Special Section on Chemical and Genetic Sensors in Biomedical Research

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

[+] Author Affiliations
Hui Zhao, Timothy C. Doyle

Stanford University School of Medicine, Department of Pediatrics, Stanford, California 94305-5427

Olivier Coquoz

Xenogen Corporation, Alameda, California

Flora Kalish

Stanford University School of Medicine, Department of Pediatrics, Stanford, California 94305-5427

Bradley W. Rice

Xenogen Corporation, Alameda, California

Christopher H. Contag

Stanford University School of Medicine, Departments of Pediatrics, Radiology, and Microbiology and Immunology, Stanford, California 94305-5427

J. Biomed. Opt. 10(4), 041210 (August 24, 2005). doi:10.1117/1.2032388
History: Received April 06, 2005; Revised May 10, 2005; Accepted May 13, 2005; Published August 24, 2005
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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°C, the in vitroλmax of these reporters are 578, 543, 615, and 480nm, respectively; at body temperature, 37°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.

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© 2005 Society of Photo-Optical Instrumentation Engineers

Citation

Hui Zhao ; Timothy C. Doyle ; Olivier Coquoz ; Flora Kalish ; Bradley W. Rice, et al.
"Emission spectra of bioluminescent reporters and interaction with mammalian tissue determine the sensitivity of detection in vivo", J. Biomed. Opt. 10(4), 041210 (August 24, 2005). ; http://dx.doi.org/10.1117/1.2032388


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