The 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 flux emission. With 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Historical reference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 experiments with fluorspar, which emitted red light when exposed to ultraviolet light. Stokes noted an interesting phenomenon. He revealed that the wavelength of fluorescent radiation is always greater than that of the excitation light flux.
To confirm the hypothesis in the 19th century, many experiments were conducted. They showed that a variety of samples fluoresce under the influence of ultraviolet radiation. Among the materials, among other things, were 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 studies of the first half of the 20th century were highly specific. Thanks to indicators that could not be achieved by contrasting methods, fluorescence microscopy 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 fluorescence microscope allows you to detect individual molecules. A variety of dyes allow you to identify multiple elements at the same time. Despite the limited spatial resolution of the equipment with a 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, and the semiconductor industry.
Features
The study of animal tissues or pathogenic microorganisms is often complicated either by too weak or very strong non-specific autofluorescence. However, the introduction of components into the material that are excited at a specific wavelength and emit a luminous flux of the necessary intensity is gaining importance in research. Fluorochromes act as dyes that can independently attach 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 profiles of the intensity of emission and excitation and were aimed at specific biological targets.
Identification of individual molecules
Often, under ideal conditions, you can register the glow of a single element. For this, among other things, it is necessary to provide a sufficiently low detector noise and optical background. The molecule of fluorescein before destruction due to photobleaching can emit up to 300 thousand photons. At 20% collection 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 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 with low fluorescence. However, subsequent experiments led to new conclusions. In particular, it was found that fluorescence microscopy based on total internal reflection, allows to achieve low background and high-intensity exciting light flux.
Mechanism
The principles of fluorescence microscopy , based on total internal reflection, are the use of a rapidly decaying or non-propagating wave. It arises at 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 adheres to an aqueous solution or glass with a low parameter. If a stream of light is directed at it at an angle that is greater than the critical, 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 a medium with a lower refractive parameter to 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 extremely insignificant 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 using epi-illumination. This requires lenses with a higher 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 apparatus. The latter is achieved with a small spot. For greater uniformity, a thin ring is used, through which a part of the flow is blocked. To obtain a critical angle, after which full reflection occurs, a high level of refraction of the immersion medium in the lenses and the microscope coverslip is needed.