Single-cell bioluminescence imaging of deep tissue in freely moving animals

Abstract

Bioluminescence is a natural light source based on luciferase catalysis of its substrate luciferin. We performed directed evolution on firefly luciferase using a red-shifted and highly deliverable luciferin analog to establish AkaBLI, an all-engineered bioluminescence in vivo imaging system. AkaBLI produced emissions in vivo that were brighter by a factor of 100 to 1000 than conventional systems, allowing noninvasive visualization of single cells deep inside freely moving animals. Single tumorigenic cells trapped in the mouse lung vasculature could be visualized. In the mouse brain, genetic labeling with neural activity sensors allowed tracking of small clusters of hippocampal neurons activated by novel environments. In a marmoset, we recorded video-rate bioluminescence from neurons in the striatum, a deep brain area, for more than 1 year. AkaBLI is therefore a bioengineered light source to spur unprecedented scientific, medical, and industrial applications.

Identification of Factors Complicating Bioluminescence Imaging

“…This result agrees with previous studies. 10 In contrast, the signals from teLuc-DTZ were often concentrated in the bladder area; the signals from Antares2-DTZ were observed in the throat, lung, and limbs areas ( Figure 4A). Signals from all groups were completely cleared one day after injection of cells to these immunocompetent mice.…”

“…injecting the corresponding substrates before image acquisition. 10 The signals from Akaluc-AkaLumine were concentrated to lungs. In contrast, the signals from Antares2-DTZ were stronger but more diffuse than Akaluc-AkaLumine.…”

“…This could be explained by the fact that Akaluc is ATP-dependent and that the intrinsic photon flux of Akaluc-AkaLumine is lower than that of teLuc-DTZ or Antares2-DTZ. 10 To further investigate whether there are other factors, we mixed recombinant teLuc, Antares2, and Akaluc proteins with serum isolated from untreated blank mice and examined luciferase activities in relation to assays in PBS. For the teLuc and Antares2 groups, the total signals in either serum or PBS within the first 10 min post substrate injection were almost identical (Figure 6A), despite that teLuc and Antares2 displayed a flash-type kinetics in PBS but a sustained light output in serum ( Figure 6B).…”

“…Notably, in early 2018, Iwano et al described an engineered luciferase-luciferin pair, namely Akaluc-AkaLumine, emitting light in the near-infrared (NIR) region for highly sensitive in vivo deep-tissue BLI (Figure 2A). 10 Conventionally, marine luciferases have limited uses for in vivo BLI, because blue photons strongly interact with mammalian tissue, leading to poor light penetration. The recent development of marine luciferase derivatives (e.g., NanoLuc and GLuc 11,12 ) that produce two orders of magnitude higher photon flux than FLuc has spurred interest in exploring these reporters for in vivo BLI.…”

An Autonomous Molecular Bioluminescent Reporter (AMBER) for voltage imaging in freely moving animals

“…The inability of the luciferin in crossing the cell membrane makes delivery inefficient and precludes their use as a neural reporter, though some have been used for reporting slow Ca 2+ signaling in neural networks [28][29][30]. Key advances have been made to improve the brightness of these bioluminescent probes using bioluminescent resonant energy transfer (BRET) [31] and directed evolution of fire-fly luciferase for a synthetic luciferin analogue [32]. LOTUS-V [33] was the first Luminescent Voltage Indicator (LuVI) created using an engineered deep-sea shrimp luciferase (Nluc) and Venus.…”