Outline of the algorithmic implementation. Multicolored boxes denote multispectral data acquisition and processing.

Autofluorescence subtraction and normalization of raw transillumination images from a mouse head (dorsal view) with tumor expressing mCherry. – (Media 1) (a) Fluorescence image at 620 nm when excited at 593 nm, (b), Autofluorescence image at 620 nm excited at 532 nm, (c), Correction with autofluorescence subtraction , (d) Normalization image of the head of the mouse at 635 nm, (e) n-Born ratio (cyan) overlaid on the reflectance image of the head (grayscale). (g) Autofluorescence corrected n-Born ratio overlaid on the reflectance image. (f) and (h) Density plots of all the uncorrected and corrected measurements, respectively, when each fluorescence pixel value is plotted vs the corresponding normalization pixel value (arbitrary units).

Representative example of intracranial U87dEGFR-mCherry glioblastoma reconstruction. (a) A 3D view of the surface of the mouse’s head, sources (red crosses) and detectors (blue circles) are projected on the surface. The black rectangle indicates the position of the axial slice 3. (b) A fluorescence epi-illumination image of histological section (slice #3) of the brain, blue – reflectance image at 593 nm, magenta – mCherry fluorescence emission at 620 nm when excited at 593 nm. (c) Single- and multi-spectral reconstructions of the mCherry fluorescence at the axial slice #3. (d) A series of axial slices of the mCherry tumor reconstructed fluorescence, and (e) Corresponding post-Gd-DTPA enhanced axial slices from the T₁-weighted MRI series. Orange arrows indicate the position of the glioma. Colorbar for 
(c) and (d) is in arbitrary units. Dashed lines in (c) and (d) are visual guides to indicate the brain area.

Schematic of the experimental tomographic system (in free-space configuration).

Characteristic fluorescence and autofluorescence emission curves in deep tissue imaging. (a) In vivo relative spectral attenuation of light transmitted through 8 mm thick mouse tissue (upper left torso). Arrows indicate key wavelengths: blue – GFP excitation at 488 nm, green – GFP emission at 514 nm, orange – RFP excitation at 593 nm, and red bars – RFP emission 610-650 nm. The red arrow indicates the laser wavelength used for the calculation of the n-Born ratio. (b) normalized absorption and emission spectra for GFP and mCherry, 
(c) Normalized fluorescence of 10⁶ U87dEGFR glioblastoma cells expressing FPs from deep tissue in transillumination mode. The orange shaded area indicates the intensity range of mCherry emission. The mCherry RFP emission was significantly higher when cells were closer to the excitation source (orange triangles pointing upward), than when they were closer to the detector (orange triangles pointing downward). GFP emission (cyan square) was essentially independent from the position. Black squares and lines indicate the autofluorescence intensity (control cells) and green squares and triangles show the emission of mCherry when excited at 532 nm. Data were normalized for relative FP brightness. (d) In vivo normalized autofluorescence emission in transillumination of 12 mm thick mouse tissue (upper center torso) when excited at 6 different laser lines ranging from 488 - 750 nm (see color legend). The measurements were normalized for spectral window bandwidth, laser power, exposure time, i.e. units are photon counts.nm⁻¹mW⁻¹.s⁻¹. The continuous lines are spline interpolations.

Multispectral fluorescence tomography reconstruction. (a) Schematic of the 3-wavelength n-Born ratio calculation. Here, s, m, and d are the source, mesh, and detector positions, respectively. G_sm, G_md, and G_sd are the Green’s functions that model the corresponding propagating photon fields between s, m, and d. (b) Drawing of the cross section of the phantom containing 1% Intralipid and 1% whole blood. Small circles indicate the position of the two tubes containing 15 and 60 pmol of Texas Red fluorochrome. (c) A 3D view of the surface of the phantom with the positions of the sources (red crosses) and the detectors (blue circles) as projected on the surface. (d) Selected axial slices of the 3D single- and multi-spectral reconstructions of the fluorescence concentration (arbitrary units) of the two tubes inside the phantom. Black discs indicate the cross section of the phantom. (b) and (d) are on the same scale.