Volume Analysis.

<p>3D (shown as xy view and orthogonal views xz, yz) autofluorescence a) and B_SHG b) from a MI sample. The region of interest selected by thresholding the autofluorescence to define the total volume of the sample is highlighted in green in a); the region of interest selected by thresholding the B_SHG to define the volume occupied by collagen is highlighted in yellow in b); c) the discretized area vs depth is plotted, black and red is from autofluorescence (AMC and MI samples respectively) grey and orange from B_SHG (AMC and MI sample respectively); the curves have been normalized so that the integral over black and red is 1, therefore representing the total volume, and over grey and orange is the fraction of volume occupied by collagen; d) average collagen presence evaluated for MI (n = 8) compared to AMC (n = 8) animals.</p

B_SHG and autofluorescence characterisation.

<p>Standard histology sections are stained with picrosirius red to highlight collagen in tissues from myocardial infarction rat model (a and b), from age-match control in c, showing the better signal to noise ratio for SHG light in discriminating collagen. In d, the spectral characterization is presented to demonstrate that the collected signals are SHG (magenta) by observing the shift in peak intensity with that of the excitation wavelength and autofluorescence (green). Scale bar 50 µm in a, 100 µm in b and c.</p

B_SHG dependence on ionic strength.

<p>B_SHG from rat tail samples has been collected at different NaCl concentrations compared to relaxing solution. From 50 mM NaCl the B_SHG reaches a plateau. Measurements performed in relaxing solution fall in the same range of intensity ensuring that in the present experimental conditions we maximize the backward light collection. Images above symbols are representative for that concentration.</p

Intensity Analysis.

<p>3D (shown as xy view and orthogonal views xz, yz) autofluorescence a) and B_SHG b) from a MI sample. The region of interest selected by thresholding the autofluorescence to define the sample edges is highlighted in green in a); the very same region is applied to the B_SHG images, highlighted in yellow in b); c) intensity profile along the thickness of the sample, MI (dark red, red, orange), AMC black and grey; d) Average intensity increase (MI/AMC) obtained by integrating B_SHG intensity over 10 µm z-steps.</p

Upper and lower B_SHG detection range.

<p>Representative 3D optical sections (xy and orthogonal view xz, yz) of a trabecula in a) and a rat tail in b); green autofluorescence, fire color scale B_SHG. Scale bar, 25 µm. In c) B_SHG intensity versus sample depth is presented for rat tail samples (black), cardiac trabeculae (dark grey) and permeabilised skeletal fibres (light grey). Intensity profiles have been normalized to the rat tail samples as they represent the upper bound of B_SHG detection. On the contrary permeabilised skeletal fibres show virtually no B_SHG (2%) thus representing the lower bound of detection. Healthy cardiac samples show a low but measurable B_SHG, thus ensuring a large range of detection for possible increases in collagen content in diseased samples.</p