In-vitro fluorescence microscopy has long been one of the foundations of life science research. Conjugate your fluorophore to an appropriate ligand, such as an antibody or quantum dot, incubate your tissue sections or cell cultures, shine a light on your sample, and the labeled molecules will shine light right back through your microscope. However, in vitro methods have an obvious drawback: They can't tell you much about biological processes in their natural environment. Increasingly, researchers are applying proven in vitro techniques to live organisms.
Any fluorescence imaging protocol requires some troubleshooting: Fluorophores can be unstable, conjugated ligands may bind nonspecifically, and signal levels can be low. With in vivo techniques, light must traverse a much greater distance through tissue to yield a good image. Because tissue absorbs and scatters light, your protocol must ensure that the excitation light reaches your fluorescent marker, and that fluorescent light makes it out of the organism. Complicating things further, many tissues are themselves fluorescent, even without a label, so whatever in vivo method you use must include a strategy to deal with this autofluorescence.
The Scientist tracked down five researchers who have overcome these challenges. Read about some of the tricks they've devised by clicking on the user profiles in the yellow box above.