Emission spectra of bioluminescent reporters and interaction with mammalian tissue determine the sensitivity of detection in vivo

Zhao et al. (2005). J Biomed Opt DOI: 10.1117/1.2032388 Citations: 341

Bioluminescence imaging depends on light surviving tissue absorption, but how spectrum affects sensitivity was unmeasured. This study compared four luciferases at 25°C and 37°C. Firefly luciferase red-shifted 34 nm at body temperature, improving deep-tissue detection.

Parts used

Firefly LuciferasehRluc

Key findings

  • All four codon-optimized luciferases showed increased activity from 25°C to 37°C, but CBLucs exhibited the greatest thermal activation (6.4-fold for CBGr68, 7.8-fold for CBRed) compared to FLuc (1.9-fold) and hRLuc (2.2-fold). FLuc uniquely displayed a 34-nm spectral red shift from 578 to 612 nm at 37°C, raising its emission fraction above 600 nm from 41% to 64%, approaching CBRed (75–80% above 600 nm).

  • Spectral imaging in liver, lung, and skin mouse models using 20-nm bandpass filters showed that blue/green-emitting luciferases (hRLuc, CBGr68) experienced the greatest tissue attenuation. Beetle luciferases transmitted 15–25% of photons through skin but only 1–2.5% through deep lung tissue. FLuc and CBRed, with peak emissions above 600 nm, showed the least attenuation and produced 307- and 315-fold greater liver bioluminescence signal than hRLuc (i.p. coelenterazine), respectively.

  • Gene fusions of CBGr68 and CBRed in both orientations (CB-GR and CB-RG) produced luciferases with broader emission spectra (480–750 nm) featuring dual peaks corresponding to the λmax of each component. The CB-RG fusion produced the highest liver bioluminescence signal (497.8-fold relative to hRLuc i.p.), exceeding all single luciferases. Differential green-light attenuation in deep tissues indicated that these dual-wavelength fusions may enable signal depth estimation in spectral imaging strategies.

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Abstract

In vivo bioluminescence imaging depends on light emitted by luciferases in the body overcoming the effect of tissue attenuation. Understanding this relationship is essential for detection and quantification of signal. We have studied four codon optimized luciferases with different emission spectra, including enzymes from firefly (FLuc), click beetle (CBGr68, CBRed) and Renilla reniformins (hRLuc). At 25 degrees C, the in vitro lambda(max) of these reporters are 578, 543, 615, and 480 nm, respectively; at body temperature, 37 degrees C, the brightness increases and the firefly enzyme demonstrates a 34-nm spectral red shift. Spectral shifts and attenuation due to tissue effects were evaluated using a series of 20-nm bandpass filters and a cooled charge-coupled device (CCD) camera. Attenuation increased and the spectra of emitted light was red shifted for signals originating from deeper within the body relative to superficial origins. The tissue attenuation of signals from CBGr68 and hRLuc was greater than from those of Fluc and CBRed. To further probe tissue effects, broad spectral emitters were created through gene fusions between CBGr68 and CBRed. These resulted in enzymes with broader emission spectra, featuring two peaks whose intensities are differentially affected by temperature and tissue depth. These spectral measurement data allow for improved understanding of how these reporters can be used in vivo and what they can reveal about biological processes in living subjects.