Multiplexed bioluminescence imaging with a substrate unmixing platform

Brennan, Caroline K.; Yao, Zi; Ionkina, Anastasia A.; Rathbun, Colin M.; Sathishkumar, Buvaneshwari; Prescher, Jennifer A.

doi:10.1016/j.chembiol.2022.10.004

Cited by 10 publications

(18 citation statements)

References 59 publications

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“…The raw images were processed using a linear unmixing algorithm to deconvolute signals produced from each pair. 63 As shown in Figure 5B, robust orthogonality was observed within this quintet set.…”

Section: ■ Results and Discussionmentioning

confidence: 60%

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Red-Shifted Coumarin Luciferins for Improved Bioluminescence Imaging

et al. 2023

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Multicomponent bioluminescence imaging in vivo requires an expanded collection of tissue-penetrant probes. Toward this end, we generated a new class of near-infrared (NIR) emitting coumarin luciferin analogues (CouLuc-3s). The scaffolds were easily accessed from commercially available dyes. Complementary mutant luciferases for the CouLuc-3 analogues were also identified. The brightest probes enabled sensitive imaging in vivo. The CouLuc-3 scaffolds are also orthogonal to popular bioluminescent reporters and can be used for multicomponent imaging applications. Collectively, this work showcases a new set of bioluminescent tools that can be readily implemented for multiplexed imaging in a variety of biological settings.

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Section: ■ Results and Discussionmentioning

confidence: 60%

“…Each substrate was added in sequence and images were acquired after substrate addition (Figure A). The raw images were processed using a linear unmixing algorithm to deconvolute signals produced from each pair . As shown in Figure B, robust orthogonality was observed within this quintet set.…”

Section: Resultsmentioning

confidence: 99%

Red-Shifted Coumarin Luciferins for Improved Bioluminescence Imaging

et al. 2023

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“…Polymerase chain reaction (PCR) was performed to amplify genes of interest. mNeptune, 36 nitroreductase (NTR), 37 blue fluorescent protein (BFP), 36 firefly luciferase (Fluc) (luc2), 36 and green fluorescent protein | 69…”

Section: General Cloning Methodsmentioning

confidence: 99%

“…(GFP) 36 genes were amplified from previously reported constructs. Human FAAH was purchased as a gene block from Genewiz©.…”

Section: General Cloning Methodsmentioning

confidence: 99%

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Caged luciferins enable rapid multicomponent bioluminescence imaging

Navarro

Brennan

Love

et al. 2023

Photochem & Photobiology

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Bioluminescence is a sensitive technique for imaging biological features over time. Historically, though, the modality has been challenging to employ for multiplexed tracking due to a lack of resolvable luciferase–luciferin pairs. Recent years have seen the development of numerous orthogonal probes for multi‐parameter imaging. While successful, generating such tools often requires complex syntheses and lengthy enzyme evolution campaigns. This work showcases an alternative strategy for multiplexed bioluminescence that takes advantage of already‐orthogonal caged luciferins and established uncaging enzymes. These probes generate unique bioluminescent signals that can be distinguished via a linear unmixing algorithm. Caged luciferins enabled two‐ and three‐component imaging on the minutes time scale. We further showed that the tools can be used in conjunction with endogenous enzymes for multiplexed studies. Collectively, this approach lowers the barrier to multicomponent bioluminescence imaging and can be readily adopted by the broader community.

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Red‐Shifted Bioluminescence Using Substrate‐Fluorophore Conjugates

Narbonne Zuccarelli,

Laxio‐Arenas,

Russo

et al. 2024

Helvetica Chimica Acta

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NanoLuc (NLuc) is one of the brightest luciferases and has been used in numerous reporter assays and sensory designs. However, its blue light emission (460 nm) is suboptimal for deep tissue imaging or some diagnostic application and red‐shifting its emission is desirable. Here, we report the design and synthesis of novel furimazine and coelenterazine analogs conjugated with fluorophores to achieve red‐shifted bioluminescence with Nluc and closely related H‐Luc via a bioluminescence resonance energy transfer (BRET). Structural insights into NLuc‐substrate interactions informed the functionalization of substrates. We report the synthesis of novel substrate‐fluorophore conjugates. We show that red‐shifted emission was achieved with high BRET ratios. The conjugates are less bright than the parent substrates, but our study highlights the importance of linker length and flexibility for optimizing bioluminescence emission at longer wavelength than 600 nm.

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Multiplexed bioluminescence imaging with a substrate unmixing platform

Cited by 10 publications

References 59 publications

Red-Shifted Coumarin Luciferins for Improved Bioluminescence Imaging

Red-Shifted Coumarin Luciferins for Improved Bioluminescence Imaging

Caged luciferins enable rapid multicomponent bioluminescence imaging

Red‐Shifted Bioluminescence Using Substrate‐Fluorophore Conjugates

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