How do Epifluorescence Microscopes Work?

Epifluorescence microscopes are a commonly used tool for studying specimens. Improved specificity and contrast provided by the application of fluorescence to the field of microscopy has stimulated the advancement of various discoveries in the biosciences.


Image Credit: D. Kucharski K. Kucharska/

In 1843, George Gabriel Stokes described fluorescence as being characterized by the wavelength of an emitted light that is longer than the wavelength of the exciting light.

This discovery led to the invention of the epifluorescence microscope, now a ubiquitous instrument in biological and medical laboratories.

The high contrast and specificity of the images produced have allowed for the increased understanding of cell structures, the location, and dynamics of gene expression and distinct interactions at a molecular level within the cell.

Principles of fluorescence

Fluorescence microscopy is a particular form of light microscopy in which, instead of utilizing visible light to illuminate specimens, a higher intensity light source excites a fluorescent molecule called a fluorophore.

The fluorophore absorbs photons leading to electrons moving to a higher energy state. When the electrons return to the ground state by losing energy, the fluorophore emits light of a longer wavelength.

The emitted light is then separated from the original excitation light via a filter and this produces a magnified image of the specimen being studied.

The use of fluorescence allows for the object of interest to be specifically targeted. As the excitation light is filtered out, the emitted fluorescence allows only the fluorescent objects of interest to remain visible.

Components of an epifluorescence microscope

An epifluorescence microscope requires particular components to separate the excitation and emission lights, acquire the image and allow for the target object to be observed. The following are important components of an epifluorescence microscope:

  1. The light source: A source of high-intensity light that can emit a broad spectrum of wavelengths is required. This is usually in the form of a xenon arc lamp or a high-pressure short arc mercury lamp.
  2. Filter cubes: They allow for the selection of specific excitation and emission wavelengths and consist of the excitation filter, dichroic mirror, and barrier filter. The transmission of the excitation light is selected via the excitation filter and the transmission of the emission light is selected via the barrier filter, by blocking the excitation light. The dichroic mirror separates the excitation light from the emission light further by reflecting the shorter wavelengths of the excitation light and transmitting the longer wavelengths of the emission light.
  3. Objective lens: The objective lens transmits light to the sample to form the image. The emission light passes down through the dichroic mirror before reaching the objective lens.
  4. Camera system: Modern epifluorescence microscopes can record the images of the specimen with high-resolution. Electron multiplying CCD cameras are often used as they are capable of imaging single-photon events without requiring an image intensifier. This means that images can be captured at high speed without loss of sensitivity.

Epifluorescence illumination or epi-illumination

Epifluorescence illumination or epi-illumination is the specific arrangement of these optical components that allow for the illumination of the specimen from above. The objective lens acts as both the source of light that excites the fluorophore and the element of the microscope that collects the emitted fluorescent light.

In comparison to other forms of fluorescence microscopy, epifluorescence illumination has the advantage of only requiring a small amount of emitted light to be blocked.

Though this approach means that the excitation light and emission light overlap, the dichroic mirror efficiently separates the excitation from the emission.

The combination of an excitation filter, dichroic mirror, and barrier filter in the filter cubes means that only one photon in 10,000 of the wrong excitation wavelength to the specimen passes, with a similar amount for the emission light. This high ratio is important for clearly imaging single fluorescent molecules.


  • Fluorophore: A chemical compound that fluoresces by emitting light upon excitation.
  • Photon: A fundamental particle of visible light.
  • Ground state: The normal, non-excited state of a molecule.
  • Excited state: Electrons can move to a higher energy level by absorbing light, therefore achieving an excited state.


  • Masters, B.R. 2010. ‘The Development of Fluorescence Microscopy’. In: Encyclopedia of Life Sciences (ELS). John Wiley & Sons.
  • Webb, D. and Brown, C. 2013. ‘Epi-Fluorescence Microscopy’, Methods in Molecular Biology, 931, pp. 29-59.
  • Lichtman, J. and Conchello, J. 2005. ‘Fluoresence microscopy’, Nature Methods, 2, pp. 910-913.

Further Reading

Last Updated: Jun 27, 2022

Shelley Farrar Stoakes

Written by

Shelley Farrar Stoakes

Shelley has a Master's degree in Human Evolution from the University of Liverpool and is currently working on her Ph.D, researching comparative primate and human skeletal anatomy. She is passionate about science communication with a particular focus on reporting the latest science news and discoveries to a broad audience. Outside of her research and science writing, Shelley enjoys reading, discovering new bands in her home city and going on long dog walks.


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

  • APA

    Stoakes, Shelley Farrar. (2022, June 27). How do Epifluorescence Microscopes Work?. AZoLifeSciences. Retrieved on May 30, 2024 from

  • MLA

    Stoakes, Shelley Farrar. "How do Epifluorescence Microscopes Work?". AZoLifeSciences. 30 May 2024. <>.

  • Chicago

    Stoakes, Shelley Farrar. "How do Epifluorescence Microscopes Work?". AZoLifeSciences. (accessed May 30, 2024).

  • Harvard

    Stoakes, Shelley Farrar. 2022. How do Epifluorescence Microscopes Work?. AZoLifeSciences, viewed 30 May 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

While we only use edited and approved content for Azthena answers, it may on occasions provide incorrect responses. Please confirm any data provided with the related suppliers or authors. We do not provide medical advice, if you search for medical information you must always consult a medical professional before acting on any information provided.

Your questions, but not your email details will be shared with OpenAI and retained for 30 days in accordance with their privacy principles.

Please do not ask questions that use sensitive or confidential information.

Read the full Terms & Conditions.

You might also like...
Cryo-EM Study Reveals Cas7-11-CRISPR Complex Regulation Mechanisms