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，在那里，单个超声换能器或换能器阵列检测到时间分辨的PA信号。这使得可以获得高分辨率、亚微米的3D图像，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软组织中基于强吸收的对比度。它被用来利用氧合和脱氧血红蛋白吸收光谱的差异来定量测量功能参数，如血氧饱和度、血流量和总血红蛋白浓度。高速PA显微镜能够直观地显示单个红细胞在繁殖过程中血氧饱和度的变化。此外，PA显微镜允许检测遗传报告，如荧光和光开关蛋白。虽然压电式超声探测器是最广泛使用的，但它们具有明显的缺点，例如共振频率响应和不透明，这会由于例如较大的源-探测器距离而对成像系统的声学灵敏度产生不利影响。法布里-佩罗涉仪(FPI)超声传感器已被证明具有衍射受限的元件尺寸、高的声学灵敏度、近乎均匀的频率响应和光学透明度，可用于高效的后模PA成像，即在目标的同一侧进行激励和检测。在PA显微镜中使用FPI传感器而不是传统的压电式探测器的另一个关键优势在于最小的源-探测器距离和声阻抗失配。与目前的方法相比，这将导致声学灵敏度的显著提高，并提高定量PA显微镜的准确性。该项目旨在通过开发新的检测几何结构和传感器读出方案，将FPI超声传感器技术与PA显微镜相结合，以实现声检测的动态聚焦，即在一个仪器中结合多尺度OR-PAM和AR-PAM能力。动态聚焦PA显微镜将与其他光学显微镜平台相结合，如扫描共焦荧光显微镜、MP和超分辨率显微镜。此外，还将开发临床前研究中血管生成的体内定量、功能性PA显微镜方法。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，如可光开关的光敏色素。这将使生长血管的同步功能和分子显微镜能够研究血流和氧合对血管生成过程中上皮细胞作用的影响。

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.