Fluorescence Microscopy-Definition Principal And Examples

Fluorescence microscopy is an optical microscope that uses fluorescence and phosphorescence instead of reflection. And absorption to study the properties of substances. Fluorescence is the emission of light by substances that absorb light or other electromagnetic radiation. While phosphorescence is a special type of photoluminescence related to fluorescence. Unlike fluorescence, phosphorus materials do not immediately reproduce the absorbed radiation.

Meaning:

“Fluorescence microscope” means any microscope that uses fluorescence to create images, either a simple configuration (such as an epifluorescence microscope) or a more complex design (such as a confocal microscope that uses optical sections to obtain fluorescence images with better resolution)

Principle:

• The sample is illuminated by a certain wavelength (or wavelengths) of light absorbed by the fluorophore, which causes them to emit light at a longer wavelength (that is, a different color than the absorbed light).
• Use a spectral emission filter to separate the illuminating light from the less diffuse fluorescence.
• Most components of the cell are colorless and cannot be seen under a microscope.
• The main purpose of fluorescence microscopy is to color the components with dyes.
• Fluorescent dyes, also known as fluorophores or fluorescent dyes, are molecules that absorb excitation light with a certain wavelength (usually ultraviolet) and emit light with longer wavelengths after a short delay.
• The delay between absorption and emission is negligible, usually within nanoseconds.
• The light emitted by the alarm light can then be filtered to locate the fluorophore.

Working of Fluorescence Microscopy:

The excitation wave light is directed at the sample through the lens. The light from the sample is directed to the detector lens. Because most of the alarm light is transmitted from the sample, only the reflected alarm light, along with the scattered light, reaches the target.

form of Fluorescence Microscopy:

“Fluorescence microscope” means any microscope that uses fluorescence to create images, either a simpler configuration (such as an epifluorescence microscope) or a more complex design (such as a confocal microscope that uses optical sections for a better resolution). Fluorescent image.

Most of the fluorescence microscopes used are epifluorescence microscopes, in which fluorophore excitation and fluorescence detection take place in the same optical path (ie through the lens).

Typical Features of a fluorescence microscope:

Fluorescent dyes:

• A fluorophore is a fluorescent compound that emits light during photoexcitation.
• Fluorophores usually contain several combined aromatic groups or several π-linked flat or cyclic molecules.
• Many fluorescent dyes are designed for many biomolecules.
• Some of these are small molecules that fluoresce and bind to related biomolecules. A prime example is a phalloidin, a nucleic acid dye used in mammalian cells to stain actin fibers, such as DAPI and Hoechst.
  it is the source of light
• Four main types of light sources are used, including xenon arc lamps or mercury vapor with alarm filters, lasers, and high-power LEDs.
• Lasers are mainly used in complex fluorescence microscopy techniques, while LEDs with xenon, mercury, and dichroic excitation filters are used in wide-field epifluorescence microscopy.

Alarm filter

The exciter is usually a band-pass filter that passes only the wavelengths absorbed by the fluorophore, thus reducing excitation by other fluorescent sources and blocking the excitation light in the fluorescence emission band.

Drain filter.

The transmitter is usually a band-pass filter that passes only the wavelengths. Emitted by the fluorophore and blocks all unwanted light from outside the band, especially the alarm light.
Filters provide the darkest background by blocking unwanted excitation energy. (including UV and IR) or autofluorescence of the sample and system.

Advantages:

• Fluorescence microscopy is the most popular method of studying the dynamic behavior displayed in living cell images.
• This is due to its ability to separate individual proteins with high specificity between non-fluorescent materials.
• The sensitivity is high enough to detect 50 molecules per cubic micron.
• Different molecules can now be colored in different colors, so you can track several types of molecules at the same time.
• These factors combine to give fluorescence microscopy distinct advantages over other in vitro and in vivo optical imaging methods.

Sample preparation of Fluorescence Microscopy:

Samples must be fluorescent to be compatible with fluorescence microscopy. There are several ways to create fluorescent patterns; the main methods are labeling with fluorescent dyes or expressing fluorescent proteins in biological samples. Alternatively, the internal fluorescence of the sample (ie autofluorescence) may be used. [1] In the life sciences, a fluorescence microscopy is a powerful tool for the specific and sensitive staining of samples to detect the distribution of proteins.

Epifluorescence:

Most fluorescence microscopes, especially those used in the life sciences, have the epifluorescence design shown in the figure. The light from the excitation wave passes through the lens to illuminate the sample. The fluorescence in the sample is directed to the detector through the same lens used for wake-up, and a larger digital aperture lens is required for higher resolution.

