Evaluation of NanoLuc substrates for bioluminescence imaging of transferred cells in mice

A Series of Furimazine Derivatives for Sustained Live-Cell Bioluminescence Imaging and Application to the Monitoring of Myogenesis at the Single-Cell Level

“…The unprotected substrates, FMZ and FMZ-OH, showed the brightest signal initially, but the signal intensity sharply decreased and reached the background level after 2 h. The strong but not lasting luminescence is consistent with the previously reported characteristic of the NLuc−FMZ system. 35,45 On the other hand, while all the C-3 protected substrates showed weaker signals than FMZ and FMZ-OH, the signal was detected for about 6 h with Piv-FMZ and Piv-FMZ-OH and 18 h with Ad-FMZ and Ad-FMZ-OH. These results supported the mechanism of their luminescence: (i) protected substrates are converted into FMZ or FMZ-OH by intracellular hydrolase, (ii) generated FMZ or FMZ-OH reacts with NLuc and emits light.…”

“…Using Piv-FMZ protected by the pivaloyl group (acyl protection), especially long-term single-cell BL imaging was achieved with a low S/B ratio compared to Boc-FMZ protected by a tert -butoxycarbonyl group (carbonate protection) despite their comparable bulkiness, which suggested the effectiveness of bulky acyl protection. In addition, other research groups also have extended FMZ derivatives mainly by the modification of C-6 and C-8 substituents of native FMZ aiming at wider application of the NLuc system and reported that the introduction of small electron-rich groups at the C-6 phenyl ring contributed to the luminescence properties in the reaction with NLuc and that modest hydrophilicity of substrates was important for BL in cells or animals. − Among them, one of the FMZ derivatives having a hydroxy group on the C-6 phenyl ring (hydrofurimazine, FMZ-OH) showed excellent luminescence properties in vivo and in practical application to the monitoring of viral infection in mice. , These successful strategies motivated us to further expand the series of FMZ derivatives toward long-term cellular BL imaging by the NLuc system.…”

Macromolecular assembly of bioluminescent protein nanoparticles for enhanced imaging

“…For example, the emission wavelength of the NLuc-E2 nanoparticle may be red-shifted by using bioluminescence resonance energy transfer (BRET) pairs involving NLuc and fluorescent proteins, including mKate2 (λ max EM ​= ​633 ​nm), CyOFP1 (Antares, λ max EM ​= ​589 ​nm), and tdTomato (ReNL, λ max EM ​= ​585 ​nm), or NLuc and fluorescent probes, including siliconrhodamine (λ max EM ​= ​670 ​nm), carbopyronine (λ max EM ​= ​645 ​nm) and tetramethylrhodamine (λ max EM ​= ​585 ​nm) [ 16 , [65] , [66] , [67] ]. Furthermore, in vivo imaging of NLuc-E2 could potentially be improved by using alternative furimazine analogues, such as fluorofurimazine, for higher luminescence, sensitivity, and bioavailability [ 68 ]. Coelenterazine analogues have also been shown to shift the NLuc maximum emission to nearly 598 ​nm, although luminescent intensities can be significantly lower [ 69 ].…”

New bioimaging avenues for organs‐on‐chips by integration of bioluminescence

“…Hikarazines-003 produced with up to 2.5 increased light intensity and signal stability lasting up to 2 h. 71 The recently developed 5-fluorofurimazine also increased light output of threefold, in living cells cultured in vitro. 72,73 Caging by addition of protecting groups, as the C-3 carbon of the imidazopyrazinone core, is a common strategy to stabilize coelenterazine-derived substrates. Other two analogues, based on caging strategy, resulted in significantly longer BL emission with higher S/N ratio than furimazine at single-cell level, namely…”

“…78 The operon, which consists of six major genes for the synthesis of the luciferase and the substrate, can be entirely inserted in the genome of the heterologous organism. 72 The independence from exogenous administration of luciferin renders their use particularly interesting, even though the photon yield of such system is low. Gregor and colleagues designed the iLUx operon with improved brightness and codon optimized it for expression in mammalian cells.…”