Photoacoustic Microscopy With High Axial Resolution In A Large Depth Of Field

The microvascular network is criss-crossed and intricate,consisting of a micro-arteriovenous vein with a diameter of 10-200 ?m and capillaries with a diameter of 5-8 ?m.Photoacoustic microscopy combines the advantages of optical and acoustic imaging,which can image capillaries with label-free.However,in order to achieve sufficiently high resolution,optical focusing is often employed,which makes the system own a narrow depth range in focus.The lateral resolution often reaches the resolution of the optical diffraction limit(hundreds of nanometers),while the axial resolution is harder to get smaller than 10 micrometers owning to the narrow bandwidth of the ultrasonic detection.This makes the system difficult to image the microvascular network with large volumetric and uniform three-dimensional spatial resolution.In this paper,we are focusing on study of the promotion of the depth of field and the axial resolution of photoacoustic microscopy,including:1.To extend the depth of field of optical-resolution photoacoustic microscopy(OR-PAM).The TAG lens was used to continuously changing the focal plane of OR-PAM by modulating its refractive power with fast-changing ultrasonic standing wave,we split a single laser pulse into three sub-pulses and introduce them into three fibers with different lengths.The sub-pulses out of the fibers were combined thereafter.We then obtained a pulse train with a time interval of 120 ns.The three laser pulses synchronize with three vibration states of the TAG lens(indicated different focal length)by synchronous circuit,respectively.And we finally achieved three focus in each A line data acquisition.The depth of focus(DoF)of the system was measured to be 360 ?m by imaging a vertically tilted carbon fiber,which is three times of that of previous systems.A mouse ear and mouse cerebral vasculature were imaged in vivo to further demonstrate the feasibility of the extended DoF of our system.2.The axial modulation is proposed for promoting the axial resolution in optical-resolution photoacoustic microscopy.The principle and reconstruction algorithm of axial modulation are illustrated through theoretical analysis.We employ series of spatial structural illumination to modulate the excited high frequency signal to a much lower frequency band.Then,we separated high spectrum information and shifted to the right position,combined them.And finally,we improved the axial resolution.The simulation model of axial modulation is built by the k-Wave simulation toolbox.To verify the ability of axial modulation,an ultrasonic probe(a center frequency of 50 MHz and a bandwidth of ~40 MHz)and a particle with diameter of 500 nm was used to image under the axial modulation with 26-ps pulse,9-ns pulse,and uniform illumination,respectively.The axial resolution is about 2.25 ?m,10.15 ?m,and 35.1?m,respectively.3.In order to demonstrate the performance of axial modulation experimentally,a photoacoustic microscopy with structured illumination along depth direction was developed.The structured illumination with different spatial frequency and phase is generated by a spatial light modulator and a cylindrical lens.The ultrasonic detection and optical excitation is in the orthogonal and confocal mode to achieve axial modulation of photoacoustic signal.The demonstration of axial modulation is carried out on the carbon nanoparticles with a diameter of 500 nm.The experimental results show that the axial resolution under axial modulation with 9-ns pulse is 7.5 ?m,which is 4.16 times that of uniform illumination.While,the axial resolution under axial modulation with 26-ps pulse is 2.22 ?m,which is 14 times that of uniform illumination.The ability of morphological analysis of axial modulation was verified by imaging a single red blood cell.Imaging of zebrafish demonstrates the ability of axial modulation in vivo.By imaging the cerebral blood vessels with intact skull,it was verified that the axial modulation can avoid the high attenuation of high frequency component caused by skull by transferring the high frequency component to low frequency one,therefore,the system can still keep high axial resolution.The experiment of imaging different levers of red blood cells aggregation verified the ability of axial modulation in photoacoustic spectral analysis.