Near-infrared dual bioluminescence imaging in mouse models of cancer using infraluciferin

Abstract

Bioluminescence imaging (BLI) is ubiquitous in scientific research for the sensitive tracking of biological processes in small animal models. However, due to the attenuation of visible light by tissue, and the limited set of near-infrared bioluminescent enzymes, BLI is largely restricted to monitoring single processes in vivo. Here we show, that by combining stabilised colour mutants of firefly luciferase (FLuc) with the luciferin (LH2) analogue infraluciferin (iLH2), near-infrared dual BLI can be achieved in vivo. The X-ray crystal structure of FLuc with a high-energy intermediate analogue, 5’-O-[N-(dehydroinfraluciferyl)sulfamoyl] adenosine (iDLSA) provides insight into the FLuc-iLH2 reaction leading to near-infrared light emission. The spectral characterisation and unmixing validation studies reported here established that iLH2 is superior to LH2 for the spectral unmixing of bioluminescent signals in vivo; which led to this novel near-infrared dual BLI system being applied to monitor both tumour burden and CAR T cell therapy within a systemically induced mouse tumour model.

Evaluating Brightness and Spectral Properties of Click Beetle and Firefly Luciferases Using Luciferin Analogues: Identification of Preferred Pairings of Luciferase and Substrate for In Vivo Bioluminescence Imaging

“…Successful multiplexing of luminescence systems with different emission spectra relies on the acquisition of images using multiple filters followed by accurate, algorithm-based spectral unmixing to resolve the contributions from each luciferase to total light output. This can be a challenge with shorter wavelength systems (e.g., CBG99/D-LH2), as they tend to shift their apparent emission peak to significantly longer wavelengths when imaged in deeper tissues or even in superficial tissue when using mice with dark fur [20][21][22]. For these more challenging imaging targets, it is therefore desirable to use bioluminescence pairs that emit in the NIR (9 650 nm), as emission peaks are essentially constant in this range of the spectrum [22,23].…”

“…This can be a challenge with shorter wavelength systems (e.g., CBG99/D-LH2), as they tend to shift their apparent emission peak to significantly longer wavelengths when imaged in deeper tissues or even in superficial tissue when using mice with dark fur [20][21][22]. For these more challenging imaging targets, it is therefore desirable to use bioluminescence pairs that emit in the NIR (9 650 nm), as emission peaks are essentially constant in this range of the spectrum [22,23]. In this regard, we found that click beetle luciferases have high photon emission with NH 2 -NpLH2 Combined data is also presented in logarithmic scale (e).…”

Shining light on the electronic structure and relaxation dynamics of the isolated oxyluciferin anion

“…18 One of the research aims of our groups is to improve the fundamental understanding of bioluminescence for the rational design of new bioluminescence tools. [19][20][21][22] The focus of the work described in this paper is to improve our understanding of the electronic structure of the lightemitting oxyluciferin (OL).…”

Porous upconversion nanostructures as bimodal biomedical imaging contrast agents

“…Fluorescent dyes, such as firefly luciferase (FLuc), is benefited by very high sensitivity in bioluminescence imaging and a broad dynamic range relative to other imaging techniques. [13,14].…”

“…Bioimaging modalities can complement with each other for a more thorough examinations. For example, optical fluorescent microscopy can achieve single molecular resolution in in vitro imaging [13,14,17], while CT is benefited on the hard tissue detection and 3D imaging with high reliability and rich details [18,19], and MRI presents higher resolution images on muscle and organs [20,21]. Furthermore, fewer possible side effects can be resulted from the application of multimodal contrast agents, due to its relative smaller necessary dose and ability of serving multi technologies [11,22].…”