Abstract

Fluorescence imaging is widely used, but the dependence on external illumination prevents its universal application. For example it cannot be used to study light dependent biological processes such as photosynthesis. Moreover it is incompatible with the non-invasive imaging of whole organisms, or other applications where the cellular substrate is autofluorescent, saturated with photopigments or extremely photosensitive. Experimental conflicts arise when external illumination is essential in other biological technologies, such as optogenetics, chromophore-assisted light inactivation, or photolysis of caged compounds, preventing simultaneous use of fluorescence imaging. Finally sub W/cm² external illumination, which is general power density for fluorescence excitation in live cell imaging under the wide field microscopic observation, sometimes causes phototoxic effects in visualized substrates which alter cellular behavior and ultimately cause cell death. By contrast chemiluminescence generates a visible light signal by a localized chemical reaction without the need for external illumination. The k_cat values of conventional luciferases are ranging from 0.1 for Fluc (quantum yield=0.5) and aprox. 4.4 for RLuc8 (quantum yield=0.053). Provided that cells express several μM Rluc8, a typical concentration of transiently expressed protein, the power density of light emitted from the Rluc8-expressing cells could be calculated as approximately 0.1 μW/cm² which is 1/10³ of general power density for fluorescence excitation in live cell imaging. It is therefore theoretically independent of the associated restrictions which limit biological application of fluorescence. However the 0.1 μW/cm² power density is not bright enough to image events on a biological scale with subsecond/micrometer accuracy. Therefore although the chemiluminescent protein including aequorin and luciferases have been used to image living cells, and organisms, the temporal and spatial resolution of this strategy is unable to match that of fluorescence.

© 2013 Japan Society of Applied Physics, Optical Society of America