Supplementary Figure 5: RS-PACT of BphP1-expressing U87 cells in an optically scattering phantom.

(a) Photograph of the phantom with BphP1-expressing U87 cells and oxygenated whole bovine blood embedded at ~12 mm depth. The phantom was made of 1% intralipid, 10% gelatin, and 2% oxygenated bovine blood in distilled water. The optical absorption coefficient was ~0.1 cm⁻¹ and the reduced scattering coefficient was ~10 cm⁻¹. (b) PACT images of the phantom at 780 nm with BphP1 in the ON state (left panel) and OFF state (middle panel), and at 750 nm with ON state BphP1 (right panel). (c) Extraction of BphP1 signals by using the single-wavelength differential method and the traditional two-wavelength spectral unmixing method. The single-wavelength differential method is the subtraction of the PACT measurements at 780 nm with BphP1 in the ON and OFF states. The two-wavelength method is based on the least-squares fitting of the PACT measurements at 780 nm and 750 nm with BphP1 in the ON state. The single-wavelength differential method has substantially higher accuracy in identifying the BphP1 signals, while the two-wavelength method suffered from the unknown optical fluence deep in the phantom. A global threshold was applied to both images with a threshold level at three times the noise level. (d) Contrast to noise ratios of extracted BphP1 images by using the two methods, showing a ~34-fold enhancement of the single-wavelength differential method. Error bars: s.d. (e) The extracted BphP1 image using differential method without thresholding. More residual artifacts due to the laser pulse energy fluctuations are visible.