Dynamic focussing photoacoustic microscopy of angiogenesis in vivo

光声（PA）显微镜依靠吸收短光脉冲在组织内产生超声波。这些波传播到目标生物体的表面，其中时间分辨的PA信号由单个超声换能器或换能器阵列检测。这允许获得高分辨率的亚微米3-D图像，其显示组织发色团的空间分布。PA显微镜结合了许多强大的属性，例如单细胞分辨率和血管化软组织中基于吸收的强对比度。这是用来利用氧和脱氧血红蛋白的吸收光谱的差异，使功能参数的定量测量，如血氧饱和度，血流量和总血红蛋白浓度。高速PA显微镜已经能够可视化繁殖单个红细胞中氧饱和度的变化。此外，PA显微镜允许检测遗传报告基因，如荧光和光开关蛋白。虽然压电超声检测器被最广泛地使用，但是它们具有明显的缺点，诸如谐振频率响应和不透明性，这由于例如较大的源-检测器距离而不利地影响成像系统的声学灵敏度。Fabry-Pérot干涉仪（FPI）超声传感器已被证明提供衍射限制的元件尺寸、高声学灵敏度、接近均匀的频率响应和光学透明性，用于有效的后向模式PA成像，即在目标的同一侧上激发和检测。使用FPI传感器的PA显微镜，而不是传统的压电检测器的其他关键优势在于最小的源检测器的距离和声阻抗失配。这将导致在声学灵敏度的主要增加相比，目前的方法和定量PA显微镜的准确性的改善。该项目旨在通过开发新的检测几何形状和传感器读出方案将联合收割机FPI超声传感器技术与PA显微镜相结合，以实现声学检测的动态聚焦，即在一台仪器中组合多尺度OR-PAM和AR-PAM功能。动态聚焦PA显微镜将与其他光学显微镜平台相结合，如扫描共聚焦荧光，MP和超分辨率显微镜。此外，将开发用于临床前研究中血管生成的体内定量、功能性PA显微镜检查的方法。多波长PA显微镜将用于血流和氧合成像，以及报告蛋白的分子成像，如光开关光敏色素。这将使功能和分子显微镜同时生长的血管，以研究血管生成过程中的血流和氧合对上皮细胞的作用的影响。

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.