In vivo bio-imaging Mice were anesthetized and placed in a custom-made bed, which allowed stable and reproducible imaging of the legs. In vivo scanning was performed using the Optix MX-2 Optical Molecular Image System (Advanced Research Technologies, Montreal, Canada), which uses time domain optical imaging. In time domain optical imaging, short pulses of light driven by pulsed laser diodes are used to illuminate the organism under study and excite fluorescent molecules. Time-of-flight distribution enables depth and concentration to be uncoupled and fluorescence lifetime to be determined. For Katushka, excitation was performed with a 635-nm (LDH-P-635) pulsing laser and emission was detected with a 650 long pass filter, while excitation of GFP was performed with a 470-nm pulsing laser and emission was detected through a 525-nm band pass filter. The scan was performed over a Cartesian grid in prioritized raster fashion, and each scan took on average 5 min.
Data analysis Data analysis was performed using the Optiview 2.2 software (ART Advanced Research Technologies Inc., Canada) supplied with the ART Optix bioimager. The software is used for background subtraction, lifetime analysis of the fluorochromes, depth and concentration analysis, and generation of 3D images.
The temporal dispersion of fluorescent photons is measured by time-correlated single photon counting (TCSPC) after excitation of a fluorophore by laser pulse. Analysis of this temporal dispersion curve—the fluorescent temporal point spread function (TPSF)—is used to obtain information of the in vivo fluorophore depth, concentration, and lifetime. Based on the Optix machine settings, the user will be able measure temporal and spatial distribution of fluorophores in regions in tissue: the light intensity is measured as a function of arrival time in nanoseconds, where the signal from deeper tissues arrives later allowing the estimation of relative concentration difference.
Results
Efficiency of Katushka expression in muscles over time
Electrotransfer of 5 µg of pTurboFP635 (‘Katushka’) plasmid resulted in a large increase in the in vivo fluorescent intensity (mean peak value = 18,695 ± 5,242 NC, n = 8) from the transfected muscle (Fig. 1). The Katushka intensity peaked 1 week after electrotransfer, where after it leveled off and returned to background level within 4 weeks (Fig. 2). To examine the sensitivity of the in vivo analysis compared with ex vivo scans, the muscles were excised at 4 weeks and scanned. Even though Katushka expression could not be detected in vivo, residual Katushka expression was present in muscles when scanned ex vivo (Fig. 3).
Fig. 1 Time course of the intensity of Katushka expression in muscles after DNA electrotransfer. The left leg was transfected, while the right leg served as untreated control. The picture series was taken of the same mouse, but is representative of seven mice.
Fig. 2 Time course of a Katushka intensity (mean ± SD) and b Katushka lifetime (mean ± SD) in a scanning series of seven mice following DNA electrotransfer of 5 µg Katushka plasmid.
Fig. 3 Four weeks after DNA electrotransfer, the muscles were scanned in vivo (left image), and then excised and scanned ex vivo with the same settings (right image).
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