Fluorescence Microscopy Principle and Working

  • In this fluorescence microscopy principle and working post we have briefly explained about definition, principle, parts, working, applications, and limitations.

Fluorescence Microscopy Principle and Working

  • The fluorescence microscope is highly effective analytical equipment that combines the magnifying capabilities of a light microscope with fluorescence visibility. Instead of using visible light to illuminate objects, a fluorescence microscope uses a higher intensity (lower wavelength) light source that excites a fluorescence molecule known as a fluorophore (also known as fluorochrome).
  • Fluorescence is a phenomenon in which a material (fluorophore) absorbs light at one wavelength and emits light at a different wavelength. As a result, fluorescence microscopy combines the light microscope’s magnifying capabilities with fluorescence technology.
  • Photons are absorbed by the fluorophore, causing electrons to move to a higher energy state (excited state). When the electrons lose energy and return to the ground state, the fluorophore emits light with a longer wavelength. Diamidino–phenylindole (DAPI) (emits blue), FITC (emits green), and Texas Red are three of the most commonly utilised fluorophores (emits red).

Principle

  • An exciter filter is used to guide light from a light source such as Xenon or Mercury Arch Lamp that emits light in a wide range of wavelengths, from ultraviolet to infrared (selects the excitation wavelength). A specific mirror called a dichroic mirror, which is intended to reflect light exclusively at the excitation wavelength, reflects this light toward the sample. The reflected light is focussed onto the fluorescent specimen by passing through the objective.
  • The emissions from the specimen are then reflected back up through the objective, where the image is magnified, and the dichroic mirror. This light is filtered by the barrier filter, which selects for the emission wavelength and filters out contaminating light from the arc lamp or other sources that are reflected off from the microscope components. Finally, the filtered fluorescent emission is sent to a detector where the image can be digitized.

Components

  • The fluorescent microscope’s principal components are similar to those of a regular light microscope. The type of light source used and the employment of specific filter components are the two key differences.

Source of light

  • A very bright light source, such as a Xenon or Mercury Arch Lamp, is required for fluorescence microscopy. The Mercury Arc Lamp emits light that is 10–100 times brighter than conventional incandescent lights and covers a wide spectrum of wavelengths from ultraviolet to infrared. Complex fluorescent microscopy techniques were usually performed with lasers or high-power LEDs.
Fluorescence microscope

Fluorescence Microscopy Principle and Working: The source of light

Filter elements

  • Excitation, emission, and the dichroic beam splitter are the three filters of a standard fluorescence microscope.

Excitation filters

  • Excitation filters are positioned within a fluorescence microscope’s illumination path. Its objective is to filter out all wavelengths of the light source save the fluorophores excitation range in the sample or specimen of interest.

Emission filters

  • A fluorescence microscope’s emission filter is positioned within the imaging path. Its function is to filter out the whole excitation range while transmitting the fluorophores emission range in the specimen.

Dichroic filter

  • The dichroic filter or beam splitter is positioned at a 45-degree angle between the excitation and emission filters. Its goal is to reflect the excitation wavelength toward the fluorophore in the specimen while transmitting the emission wavelength to the detector.

Working Mechanism

  • The specimens to be studied are stained or labelled with a fluorescent dye and then lit with a mercury arc lamp that emits high-intensity ultra violet light. The light travels via the exciter filter, which only permits blue light to pass. The blue light is then reflected downward to the specimen via a dichroic mirror.
  • The fluorescent dye-labeled material absorbs blue light (of a shorter wavelength) and emits green light. The produced green light travels upward, passing through a dichroic mirror, which reflects back blue light and lets only green light to pass through the objective lens, before arriving at a barrier filter that allows only green light to pass through.

Applications

  • In biological research and clinical pathology, the fluorescence microscope has become one of the most powerful tools (To identify the Mycobacterium tuberculosis).
  • Multicolour staining, labelling of structures within cells, and evaluation of a cell’s physiological status are all possible with a fluorescence microscope.
  • A fluorescence microscope is useful for analysing coal texture and structure and using a fluorescent dye, investigates porosity in ceramics.

Further Readings

Reference