Dynamic focussing photoacoustic microscopy of angiogenesis in vivo

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Photoacoustic (PA) microscopy relies on the absorption of short optical pulses to generate ultrasound waves within the tissue. These waves propagate to the surface of the target organism where time-resolved PA signals are detected by single ultrasound transducers or transducer arrays. This allows high resolution, sub-micron 3-D images to be obtained, which show the spatial distributions of the tissue chromophores. PA microscopy combines a number of powerful attributes, such as single cell resolution and strong absorption-based contrast in vascularised soft tissues. This is used to exploit the differences in the absorption spectra of oxy- and deoxyhaemoglobin for making quantitative measurements of functional parameters, such as blood oxygen saturation, blood flow, and total haemoglobin concentration. High-speed PA microscopy has enabled the visualisation of the change in oxygen saturation in propagating single red blood cells. In addition, PA microscopy allows the detection of genetic reporters, such as fluorescent and photoswitchable proteins. While piezoelectric ultrasound detectors are the most widely used, they have distinct drawbacks, such as a resonant frequency response and opacity, which adversely affect the acoustic sensitivity of the imaging system due to, for example, large source-detector distances. Fabry-Pérot interferometer (FPI) ultrasound sensors have been shown to provide diffraction-limited element sizes, high acoustic sensitivity, near-uniform frequency response, and optical transparency for efficient backward mode PA imaging, i.e. excitation and detection on the same side of the target. Additional key advantages of using FPI sensors for PA microscopy, rather than conventional piezoelectric detectors, lie in minimal source-detector distances and acoustic impedance mismatches. This will result in major increases in acoustic sensitivity compared to current methods and an improvement in the accuracy of quantitative PA microscopy. This project aims to combine FPI ultrasound sensor technology with PA microscopy by developing novel detection geometries and sensor readout schemes to enable dynamic focussing of the acoustic detection, i.e. the combination of multi-scale OR-PAM and AR-PAM capability in one instrument. Dynamic focussing PA microscopy will be combined with other optical microscopy platforms, such as scanning confocal fluorescence, MP and super-resolution microscopy. In addition, methods for in vivo quantitative, functional PA microscopy of angiogenesis in preclinical studies will be developed. Multiwavelength PA microscopy will be used to image blood flow and oxygenation, and molecular imaging of reporter proteins, such as photoswitchable phytochromes. This will enable simultaneous functional and molecular microscopy of growing blood vessels to study the effect of blood flow and oxygenation on the role of epithelial cells during angiogenesis.