Multiplexed in vivo photoacoustic imaging of photoswitchable chromoproteins GAF2 and BphP1 with difference spectra differentiation (Conference Presentation)

Photoswitchable chromoproteins allow for molecular photoacoustic images with reduced hemoglobin background signal. We have previously introduced GAF2 a far-red photoswitchable chromoprotein similar to BphP1 [Yao et al., Nat. Meth. 13, 67–73 (2016)], but one-tenth the size. We introduce a new strategy for differentiating between spectrally similar photoswitchable chromoproteins. This strategy is based on exploiting relative spectral differences between two photoswitchable absorption states. We present in vivo photoacoustic images of GAF2 and BphP1 with differentiation based on their relative intensity change. We imaged using a custom photoswitchable photoacoustic imaging system that allows simultaneous background-free photoacoustic signal acquisition and difference spectra differentiation. E.coli expressing BphP1 and GAF2 are injected at various depths in hairless SCID mice. Background-free photoacoustic images are obtained and the chromoproteins are differentiated based on their relative intensity change. We photoconvert GAF2 and BphP1 using 607.5nm (5.2mJ/cm2) and 710nm (8.3mJ/cm2) light. The cycle of imaging and photoconversion is 20s. We are able to obtain background-free photoacoustic images 1.2cm deep in vivo and clearly differentiate between GAF2 and BphP1 via relative intensity changes despite similar imaging spectra. Photoswtichable chromoproteins and difference spectra differentiation could prove promising for deep multiplexed molecular background-free photoacoustic imaging.