sharper images.

In older models of epifluorescent microscopy, carbon arc lamps were used and later, stronger mercury lamps.  These days, however, smaller lamps with lower wattages (maximum of 100 watts) are used.  The source of light often used in epifluorescent microscopy is a lamp, usually a tungsten, mercury or xenon lamp.  It also contains essential components such as the dichroic mirror or beamsplitter, the barrier filter, the emission filter and the excitation filter.

Aiding fluorescence chemically
The challenge in using microscopy techniques such as epifluorescence is that a fluorescing organism can sometimes lose its ability to emit light.  Ironically, this often occurs because of illumination, which leads to photobleaching.  To prevent this from happening, either the amount of illumination is reduced or certain chemicals are used to enhance the specimens and encourage better fluorescence.

To ensure that images are viewed effectively, epifluorescence microscopy requires that specimens are stained using a chemical dye, usually a calcein/AM dye.  The dye encourages specimens to produce a fluorescent image thanks to the AM component of the chemical, which binds calcium.  Calcium is a fluorescent mineral and when the calcein/AM dye is used on a living cell, enzymes within that cell eliminate the AM component and keep the calcein content.  This produces a fluorescence, which usually appears green under UV light.

While this technique works best with living cells, dead cells are best used with a chemical dye known as propidium iodide.  This dye is capable of penetrating dead cells, an action that produces a red fluorescence when viewed under UV light.

Although chemical stains are often used in microscopy, there are also certain specimens where dyes are not required, such as when primary or auto fluorescence occurs in materials like chlorophyll.  With other materials, chemical stains (such as those mentioned earlier) are required.

Epifluorescence is also referred to as incident light fluorescence.  It is the type of microscopy often used by biologists, toxicologists, chemists and biotechnologists, primarily to observe living cells.  It is often used to view changes in cell populations and cell cultures in order to observe physiological and biochemical changes. More on this topic