What are the basic principles of fluorescence microscopy?

The basic premise of fluorescence microscopy is to stain the components with dyes. Fluorescent dyes, also known as fluorophores or fluorochromes, are molecules that absorb excitation light at a given wavelength (generally UV), and after a short delay emit light at a longer wavelength.

What is the principle of atomic fluorescence spectroscopy?

Atomic fluorescence spectrometry (AFS) is an analytical method used to determine the concentration of elements in samples. The sample is converted to gaseous atoms, and the element of interest is excited to a higher electronic energy level by a light source.

What is the principle of confocal microscopy?

The basic principle of confocal microscopy is that the illumination and detection optics are focused on the same diffraction-limited spot, which is moved over the sample to build the complete image on the detector.

What is the working principle of electron microscope?

An electron microscope uses an ‘electron beam’ to produce the image of the object and magnification is obtained by ‘electromagnetic fields’; unlike light or optical microscopes, in which ‘light waves’ are used to produce the image and magnification is obtained by a system of ‘optical lenses’.

How does fluorescence spectroscopy work?

Fluorescence spectroscopy uses a beam of light that excites the electrons in molecules of certain compounds, and causes them to emit light. That light is directed towards a filter and onto a detector for measurement and identification of the molecule or changes in the molecule.

What are the applications of fluorescence spectroscopy?

Examples of the use of fluorescence spectroscopy include the study of fluorescent dyes that are widely used with biological samples, both in routine assays and in advanced research. It is also employed in material science to characterize luminescent materials.

What is the principle of transmission electron microscope?

The TEM operates on the same basic principles as the light microscope but uses electrons instead of light. Because the wavelength of electrons is much smaller than that of light, the optimal resolution attainable for TEM images is many orders of magnitude better than that from a light microscope.

What is the principle of interference microscopy?

Interference microscopy uses a prism to split light into two slightly diverging beams that then pass through the specimen. It is thus based on measuring the differences in refractive index upon recombining the two beams. Interference occurs when a light beam is retarded or advanced relative to the other.

What is the principle of electron?

Principle of electron microscopy Electrons are such small particles that, like photons in light, they act as waves. A beam of electrons passes through the specimen, then through a series of lenses that magnify the image. The image results from a scattering of electrons by atoms in the specimen.

What are the characteristics of fluorescence?

Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. It is a form of luminescence. In most cases, the emitted light has a longer wavelength, and therefore a lower photon energy, than the absorbed radiation.

Why is fluorescence used?

Fluorescence is often used to analyze molecules, and the addition of a fluorescing agent with emissions in the blue region of the spectrum to detergents causes fabrics to appear whiter in sunlight. X-ray fluorescence is used to analyze minerals.

Why does fluorescence quenching occur?

Fluorescence quenching refers to any process that decreases the fluorescence intensity of a sample. A variety of molecular interactions can result in quenching. These include excited-state reactions, molecular rearrangements, energy transfer, ground-state complex formation, and colli-sional quenching.

What are examples of fluorescence?

Common materials that fluoresce Vitamin B2 fluoresces yellow. Tonic water fluoresces blue due to the presence of quinine. Highlighter ink is often fluorescent due to the presence of pyranine. Banknotes, postage stamps and credit cards often have fluorescent security features.

How was fluorescence first discovered?

In 1845, Sir Frederik William Herschel noted that a quinine solution, although itself colorless and transparent, exhibits a “vivid and beautiful celestial blue color,” when illuminated and observed under certain incidences of sunlight 1. This is the first reported observation of fluorescence.

How does fluorescein work?

In pure state, fluorescein is a red or orange powder which is dissolved in alkaline solutions to make up a yellow solution which will glow green. In addition to using straight fluorescein, researchers also work with various derivatives of the compound which are designed to conjugate to specific types of molecules and perform other functions.

How does fluorescence work?

Fluorescence occurs when a substance releases absorbed energy in the form of light. A fluorescent substance absorbs electromagnetic radiation (often ultraviolet light), which promotes the electrons to a higher energy level. While in the higher energy state, the electron loses some energy in the form of heat.