Bioluminescent methods are gaining more and more attention among scientists due to their sensitivity, selectivity and simplicity; coupled with the fact that the bioluminescence can be monitored both in vitro and in vivo. Since the discovery of bioluminescence in the 19th century, enzymes involved in the bioluminescent process have been isolated and cloned. The bioluminescent reactions in several different organisms have also been fully characterized and used as reporters in a wide variety of biochemical assays. From the 1990s it became clear that bioluminescence can be detected and quantified directly from inside a living cell. This gave rise to numerous possibilities for the in vivo monitoring of intracellular processes noninvasively using bioluminescent molecules as reporters. This review describes recent developments in the area of bioluminescent imaging for cell biology. Newly developed imaging methods allow transcriptional/translational regulation, signal transduction, protein-protein interaction, oncogenic transformation, cell and protein trafficking, and target drug action to be monitored in vivo in real-time with high temporal and spatial resolution; thus providing researchers with priceless information on cellular functions. Advantages and limitations of these novel bioluminescent methods are discussed and possible future developments identified.
Keywords: cellular physiology, bioluminescent imaging
Overview of bioluminescence
Bioluminescence, that is the production of light by living organisms, is a result of enzymatic oxidative reactions. Enzymes that catalyze these reactions are called luciferases and their respective substrates are referred to as luciferins.
Mysteries surrounding this beautiful natural phenomenon attracted the attention of scientists thousands of years ago (1). As far back as 350 BC, the famous Greek philosopher, Aristotle, wrote about the strange phenomenon that sometimes was seen when "you strike the seas with a rod by night and the water seems to shine". He was, in fact, observing bioluminescence. However, it was not until the 19th century that the French scientist Raphael Dubois proved that light emission from the bioluminescent crustacean Cypridina was the result of metabolic processes in living cells that needed oxygen and two other components, one of which was thermo-stable and the other was thermo-labile. Later these substances were isolated, characterized and named luciferin and luciferase, respectively. Though the biological function of the luciferin-luciferase reaction is the same for all bioluminescent systems, viz. to produce light, the molecular structures of luciferins and luciferases from different organisms are different.
Several bioluminescent systems of different organisms have been fully characterized in terms of component structure and reaction mechanisms. These include the luciferin-luciferase system of bacteria, insects (fireflies and click-beetles) and the jellyfish Aequorea victoria (2). A schematic representation of the reaction mechanisms for these bioluminescent systems is presented in Figure 1.
A major advantage of using bioluminescent systems as analytical tools is that extremely low levels of enzyme activity can be detected by measuring the emitted light. Modern instruments are capable of detecting single photons with both temporal and spatial distribution; thus providing accurate information on the location and intensity of the light source. Another feature of bioluminescent systems that makes them an excellent investigative tool is an almost absolute specificity for their substrates. For example, for firefly luciferase even minor changes in the structure of ATP and firefly luciferin result in a total loss of enzyme activity and this consequently results in a loss of light emission. This specificity for substrates allows the real-time measurement of luciferase activity in situ in very complex samples without the need for any pretreatment. …