Abstract
Background/Aim: Genetic reporters encoding fluorescent proteins or luciferase have been used in vivo for the last three decades with claims about their superiority or inferiority over each other. In the present report, a head-to-head in vivo comparison of green fluorescent protein (GFP) fluorescence imaging and luciferase-luciferin imaging, using single-nanometer laser-excitation tuning of fluorescence excitation and an ultra-low-light-detection camera and optics was performed. Materials and Methods: Mouse Lewis-lung carcinoma cells labeled with GFP (LLC-GFP) or luciferase (LL/2-Luc2) were injected subcutaneously into the flank of nude mice. One week after injection, GFP-fluorescence imaging and luciferase-luciferin imaging was performed using the UVP Biospectrum Advanced system with excitation at 487 nm and peak emission at 513 nm for GFP, and with emission at 560 nm for luciferase-luciferin. GFP fluorescence images were obtained at 0, 10, and 20 min. Luciferase-luciferin images were obtained 10 and 20 min after the injection of D-luciferin. Results: The intensity of GFP images was 55,909 at 0 min, 56,186 at 10 min, and 57,085 at 20 min, and maintained after 20 min. The intensity of luciferase-luciferin images was 28,065 at 10 min after the injection of D-luciferin and 5,199 at 20 min after the injection. The intensity of luciferase-luciferin images decreased by approximately 80% at 20 min compared to 10 min. An exposure time of 30 s for luciferase-luciferin imaging was needed compared to 100 ms for GFP fluorescence imaging in order to detect signals. Conclusion: An imaging system with single-nanometer tuning fluorescence excitation and an ultra-low-light detection camera and optics was able to directly visualize both GFP and luciferase-luciferin images in vivo. The intensity and stability of the signals were both greater for GFP than for luciferase-luciferin, and the exposure time for GFP was 300 times faster, demonstrating the superiority of GFP.
Imaging with genetic reporters expressing green fluorescent protein (GFP) or luciferase-luciferin is widely used in vivo and has been adapted for cancer research to measure tumor growth and cell behavior in mouse models (1-16). Claims of the superiority and inferiority of both types of genetic reporters have been made (1, 17-19). In the present report, an in vivo head-to-head comparison was made of directly-visualized GFP fluorescence imaging and directly-visualized luciferase-luciferin imaging, using an imaging system with single-nanometer laser-excitation tuning and an ultra-low-light detection camera and optics.
Materials and Methods
Cell lines. The Lewis lung carcinoma mouse cell line expressing green fluorescent protein (LLC-GFP) has been previously described (20). The Lewis lung carcinoma mouse cell line expressing luciferase (LL/2-Luc2) was obtained from the American Type Culture Collection (Manassas, VA, USA). Both LLC cell lines were cultured in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 with GlutaMAX™ supplement (GIBCO, Grand Island, NY, USA), 10% fetal bovine serum, and 100 IU/ml of penicillin/streptomycin.
Mice. Nude nu/nu mice aged 6-8 weeks (AntiCancer, Inc., San Diego, CA, USA) were used in the present study. All mice were housed in a barrier facility with a high efficacy particulate air (HEPA)-filtered rack and kept in standard settings with 12-h light/dark cycles. This study was approved by the AntiCancer Inc. Institutional Animal Care and Use Committee and the protocol outlined in the National Institutes of Health Guide for the Care and Use of Animals was followed.
Procedure to establish LLC xenografts in nude mice. LLC-GFP and LL/2-Luc2 cells (1.0×106 cells/100 μl phosphate-buffered saline [PBS]) were injected subcutaneously (SC) into the mouse flank. The tumor grew for one week before imaging was performed.
Imaging. The BioSpectrum Advanced 900 (Analytik Jena US LLC, Upland, CA, USA) is an optimized imaging system for luciferase and GFP detection. The system camera uses ultra-low-light f0.95 optics and an ultra-cooled (−70°C) back-thinned 13 μm m-sized pixel CCD camera to collect low-intensity light. The sample platform is mounted on a motorized lift. This reproducibly changes the magnification by placing the sample closer to the lens, thus improving resolution while maintaining ultra-low-light signal detection. GFP fluorescence imaging was performed with excitation at 487 nm and peak emission at 513 nm. Luciferase-luciferin imaging was performed following intravenous (IV) injection of D-luciferin potassium salt (150 mg/kg) (Gold Biotechnology, St. Louis, MO, USA) into the mouse tail vein for emission at 560 nm. For GFP fluorescence, the images were obtained at 0, 10, and 20 min. For luciferase-luciferin, the images were obtained at 10 and 20 min after the injection of D-luciferin. The exposure time for GFP fluorescence was 100 ms and for luciferase-luciferin was 30 s. Quantification was performed using the light intensity of the GFP fluorescence and intensity of the luciferase-luciferase light emission measured with the UVP Biospectrum Advanced system.
