Fluorescence microscopy: principles of the method

The absorption and further reradiation of light by inorganic and organic media is the result of phosphorescence or fluorescence. The difference between the phenomena consists in the duration of the interval between the light absorption and the emission of the flux. Under fluorescence, these processes occur almost simultaneously, and with phosphorescence, with some delay.

Historical reference

In 1852 the British scientist Stokes first described fluorescence. He introduced a new term as a result of performed experiments with fluorspar, which emitted red light under the influence of ultraviolet. Stokes noted an interesting phenomenon. He found that the wavelength with fluorescent radiation is always greater than that of the excitation light flux.

To confirm the hypothesis in the 19th century, many experiments were carried out. They showed that a variety of samples fluoresce under the action of ultraviolet. Among the materials, among others, were crystals, resins, minerals, chlorophyll, medicinal raw materials, inorganic compounds, vitamins, oils. Direct application of dyes for carrying out biological analyzes only began in 1930.

Fluorescence microscopy: description

Some of the materials used in the studies of the first half of the 20th century had high specificity. стал важнейшим инструментом и в биомедицинских, и в биологических исследованиях. Due to indicators that could not be achieved by contrast methods, the method of fluorescence microscopy has become an important tool in both biomedical and biological research. The obtained results were of no small importance for materials science.

? What are the advantages of fluorescence microscopy ? With the help of new materials it became possible to isolate highly specific cells and submicroscopic components. A fluorescent microscope can detect individual molecules. A variety of dyes allow the identification of several elements at the same time. Despite the limited spatial resolution of the equipment by the diffraction limit, which in turn depends on the specific properties of the sample, it is also possible to detect molecules below this level. Various samples after irradiation exhibit autofluorescence. This phenomenon is widely used in petrology, botany, semiconductor industry.

Features

The study of animal tissues or pathogenic microorganisms is often complicated by either too weak or very strong nonspecific auto fluorescence. However, the significance in the studies is the introduction into the material of components excited at a particular wavelength and emitting a light flux of the required intensity. Fluorochromes act as dyes capable of attaching themselves to structures (invisible or visible). At the same time, they are highly selective with respect to targets and quantum yield.

стала широко применяться с появлением естественных и синтетических красителей. Fluorescence microscopy has become widely used with the advent of natural and synthetic dyes. They had definite profiles of intensity of emission and excitation and were aimed at specific biological targets.

Identification of individual molecules

Often, under ideal conditions, it is possible to register the glow of an individual element. For this, among other things, it is necessary to ensure a sufficiently low noise of the detector and an optical background. The molecule fluorescein before destruction due to photobleaching can emit up to 300 thousand photons. At 20% of the collection and efficiency of the process, they can be registered in the amount of about 60 thousand.

, основанная на лавинных фотодиодах или электронном умножении, позволяла исследователям наблюдать поведение отдельных молекул на протяжении секунд, а в ряде случаев и минут. Fluorescence microscopy , based on avalanche photodiodes or electron multiplication, allowed researchers to observe the behavior of individual molecules for seconds, and in some cases, minutes.

Difficulties

The key problem is the suppression of noise from the optical background. Due to the fact that many of the materials used in the design of filters and lenses exhibit some autofluorescence, the efforts of scientists at the initial stages were focused on the production of components that possess low fluorescence. However, subsequent experiments led to new conclusions. , основанная на полном внутреннем отражении, позволяет достичь низкого фона и высокоинтенсивного возбуждающего светового потока. In particular, it was found that fluorescence microscopy , based on total internal reflection, achieves a low background and a high-intensity exciting light flux.

Mechanism

, основанной на полном внутреннем отражении, заключаются в использовании быстрозатухающей или нераспространяющейся волны. The principles of fluorescence microscopy , based on total internal reflection, consist in the use of a rapidly decaying or non-propagating wave. It arises on the boundary of media with different refractive indices. In this case, the light beam passes through the prism. It has a high refractive index.

The prism is adjacent to an aqueous solution or a glass with a low parameter. If the stream of light is directed at it at an angle that is greater than the critical one, the beam is completely reflected from the interface. This phenomenon, in turn, causes a non-propagating wave. In other words, an electromagnetic field is generated that penetrates into the medium with a smaller refractive index for a distance of less than 200 nanometers.

In a non -propagating wave, the intensity of light will be quite sufficient to excite the fluorophores. However, due to its extremely insignificant depth, its volume will be very small. As a result, a low-level background appears.

Modification

Fluorescence microscopy, based on total internal reflection, can be realized with the help of epi-illumination. This requires lenses with an increased numerical aperture (at least 1.4, but it is desirable that it reaches 1.45-1.6), as well as partially illuminated field of the device. The latter is achieved by using a spot of small size. For greater uniformity, a thin ring is used, through which part of the flow is blocked. To obtain a critical angle, after which a complete reflection occurs, a high level of refraction of the immersion medium in the lenses and the cover glass of the microscope is needed.