What is the Difference Between Fluorescence and Epifluorescence Microscopy

The main difference between fluorescence and epifluorescence microscopy is that fluorescence microscopy is a type of optical microscopy that uses fluorescence instead of the visible spectrum to generate an image, whereas epifluorescence microscopy is a type of fluorescence microscopy in which excitation of the fluorophore and the detection of the fluorescence occur through the same light path.

Fluorescence and epifluorescence microscopy are two types of optical microscopy that use fluorescence instead of visible light to make a specimen visible.

Key Areas Covered

1. What is Fluorescence Microscopy
   • Definition, Characteristics, Importance
2. What is Epifluorescence Microscopy
   • Definition, Characteristics, Importance
3. Similarities Between Fluorescence and Epifluorescence Microscopy
   • Outline of Common Features
4. Difference Between Fluorescence and Epifluorescence Microscopy
   • Comparison of Key Differences

Key Terms

Epifluorescence Microscopy, Fluorescence Microscopy

What is Fluorescence Microscopy

Fluorescence microscopy is a type of optical microscopy which use fluorescence to make an object visible. More importantly, the principle of fluorescence microscopy includes the illumination of the specimen with the light of a specific wavelength (or wavelengths) that is absorbed by fluorophores, causing them to emit light of longer wavelengths (i.e., of a different color than the absorbed light).

Components of Fluorescence Microscopy

The components of the fluorescence microscope are the light source, excitation filter, dichroic mirror, and emission filter. The light source is either a xenon arc lamp or a mercury-vapor lamp; more advanced forms are high-power LEDs and lasers. Moreover,  filters and dichroic beamsplitter match the spectral excitation and emission characteristics of the fluorophore. The separation of the illumination light is from the much weaker emitted fluorescence through the use of a spectral emission filter. Imaging is the distribution of a single fluorophore at a time and images with multiple colors can be combining single-color images.

Figure 1: Endothelial Cell

Furthermore, there are several types of fluorescence microscopes: epifluorescence microscope, confocal microscope, and total internal reflection fluorescence microscope. However, the most common fluorescence microscope in use is the epifluorescence microscope. In these microscopes, excitation of the fluorophore and detection of the fluorescence happen through the same light path (i.e. through the objective). It is also the basis of advanced fluorescence microscope designs such as a confocal microscope in which capturing multiple two-dimensional images at different depths in a sample enables the reconstruction of three-dimensional structures.

What is an Epifluorescence Microscopy

Epifluorescence microscopy is a type of optical microscopy in which the excitation of the fluorophore and detection of the fluorescence are done through the same light path (i.e. through the objective). The majority of fluorescence microscopes, especially those used in the life sciences, are of the epifluorescence design. Here, the light of the excitation wavelength illuminates the specimen through the objective lens. From the same objective, the emitting fluorescence by the specimen focuses on the detector.

Figure 2: Epifluorescence Microscopy

Moreover, the dichroic beamsplitter acts as a wavelength-specific filter, transmitting fluoresced light through to the eyepiece or detector, but reflecting any remaining excitation light back towards the source. Though the epifluorescence microscope is the most common fluorescence microscope in use, the signal-to-noise ratio of the epifluorescence microscope is high since most of the excitation light is transmitted through the specimen.

Similarities Between Fluorescence and Epifluorescence Microscopy

• Fluorescence and epifluorescence microscopes are two microscopic designs that use fluorescence instead of the visible spectrum.
• In both microscopes, the specimen is illuminated with the light of a specific wavelength (or wavelengths) that is absorbed by the fluorophores, causing them to emit light of longer wavelengths (i.e., of a different color than the absorbed light).

Difference Between Fluorescence and Epifluorescence Microscopy

Definition

Fluorescence microscopy refers to an optical microscope that uses fluorescence instead of, or in addition to, scattering, reflection, and attenuation or absorption, to study the properties of organic or inorganic substances while epifluorescence microscopy refers to a parallel beam of light is passed directly upwards through the sample, maximizing the amount of illumination.

Types

Usually, fluorescence microscopy is a type of optical microscopy while epifluorescence microscopy is a design of fluorescence microscopy.

Importance

Fluorescence microscopy is important to study the properties of organic and inorganic molecules while epifluorescence microscopy is important as the basis of the fluorescence microscopy designs.

Significance

Fluorescence microscopy has more enhanced capabilities than light microscopes while epifluorescence microscopy has a high signal-to-noise ratio.

Conclusion

In brief, fluorescence and epifluorescence microscopy are two types of optical microscopy that use fluorescence instead visible light to illuminize an image. However, fluorescence microscopy has enhanced capabilities while epifluorescence microscope has a high signal-to-noise ratio. In an epifluorescence microscope, the excitation of the fluorophore and detection of the fluorescence occurs through the same light path. Therefore, the main difference between fluorescence and epifluorescence microscopy is their microscopic design.

References:

1. Wikimedia Foundation. (2022, July 1). Fluorescence microscope. Wikipedia. Retrieved September 2, 2022.
2. Stoakes, S. F. (2022, June 27). How do epifluorescence microscopes work? News-Medical.net. Retrieved September 2, 2022.

Image Courtesy:

1. “FluorescentCells ” (Public Domain) via Commons Wikimedia
2. “FluorescenceFilters 2008-09-28 ” By R.J. Reynolds Tobacco Company Slide Set – Own Work (CC BY-SA 3.0) via Pixabay