Fluorescence Microscopy

Introduction image

The fluorescence microscopy makes the use of fluorescence mechanism to generate an image and optical sectioning for the high resolution. It is available in different designs. The most common and simple set-up in use is epifluorescence microscopes, while confocal microscope has a sophisticated set-up. Epifluorescence microscope is commonly used in different laboratories that allows excitation of reactive dye and detection of the fluorescent emitted light through the same objective.

Therefore, the working of a fluorescent microscope depends upon the reactive species within the fluorescent dye that absorbs a photon of the exciting light ( low wavelength) and later transmits fluorescent light (high wavelength). Thus, there is a short delay between the excitation and emission of fluorescent light that is generally negligible (takes nanoseconds).

Content: Fluorescence Microscopy

  1. Definition
  2. Working
  3. Fluorescence
  4. Components
  5. Advantages
  6. Limitations
  7. Conclusion

Definition of Fluorescence Microscopy

It can define as one of the microscopic techniques that use chemical substances, which stains the specimen, absorbs white light of specific wavelength and finally emits light of longer wavelength to form an image. The chemical substance used in this advanced microscopic method refers to as fluorescent dyes that come into an action under the visible light.

The specimen is first stained with fluorescent dyes, where some of the portions may take up the stain, while few may not. Therefore, the portion stained with fluorescent dye will appear fluorescent green against the dark black background, while the unstained portions will remain invisible.

Working

The working of fluorescence microscopy is explained below:

working of fluorescence microscopy

  1. Firstly a light source falls onto the excitation filter.
  2. The function of the excitation filter is to only pass the light of a particular wavelength that can excite the fluorescent molecules tagged the specimen.
  3. The excitation light will then fall onto the dichroic mirror.
  4. A diachronic mirror reflects this emitted light towards the objective lens and then onto the specimen.
  5. As the specimen is stained with a fluorescent dye, the fluorescent molecules will excite and emits a high wavelength light in a reverse manner.
  6. The emitted light will first go through the dichroic mirror that will pass the majority of the green light to pass along with some blue light towards the emission filter.
  7. The role of the emission filter is to only permit the green light of longer wavelength and totally rejects the blue light.
  8. Finally, the green light goes to the ocular lens, where the detector detects this light and allow it to fall back onto the specimen.
  9. Thus, the majority of specimens appear fluorescent green against a jet black background.

Fluorescence

Its working depends upon the principle of fluorescence, rather than scattering and reflection of light. Fluorescence is the phenomena carried out by certain chemical substances or reactive dyes that possess a property of light absorption and emission. A scientist named George G. Stokes was the first who studied the mechanism of fluorescence and also coined the term, in the year 1852.

Jablonski diagram illustrating fluorescence mechanism

When the fluorescent molecules absorb the light of short wavelength and get excited by the high energy photons, the process termed as excitation, the light triggering the fluorescent particles will refer as “Exciting light” that are generally ionizing X-rays and UV-rays.

The fluorescent molecules emit high wavelength light, the process termed as emission. The light reflected by these chemical substances after absorption of photons will refer to as “Emission light” that are generally in the form of visible light.

The fluorescence is achieved when the high energy photon molecules in the excited state will go back to their normal ground state by releasing some energy in the form of fluorescence. The difference between the phase of excitation and emission is known as “Strokes shift”, which can be relatively short and sometimes far apart.

fluorescence spectrum

Components

Typical components of a fluorescence microscope are:

Fluorescent dyes

These are the chemical compounds that are also called fluorophore or reactive dyes. Fluorescent dye possesses a property of fluorescence, by which it can form a fluorescent image by emitting highly contrast visible green light after getting excited by the highly illuminating UV-light.

A fluorophore is designed to highlight a wide range of biomolecules like antibodies, nucleic acids, proteins, etc. For instance, Hoechst and phalloidin are used to stain actin fibres in mammalian cells.

Light Source

It includes xenon arc lamps, mercury-vapour lamps, lasers, and high- power LEDs. Laser light is mostly used in sophisticated confocal fluorescence microscopy. On the contrary, a light source from xenon lamps, mercury lamps, and LEDs are used in the simple epifluorescence microscopy that is widely accepted by the different laboratories.

Excitation Filter

The excitation filters are designed with high-resolution capacity and interference optics. It is explicitly constructed to pass the light of a shorter wavelength, which could be typically absorbed by the fluorescent dye. It blocks the other sources of exciting light.

Dichroic Mirror

It is a dichromatic beam splitter works as an interference colour filter that selectively reflects or transmits light of determined wavelengths. In the fluorescent microscope, it is placed at an angle of 45 degrees along the path of light coming from the exciter filter.

Emission Filter

The emitter filters are designed with coloured glass or interference coating or the combination of both. It works as a barrier filter that only permits the passage of light emitted by the fluorophore and blocks excitation light.

Advantages

  • Fluorescence microscopy helps in the study of cell behaviour.
  • It is the specific microscopic method that highlights the biomolecule of interest.
  • The fluorescent microscope can be used to get the image of particular structural components found within the microscopic organisms.
  • It is a highly sensitive technique that can detect around 50 molecules/µm3.
  • Different biomolecules can be tagged explicitly with different fluorescent dyes, by which we can analyze or track the physiochemical properties of multiple biomolecules simultaneously.

Limitations

  • It only allows the observation of specific structures inside a cell that are tagged with a fluorescent dye.
  • In this microscopy technique, the reactive molecules in the fluorescent dyes can be affected by the photobleaching caused due to the electron excitation during the process of fluorescence. As a result, the reactive dyes might lose their chemical property of fluorescence emission intensity.
  • The cells stained with fluorescent light might be susceptible to phototoxic effect, as the reactive dye absorbs the high energy photons from the short wavelength light.

Conclusion

Therefore, fluorescence microscopy uses fluorescent stain in place of common dyes used by the other microscopy techniques. Its working depends upon the excitation of the absorbed light and the emission of the fluorescent light to get an image with high contrast and high resolution.

In a fluorescent microscope, the first light of low wavelength having much energy molecules illuminates the sample that in turn excites the reactive species in the specimen to emit a second light of longer wavelength having lesser energy content. It is important to note that an image produces by the filtered emitted light out of total exciting light.

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