Results
LLC tumors formed one week after SC implantation of LLC cells in nude mice. For GFP imaging, the intensity of the fluorescence was 55,909 at 0 min, 56,186 at 10 min, and 57,085 at 20 min with a 100 ms exposure time (Figure 1, Table I). The GFP intensities were constant for at least 20 min. In contrast, the intensity of the luciferase-luciferin light emission was 28,065 at 10 min after the injection of D-luciferin and 5,199 at 20 min after the injection with a 30 s exposure time (Figure 2). The light intensity decreased by approximately 80% between 10 and 20 min after luciferin injection. For luciferase-luciferin imaging, a longer exposure times (30 s) were needed to detect signals than for GFP fluorescence imaging (100 ms) (Figure 3, Table I).
Green fluorescent protein (GFP) fluorescence images at 0, 10, and 20 min, from the Lewis-lung carcinoma tumor expressing GFP, in nude mice. The tumor is located on the right flank. Left; 0 min. Middle; 10 min. Right; 20 min. Exposure time was 100 ms. Images were obtained with a UVP Biospectrum Advanced system with a white-light camera. Images were pseudo-colored green.
Intensity and exposure times of GFP fluorescence and luciferase-luciferin images. Images were obtained with a UVP Biospectrum Advanced system.
Luciferase-luciferin images 10 and 20 min after the injection of D-luciferin in the Lewis-lung carcinoma tumor expressing luciferase in nude mice. The tumor is located on the right flank. Left; 10 min. Right; 20 min. Exposure time was 30 s. Images were obtained with a UVP Biospectrum Advanced system with a white-light camera. The image on the left is pseudo-colored red to indicate a high intensity of 28,066 and the image on the right is pseudo-colored green to indicate low intensity of 5,199.
Time-course of the tumor GFP fluorescence intensity and luciferase-luciferin intensity relative values. Images were obtained with a UVP Biospectrum Advanced system.
Discussion
The present study compared direct GFP fluorescence imaging and direct luciferase-luciferin imaging in vivo, with the Lewis-lung carcinoma (LLC) in nude mice. The intensity of the GFP fluorescence image was initially twice that of the luciferase-luciferin image. The GFP images were stable for at least 20 min. The luciferase-luciferin images decreased by 80% from 10 min to 20 min after the injection of D-luciferin.
The intensity of the luciferase-luciferin image decreases rapidly over time due to the clearance of D-luciferin from the mouse. The intensity of the GFP fluorescence images was constant with small changes in the present study. Fluorescence intensity has been previously reported to decrease over time due to photobleaching (17-19) which was not an issue in the present study. In the present study, compared to fluorescence imaging, luciferase-luciferin imaging required a 300x longer exposure time in order to detect signals. The UVP Biospectrum Advanced system eliminates background fluorescence with single-nanometer tuning of laser excitation and directly visualized the luciferase-luciferin image with an ultra-low-light detection camera and optics.
Conclusion
The current study demonstrated the distinction between in vivo GFP fluorescence imaging and luciferase-luciferin imaging in a head-to-head comparison. The intensity of the GFP fluorescence image was twice that of the intensity of the initial luciferase-luciferin image. The intensity of the luciferase-luciferin images sharply decreased over time, while that of the fluorescence images did not change over 20 min. A much longer exposure time (300x) was needed to obtain the luciferase-luciferin images, precluding real-time in vivo imaging that is possible with GFP, even at the sub-cellular level (8, 12, 21-23). The present report shows the superiority of GFP. The present study and two recent studies (4, 5) should overcome confusion in the field of small-animal imaging due to misinformation that GFP imaging is inferior to luciferase-liciferin imaging in vivo (24-26).
Acknowledgements
This paper is dedicated to the memory of A. R. Moossa, MD, Sun Lee, MD, Professor Li Jiaxi, Masaki Kitajima, MD, Shigeo Yagi, PhD, Jack Geller, MD, Joseph R. Bertino, MD, J.A.R. Mead PhD, Eugene P. Frenkel, MD, Professor Lev Bergelson, Professor Sheldon Penman, Professor John R. Raper, and Joseph Leighton, MD.
Footnotes
Authors’ Contributions
KM, SG, AW, and NC performed experiments. KM and RMH wrote the article. SM, MS, and BMK critically reviewed this article.
Conflicts of Interest
AW, NC, and SG are employees of Analytik Jena. KM, SM, MS, BMK, and RMH are non-salaried associates of AntiCancer Inc. AntiCancer Inc. uses mouse models of cancer for contract research.
Funding
The Robert M. Hoffman Foundation for Cancer Research contributed to the funding of this study.
- Received April 30, 2024.
- Revision received May 22, 2024.
- Accepted May 23, 2024.
- Copyright © 2024 The Author(s). Published by the International Institute of Anticancer Research.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