Because most of the excitation light is transmitted from the sample and only the reflected excitation light reaches the target with the emitted light, the epifluorescence has a high signal-to-noise ratio.

What Is Light Source On A Microscope:

Fluorescence microscopy light source requires intense, almost monochromatic illumination, which is some ordinary light. Sources, such as halogen lamps, cannot provide. Main types of light sources are used. lamps or mercury vapor with alarm filters, lasers, ultra-strong light sources, and high-power LEDs.

Lasers are most commonly used in more complex fluorescence microscopy techniques, such as confocal microscopy and internal reverse fluorescence general microscopy. While xenon lamps and LEDs with mercury lamps and dichroic excitation filters are used in wide-field epifluorescence microscopy.

Filter Terminology:

As various manufacturers use various capital letters and codes to describe their filters. The generic terminology applied to filter combinations for fluorescence microscopy has become confusing for light source microscopes. Basically, filters fall into three main categories: excitation (often called exciters), obstacles (emission), and dichroic beam separators (or dichroic mirrors).

The fluorescence filters were made almost entirely of stained glass or gelatin, pressed between two layers of glass. However, the current trend is to produce high-resolution filters with interference optics. So that excitation filters pass or reject light wavelengths with high specificity and high transmittance. A dichroic beam separator is an interference filter specially designed to reflect or transmit certain wavelengths of light when placed at a 45-degree angle to the optical path.

Detecting Single Molecules:

When the optical background and detector noise are low enough, the fluorescence emission of the molecules can often be detected under ideal conditions. As mentioned above, a single fluorescent molecule can emit up to 300,000 photons before being destroyed by photobleaching. Assuming a collection and detection efficiency of 20%, approximately 60,000 photons will be detected.

Using avalanche photodiodes or electronically amplified CCD detectors in these experiments. The researchers were able to track the behavior of individual molecules in seconds or even minutes. The biggest problem is the proper suppression of background optical noise. Because most materials used to build microscope lenses and filters have some autofluorescence, initial efforts focused on producing components with very low levels of fluorescence.

Immunofluorescence:

Immunofluorescence is a technique that uses highly specific binding of antibodies to antigens to label specific proteins or other molecules inside cells. The sample is treated with a base antibody specific to the target molecule.

The fluorophore can bind directly to the primary antibody. Alternatively, a fluorophore-conjugated secondary antibody specific for the primary antibody may be used. For example, primary antibodies raised in tubulin-recognizing mice and mouse secondary antibodies derived from fluorophores

Fluorescent proteins:

The modern understanding of genetics and the methods available for DNA modification allow scientists to genetically modify proteins, as well as to carry homologous fluorescent proteins. In biological samples, this allows scientists to directly fluoresce the proteins of interest. The location of the protein can then be tracked directly, including in living cells.

How does Fluorescent Microscopy Work?

In most cases, the sample of interest is labeled with fluorescent substances called fluorophores. Which are then illuminated by a lens with a higher energy source. Light is absorbed by fluorophores (now added to the sample), causing them to emit light at longer and shorter wavelengths. This fluorescence can be separated from the surrounding radiation by a filter designed for that wavelength and allows the observer to see only the fluorescence.

The main task of a fluorescence microscope is to allow the excitation light to propagate through the sample and then separate the dim diffuse light from the image. First of all, microscopes have a filter that allows specific wavelengths of radiation to pass only through fluorescent materials. The radiation collides with the atoms in the sample, and the electrons are excited at higher energy levels. When they rest at a lower level, they shine. To be detectable (visible to the human eye), the fluorescence emitted by the sample is separated from the brighter excitation light by a second filter. This is because scattered light has less energy and has a longer wavelength than the light used for lighting.

Limitations:

• Fluorophores lose their fluorescent properties when exposed to a process called photovoltaics. Photobleaching occurs because fluorescent molecules accumulate chemical damage from excited electrons during fluorescence.
• Cells are sensitive to phototoxicity, especially in short-wavelength light. In addition, fluorescent molecules form reactive chemicals that increase the phototoxic effect of light exposure.
• Unlike transmitted and reflected light microscopy methods, fluorescence microscopy only allows the observation of certain structures that are labeled as fluorescent.
• It is suggested that calculation methods for estimating fluorescent signals in non-fluorescent images.
• In general, these methods involve the study of deep convolutional neural networks of stained cells.
• The subsequent evaluation of fluorescence in non-stained samples.
• Therefore, by separating the studied cells from the cells used to drive the network, imaging can be performed faster and with less phototoxicity.