Fluorescence microscopy - a brief explanation

In fluorescence microscopy visualization of membranes and cells is accomplished by either labelling with fluorescent molecules or by using the intrinsic fluorescent properties of molecules already present in the biological system. Fluorescence is the optical phenomenon of illuminating a fluorescent compound with a particular wavelength of light that gets absorbed by the fluorescent compound and observing the emission of light with a longer wavelength from the sample. The energy diagram below shows the process of the absorption of a photon at the ground state of the fluorescent molecule. The photon absorption excites the fluorescent molecule, which enters a high-energy state. This state is not favourable for the fluorescent molecule to be in for longer periods of time. Therefore in order to re-enter the ground state again the additional energy gained from the photon absorption has to be removed. This can happen in several ways for instance by bond cleavage or heat formation. The most common way of getting rid of the excess energy in fluorescent molecules is to emit a photon with an energy equivalent of the energy difference between the lowest excited state and the ground state. Hence the emitted photon loses some energy compared with the absorbed energy thus is less energetic and has a longer wavelength than the absorbed photon.

: Schematic representation of an energy diagram (Jablonski diagram) that shows how fluorescence occours. The colored circles represent the energy state of the fluorophore, where green depicts the nomal energy level and red the maximum energy level

Fluorescent molecules absorb only at specific wavelengths therefore a fluorescence microscope must have a light source able to produce various wavelengths for excitation. Having a xenon arc lamp or mercury-vapour lamp that generates white light, which is a mixture of all visible wavelengths, usually solves this problem. A special optical filter called an excitation filter removes any other wavelength of light other than the wavelength used to excite the fluorescent molecule. The next element in the optical pathway is called a dichroic mirror, which is a special mirror that is able to reflect certain wavelengths of light and let other wavelengths pass through. When the filtered wavelength exits the excitation filter it gets reflected onto the sample containing the fluorescent molecules. This leads to the absorption of photons and the emission of photons of a shorter wavelength. Because the emitted photons have a shorter wavelength than the absorbed photons the design of the dichroic mirror permits them to pass through the dichroic mirror onto the ocular or detector of the microscope

: A simple illustration showing the main components of a fluorescent microscope.

Several different types of fluorescent molecules (fluorophores) exist. Most fluorophores tend to be organic molecules containing double, triple or aromatic systems. In order to label specific environments these fluorophores are coupled to other molecules such as lipids, proteins, antibodies etc. These different fluorescent probes tend to accumulate in the environment for which they were designed e.g. lipid probes will accumulate in membrane structures.

: A small selection of commonly used fluorophores used to label for instance DNA (DAPI), cells (fluorescein & green fluorescent protein, GFP) and lipid membranes (perylene & DiI). Green fluorescent protein taken from PDB file 1GFL.

Some fluorophores are more susceptible to photobleaching than others. Photobleaching occurs when a fluorephore is overexposed to the light used to stimulate them into fluorescing. This disadvantage of organic fluorophores reduces the exposure time while studying fluorescent labelled samples. A solution to this problem could be the usage of Quantum Dots – semiconducting nanocrystals, because these are more resilient towards photobleaching. In contrast to organic fluorophores the emission wavelength of Quantum Dots does not depend on their composition but rather the size of the Quantum Dots. This effect is due to quamtum mechanics, which will not be discussed here. Nevertheless by changing the size of the Quamtum Dot rather than its composition leads to a change in emission wavelength. Although Quantum Dots have a lot of superior qualities compared with normal fluorephores, they have the tendency of blinking on and off, which is their main disadvantage.