In Vivo Photoacoustic Microscopy Based On Surface Plasmon Sensing

Based on unique optical absorption contrast,photoacoustic imaging technology demonstrates the capability of multi-scale imaging across cells,tissues,and organs.To date,it has shown broad applications in the fields of life science and clinical medicine.Taking advantage of the intrinsic optical absorption spectra of biomolecules,photoacoustic microscopy can achieve high-contrast and high-resolution imaging of three-dimensional morphological structure of biological tissues without external labels.Generally,the traditional photoacoustic microscopy adopts piezoelectric transducer as the ultrasonic detector.Due to the physical properties of the piezoelectric materials,the detection bandwidth of such transducer is insufficient,thus causing a series of problems,such as inaccurate depth positioning,distortion of 3D image reconstruction,and saturation effects.In order to overcome the above limitations,this work proposes a photoacoustic detection technology based on surface plasmon resonance(SPR)approach.Relying on the characteristics of fast response and high-sensitivity refractive index sensing of surface plasmon polaritons,the polarization-differential SPR sensing realizes broadband and highly sensitive detection of pulsed photoacoustic signals.Further,a reflectionmode photoacoustic microscopy is developed by constructing a compact photoacoustic excitation-detection device that integrates a miniature SPR sensor with a reflective microscopic objective.In consequence,we obtained the three-dimensional microscopic structure of microvessels in living mice.This work mainly consists of three parts.Firstly,the principle of responding the ultrasonic excitation based on an SPR sensor is explained theoretically.The spatial distribution characteristics of SPR electromagnetic field are simulated by finite-different time-domain method.We determine the light reflectance of the SPR sensing with respect to the incident light wavelength,laser polarization,incident angle,structural properties of the sensing layer,and environmental refractive index.As a result,the optimal parameters are derived for enhancing the performance of the SPR sensor.Secondly,a reflection-mode photoacoustic microscopy incorporating an SPR sensor is built.In order to realize the reflection-mode detection of the photoacoustic signals,we integrate a miniature SPR sensor and a reflective microscopic objective for high-fluence optical illumination,high-resolution optical focusing,and highefficiency detection to the backwards emission acoustic waves.The photoacoustic microscopy system possesses the detection bandwidth of up to 173 MHz,giving an estimated axial resolution of ～7.6 μm.In addition,the system features a linear acoustic pressure response ranging from 3 k Pa to 107 k Pa,defining the noise-equivalent-acoustic pressure sensitivity of about 477 Pa.We measure the microscopy’s lateral resolution at ～4.5 μm.Thirdly,we perform the photoacoustic imaging of biological samples in vivo.Utilizing the strong optical absorption of hemoglobin in the visible-light spectrum,we acquire the morphological images of blood vessel network in the ears of living mice.More importantly,because of the isometric spatial resolution at micrometer scale and sufficient ultrasonic detection sensitivity of our photoacoustic microscopy,the three-dimensional anatomic features of the microvascular networks are visible at high imaging contrast,delineating the main blood vessels,capillaries,and even a single red blood cell.