Single-cell imaging of cells in living matrices is essential for tracking the etiology of diseased versus normal cells. Until recently, optical imaging was limited by the strong absorbance of components of the tissue and cellular matrix, particularly water and hemoglobin. Both have very strong absorbance, except for the window between the NIR wavelengths 650–880 nm. This mismatch is an experimental problem for optical in-vivo imaging of single cells, especially in animals larger than small rodents.
Scientists seeking to image single cells in vivo thus needed an improved reporter system to image events deeper in living animals. The luciferase bioluminescence assay, which was adapted from the firefly, has been the best available technology. When oxygen combines with calcium, adenosine triphosphate (ATP) and the chemical luciferin in the presence of luciferase, a bioluminescent enzyme, light is produced. The emitted light from a firefly is about 570 nm, which is inadequate for imaging single cells in thick tissue.
Researchers who wanted to image single cells in vivo needed improved markers to reach deeper into living animals. An engineered luciferase called Aka Lumine (Fujifilm Wako Pure Chemical Corp.) emits light at 670–680 nm, which is just in the near-infrared window (650–880 nm) of live animals. In this spectral window, the absorbance of water and hemoglobin is lower, which improves imaging depth.
Nasu et al. described the development of Aka Lumine starting with D-luciferin/insect luciferase, which emits at about 570 nm (Nasu, Y.; Campbell, R.E. et al. Naturally aglow with a bright inner light. Science Feb 2018, 359[6378], 868–9). Other natural variants such as the railroad worm were better, but not adequate. The developers used 21 cycles of directed evolution to shift the emission wavelength to longer while also improving emitted intensity. The original paper (Iwano, S.; Sugiyama, M. et al. Single-cell bioluminescence imaging of deep tissue in freely moving animals. Science 2018, 359[6378], 935–9) reports on the use of AKaBLI (an engineered bioluminescence in-vivo imaging system) for imaging single tumor cells in mouse lungs and video imaging of cells deep in the brain.
Robert L. Stevenson, Ph.D., is Editor Emeritus, American Laboratory/Labcompare; e-mail: [email protected]
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