Fluorescence Microscope and Microscopy

Fluorescence Microscopy is an optical technique that continues to evolve and develop into newer techniques as better probes and equipment is developed. Fluorescent microscopes adapt this technique which has helped in a lot of microscope applications such as the medical and biological sciences. To understand more about fluorescent microscopy, we will be discussing the nature of fluorescence first.

In order to fulfil its function, fluorescent microscopes need their specimens to fluoresce. Fluorescence is a phenomenon wherein light is emitted by an organic or inorganic substance after it absorbs light with a shorter wavelength. This shorter wavelength is also known as the excitation light which brings about the emission of light from the specimen. The difference between these excitation wavelengths and the emitted wavelengths is an important property in fluorescence. The exciting light is completely filtered out without blocking the emitted light and this makes the fluorescing parts of the specimen visible.

This principle enables fluorescing microscopic structure to become clearly visible and highly specific against non-fluorescing background. This led in developing the fluorescent microscope which became an important tool in biological applications as well as in the materials sciences.

There are substances that have intrinsic fluorescent or auto-fluorescence properties that enable them to emit light without any aid. These are called fluorochromes and are usually helpful in certain applications such as petrology, botany as well as the semiconductor industry. However, there are also substances that need to be stained in order for their molecules to fluoresce. These stains are fluorochrome-containing dyes or fluorophores which attached itself to highly specific structure and substructure of the specimen. These are very essential especially in the study of plant and animal tissues as well as pathogens and antibodies.

Fluorescence microscopes expose the specimen under a specific wavelength or excitation light and sort out the weaker emitted light to form an image in a very high contrast against a dark background. Exciting the fluorochrome requires a very precise wavelength as the optimal wavelength among each substance with fluorochrome is different. The modern fluorescence microscope utilizes epi-illumination. The excitation light comes through from the objective and onto the specimen. When the specimen fluoresces, the emitted light rises towards the detector of the same objective that released the excitation light. This excitation light is blocked out by a filter located between the objective and the detector filters and the fluorescent light is allowed to enter.

There is also the transmitted illumination wherein the exciting light comes from the condenser to the specimen then into the objective until it reaches the eyepiece. This kind of lighting, however, has a lesser type of illumination than that of an incident lighting or epi-fluorescence microscope.

A fluorescence microscope requires special parts in order to function properly. This high power microscope has an epi-fluorescence attachment that is situated between the microscope head and the body tube. A mercury lamp is attached in a slotted box behind the microscope and its light passes through a bridge towards the objectives. Inside the microscope objectives is a modular unit that is composed of the dichroic filter, exciter filter and the barrier filter. These filters are present in order to precisely sort out the emission wavelengths from the excitation wavelengths. When these filters are removed, the microscope can function like any other brightfield microscope.

Barrier filters are an important part in fluorescence microscopes as it blocks the ultra-violet light from passing through the object from the specimen. Only a fraction of the UV light is absorbed by the fluorochrome and the rest of it passes into the objective lenses. Located in a slider mechanism beneath the microscope head, the barrier filter removes the ultra-violet radiation from the light beam before it reaches the observer’s eyes otherwise it will cause severe burns in the cornea.

Exciter filters blocks all wavelengths except those wavelengths which are necessary to excite the fluorochrome or the specimen. Light from the source passes through the exciter filter in order to achieve a maximum contrast and at the same time minimal non-specific fluorescence of the specimen. There are many types of exciter filter. A “narrow band” exciter filter only allows specific or short range wavelengths while “broad band” filters are for wavelengths with broader ranges. The dichroic filter is also called the dichroic beam splitter which reflects the short wavelength light downwards from the exciter filter.

There is no condenser for epi-fluorescence microscopes as its function is also performed by the microscope objective. The microscope objective is the source of illumination that excites the specimen and at the same time the optical element that gathers the emitted light of the specimen. The emitted light then passes through the dichroic beam splitter, a suppression filter and finally the barrier filter before it reaches the observer’s eyes.

Fluorescence microscopy has been a big help to the field of science as well as other applications. Microscopic structures and even the tiniest substratum can be seen easily because of the high contrast of the fluorescing substance against a very dark background. This contrast is further enhanced when incident lighting is used. In the medical sciences, fluorescence microscopes are used to observe antibodies as well as identify diseases or impurities in the cells or cellular components of inorganic materials.

Learning more about fluorescence microscopes is a big advantage especially when the use of fluorescence to examine specimens is becoming more and more popular particularly in the cell and molecular biology fields. Students can start with learning the principles of fluorescent microscopes so that when the time comes that they can get hold of one, they are well aware of the things that they can do and explore with fluorescence microscopes.