Absorption and further re-emission of light by inorganic and organic media is the result of phosphorescence or fluorescence. The difference between the phenomena is the length of the interval between light absorption and emission of the stream. With fluorescence, these processes occur almost simultaneously, and with phosphorescence, with some delay.
Historical background
In 1852, the British scientist Stokes first described fluorescence. He coined the new term as a result of his experiments with fluorspar, which emitted red light when exposed to ultraviolet light. Stokes noted an interesting phenomenon. He found that the wavelength of fluorescent light is always longer than that of the excitation light.
Many experiments were carried out in the 19th century to confirm the hypothesis. They showed that a variety of samples fluoresce when exposed to ultraviolet light. Materials included, among others, crystals, resins, minerals, chlorophyll,medicinal raw materials, inorganic compounds, vitamins, oils. The direct use of dyes for biological analysis began only in 1930
Fluorescence microscopy description
Some of the materials used in research in the first half of the 20th century were highly specific. Thanks to indicators that could not be achieved by contrast methods, the fluorescence microscopy method has become an important tool in both biomedical and biological research. The results obtained were of no small importance for materials science.
What are the benefits of fluorescence microscopy? With the help of new materials, it became possible to isolate highly specific cells and submicroscopic components. A fluorescent microscope allows you to detect individual molecules. A variety of dyes allow you to identify several elements at the same time. Although the spatial resolution of the equipment is limited by the diffraction limit, which, in turn, depends on the specific properties of the sample, the detection of molecules below this level is also quite possible. Various samples exhibit autofluorescence after irradiation. 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 too strong non-specific autofluorescence. However, the value inresearch acquires the introduction into the material of components excited at a specific wavelength and emitting a light flux of the required intensity. Fluorochromes act as dyes capable of self-attachment to structures (invisible or visible). At the same time, they are distinguished by high selectivity with respect to targets and quantum yield.
Fluorescence microscopy has become widely used with the advent of natural and synthetic dyes. They had specific emission and excitation intensity profiles and were aimed at specific biological targets.
Identification of individual molecules
Often, under ideal conditions, you can register the glow of a single element. To do this, among other things, it is necessary to ensure sufficiently low detector noise and optical background. A fluorescein molecule can emit up to 300,000 photons before destruction due to photobleaching. With a 20% collection rate and process efficiency, they can be registered in the amount of about 60 thousand
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 noise suppression from the optical background. Due to the fact that many of the materials used in the construction of filters and lenses exhibit some autofluorescence, the efforts of scientists in the initial stages were focused on issuingcomponents with low fluorescence. However, subsequent experiments led to new conclusions. In particular, fluorescence microscopy based on total internal reflection has been found to achieve low background and high excitation light output.
Mechanism
The principles of fluorescence microscopy based on total internal reflection are to use a rapidly decaying or non-propagating wave. It arises at the interface between media with different refractive indices. In this case, the light beam passes through a prism. It has a high refractive index.
The prism is adjacent to an aqueous solution or low parameter glass. If the beam 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, gives rise to a nonpropagating wave. In other words, an electromagnetic field is generated that penetrates a medium with a lower refractive index at a distance of less than 200 nanometers.
In a non-propagating wave, the light intensity will be quite sufficient to excite fluorophores. However, due to its exceptionally shallow depth, its volume will be very small. The result is a low-level background.
Modification
Fluorescence microscopy based on total internal reflection can be realized with epi-illumination. This requires lenses with increased numerical aperture (at least 1.4, but it is desirable that it reaches 1.45-1.6), as well as a partially illuminated field of the device. The latter is achieved with a small spot. For greater uniformity, a thin ring is used, through which part of the flow is blocked. To obtain a critical angle after which total reflection occurs, a high level of refraction of the immersion medium in the lenses and the microscope cover glass is needed.
