Bioluminescence imaging and fluorescence imaging are two very common preclinical optical imaging modalities (way in which imaging occurs). Both offer important advantages and disadvantages to consider. Therefore, this article briefly highlights the difference between BLI and FLI as well as their advantages and disadvantages.

What is bioluminescence and fluorescence imaging?

Both bioluminescence and fluorescence imaging are types of optical imaging techniques that use two different sources to generate light.

Bioluminescence occurs when the luciferin substrate is oxidized in a reaction catalyzed by the enzyme luciferase, resulting in the visible production of light. Simply put, the bioluminescence event that produces light is the result of an enzyme-substrate reaction (Brovko, 2010, p. 2).

Bioluminescence imaging is an optical imaging method that allows noninvasive in vivo study of molecular events and biological processes. Not only is this imaging method noninvasive, but it also allows real-time study of biological processes (Sadikot & Blackwell, 2005). This technique has allowed researchers to study disease progression, protein-protein interaction, treatment efficacy, and so much more.

Like bioluminescence imaging, fluorescence imaging is a method that allows researchers noninvasive study of biological processes.

Unlike bioluminescence, however, fluorescence imaging requires light excitation of a particular molecule (fluorophore) in order to produce light of a lower energy. Therefore, rather than an enzymatic reaction, fluorescence is dependent on excitation to reemit light.

Bioluminescence reaction vs. fluorescence reaction- bioluminescence is an enzyme-substrate reaction involving luciferin and luciferase while fluorescence requires excitation light for light emissionFigure 1 Bioluminescence results when the substrate luciferin is oxidized in a reaction catalyzed by the enzyme luciferin. Fluorescence results when light of a higher energy is absorbed, exciting a fluorophore, which causes light of a lower energy to be reemitted.

What is bioluminescence and fluorescence imaging used for?

When it comes to preclinical optical imaging, researchers use bioluminescence and fluorescence imaging for several purposes:

  • Cell viability and proliferation
  • Environmental toxicity
  • Drug development
  • Oncology research
  • Gene expression
  • Protein-protein interactions

Keep in mind, this is list only details some of the popular purposes and is in no way comprehensive. Researchers use these imaging modalities for many more purposes than what is provided here.

Bioluminescence imaging vs. fluorescence imaging – the advantages and disadvantages

Both bioluminescence and fluorescence allow researchers powerful insights into molecular processes. But they also come with certain limitations, advantages and drawbacks which are important for consideration.

compares bioluminescence imaging BLI to fluorescence imaging FLI depth - illustration of BLI and FLI depth measurementsFigure 2 Conceptual example comparing imaging depth (depth of the object or tissue being imaged) of bioluminescence (BLI) to fluorescence (FLI). Keep in mind, imaging depth can depend on other factors such as tissue type, location, etc.


Advantages of bioluminescence imaging include high signal-to-noise ratio (low background), the ability to simultaneously image multiple organisms (instrument dependent), high sensitivity, high specificity, no phototoxicity (damaged caused by high and low wavelength light), and its noninvasive nature.

One of the major limitations to bioluminescence imaging is its limited imaging depth (depth of tissue that can be imaged), which is about 1-2 cm. Bioluminescence also requires a substrate and takes minutes to visualize whereas fluorescence acquisition can take seconds.


Fluorescence offers the benefit of not requiring a substrate. Furthermore, image acquisition can occur within seconds. And like BLI, FLI allows researchers to image multiple animals at once (instrument dependent).

Fluorescence imaging, however, does require excitation light, which can cause photobleaching and phototoxicity (Tung, Berglund, Gutekunst, Hochgeschwender, Gross, 2016). Fluorescence does cause more background. And like BLI, FLI also has limited imaging depth (about 1 cm).

Table 1.1 Comparison between bioluminescence and fluorescence imaging.

comparison between bioluminescence optical imaging BLI and fluorescence optical imaging FLI to determine whether to use FLI or BLI for your experiment


Brovko, L. (2010). Bioluminescence and fluorescence for in vivo imaging. In Bioluminescence and Fluorescence for in Vivo Imaging (pp. 1-149).

Sadikot, R., & Blackwell, T. (2005). Bioluminescence Imaging. Proceedings of the American Thoracic Society. doi:10.1513/pats.200507-067DS

Tung, J. K., Berglund, K., Gutekunst, C. A., Hochgeschwender, U., & Gross, R. E. (2016). Bioluminescence imaging in live cells and animals. Neurophotonics, 3(2), 025001.