A few months ago I wrote this post about using two photon fluorescence to study the capillary system. The basic idea being to use infrared light, which passes through flesh easily, to excite the fluorescent dye but to use the process of two photon absorption so that the remitted photon can be of greater frequency than infrared. Now although this can be done with a chemical dye, there is another option.
Aspects of our body such as the mitochondria and lysosomes are themselves autofluorescent. Called endogenous fluorophores these organic components can also undergo two photon absorption and although they normally act to interfere with the precise monitoring of a proper dye, if information about the working of cell is required it can be possible to look straight to them as a source of this information. These techniques have been developed and improved for years with methods such as fluorescence lifetime imaging (watching the decay of light output) and spectral detection being implemented with notable success.
The main things hampering these techniques is the maximum speed of data which can be gathered per second and multicolour excitations which are harder to analyse. Ideally it would be possible to image multiple natural fluorescent chemical compounds at once as to dynamically monitor physiological processes. Currently, two if the most common fluorophores found naturally are nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) which are only excited by first applying light at 750 nm for NADH and then 880 nm for FAD.
Some suggested improvements have been to use a midway wavelength which can simultaneously excite both chemicals but with a reduced efficiency as the wavelength strays from the optimum for either chemicals excitation. This paper has came up with, perhaps, the seemingly simple idea of using a mix of wavelengths in order to excite both fluorophores simultaneously. This process was demonstrated on zebrafish embryos to analyse the redox reactions occurring during the development. Generally this method can be used for studying metabolic changes in living cells with new levels of efficiency.