Advances in Fluorescence Microscopy

In fluorescence microscopy, a specimen emits energy when activated by light of a certain wavelength. Some substances such as chlorophyll and some minerals will do this naturally, while others will need the help of other chemical triggers.

Fluorescence microscope components

A fluorescence microscope is typically made up of the following components:

  • A light source, which may be a xenon or mercury burner, an LED, or a laser.
  • An excitation filter, which selects from the light source only the excitation wavelengths that are able to excite the specimen.
  • A dichroic beam splitter or mirror transmits and reflects light as a function of wavelength.
  • An emission filter, which ensures only light emitted from the sample is transmitted, by selecting for the light of a distinct wavelength.
  • A CCD camera, which is the most widely used device for detecting the emitted light. It is usually linked to a computer screen which displays the images generated
Modern microscope station with atibody-stained tissue sample image on the screen. Image Copyright: anyaivanova / Shutterstock
Modern microscope station with antibody-stained tissue sample image on the screen. Image Copyright: anyaivanova /

Types of fluorescence microscopy

Epifluorescence microscopy

The Greek word “epi” means the same. In epifluorescence microscopy, the illuminated and emitted light both travel through the same objective lens.

The light source is often a xenon or mercury burner, but more recently, LEDs have become popular. A filter is used to select wavelengths coming from the light source.

The light reaches the specimen via a microscope objective lens and is absorbed by the fluorophores. The emitted light from the sample travels back through the objective lens, towards the emission filter and detector.

Total internal reflection fluorescence (TIRF)

This technique ensures that the excitation light does not make it very far into the sample. For example, if neurons are placed in a solution on a glass slide, some of them will stick to the surface of the glass.

In TIRF, the light is sent sideways into the slide, which means it does not get very far into the solution containing the specimen; it just leaks into the solution, but only very near to the glass surface.

Light is then only emitted in a very thin area against the surface of the glass. This can provide very clear images for a sample such as neurons, where a lot happens on the cell surface.

Confocal fluorescence

Here, a laser spot is focused in the same plane as the focus of the microscope. The light that is not from the microscope focus is blocked by a pinhole in front of the detector.

Ordinarily, with epifluorescence, the microscope would collect all light within the microscope field of view, including light that is not from the microscope focus. This process means the out-of-focus light is blocked and does not reach the detector.

Multiphoton fluorescence

The arrangement of the objective, collection optics, and pinhole in confocal microscopy has to be very precise for the microscope to work properly. Multiphoton fluorescence uses laser light of a wavelength that only has half the energy it needs to be in order to excite the sample.

Fluorophores in the sample will only become excited and emit light when the laser is bright enough for photons to reach the fluorophores very quickly, which only happens when the laser light is directed to a very small spot. Therefore, light is only emitted from the area of the sample where the laser is focused. This eradication of background light results in very clean, sharp images.

Super-resolution fluorescence

Structured illumination microscopy (SIM) and stimulated emission depletion microscopy both work by restricting the size of the spot that emits light by reducing the size of the excitation spot. This is because a focused spot of light can only not get any smaller than around 200nm and single molecules are much smaller than this.


Further Reading

Last Updated: Jan 12, 2023

Deborah Fields

Written by

Deborah Fields

Deborah holds a B.Sc. degree in Chemistry from the University of Birmingham and a Postgraduate Diploma in Journalism qualification from Cardiff University. She enjoys writing about the latest innovations. Previously she has worked as an editor of scientific patent information, an education journalist and in communications for innovative healthcare, pharmaceutical and technology organisations. She also loves books and has run a book group for several years. Her enjoyment of fiction extends to writing her own stories for pleasure.


Please use one of the following formats to cite this article in your essay, paper or report:

  • APA

    Fields, Deborah. (2023, January 12). Advances in Fluorescence Microscopy. AZoLifeSciences. Retrieved on February 26, 2024 from

  • MLA

    Fields, Deborah. "Advances in Fluorescence Microscopy". AZoLifeSciences. 26 February 2024. <>.

  • Chicago

    Fields, Deborah. "Advances in Fluorescence Microscopy". AZoLifeSciences. (accessed February 26, 2024).

  • Harvard

    Fields, Deborah. 2023. Advances in Fluorescence Microscopy. AZoLifeSciences, viewed 26 February 2024,


The opinions expressed here are the views of the writer and do not necessarily reflect the views and opinions of AZoLifeSciences.
Post a new comment
You might also like...
Scientists Capture Microtubule Birth in Human Cells