IVIS Spectrum in vivo imaging

The IVIS SpectrumCT (PerkinElmer, Waltham, MA, USA) instrument was used to image bioluminescent MRSA and macrophages in the skin of the animal.

For bioluminescence imaging, the mice were scanned without emission filtration for 1–4 min/image. Mice were scanned longitudinally once a day for 10 days to monitor the course of MRSA skin infection. For analysis, regions of interest were drawn around each site of infection and the total photon flux (photons/second) was measured. To account for background signal resultant from camera thermal noise and emission scatter, a background region was drawn outside the mouse to determine the background signal of each scan. To establish the relationship between bacterial CFU and background-free total photon flux, bacterial standard curves were prepared and imaged by spotting known bacterial CFU onto TSA plates (for in vitro studies) or from subcutaneously infected mice (for in vivo studies). By establishing the luminescence and CFU relationship, and fitting these with linear regression, it was possible to quantify the bacterial burden in the skin or each mouse.

For DsRed and mCherry fluorescence imaging, in the presence of bioluminescent MRSA, groups of 4 mice were imaged at 6–8 distinct emission wavelengths over the (excitation: 535, 570, 605) 560-680nm and (excitation: 500, 535, 570) 580-720nm bandwidths, respectively, with an exposure range of 1–5 sec/group. To provide negative controls, WT mice that were DsRed negative (or mCherry negative) with and without bioluminescent MRSA were imaged as above, and served as bioluminescent MRSA and auto-fluorescence controls, respectively. Because the total emission in each mouse is a linear combination of DsRed (or mCherry) fluorescence combined with MRSA bioluminescence, DsRed (or mCherry) the fluorescence emission must be spectrally deconvolved. Individual basis functions for each spectral series were constructed as follows:

Where λ, i, E_Auto, E_WT, E_BL, E_MRSA, E_Total, and E_FL are the wavelength, subject, autofluorescence emission, wild-type emission (i.e. no DsRed or mCherry), bioluminescence emission, MRSA emission (i.e. bioluminescence + autofluorescence), total emission (i.e. bioluminescence + autofluorescence + mCherry), and mCherry emission only. Wavelength-dependent spectral basis functions were manually constructed and loaded into LivingImage (PerkinElmer, Waltham, MA, USA), where mCherry spatially dependent signals for each pixel were spectrally deconvolved using a multi-linear least squares approach. Once the images were spectrally decomposed, a region of interest was drawn around the mCherry signal and was the average pixel intensity was reported in total radiant efficiency ([photons/second]/[μW/cm²]).