Three-dimensional Optical-resolution Photoacoustic Microscopy

We present a dual modality ultra-thin imaging system based on a optical multimode fiber and a optical fiber hydrophone that combines optical resolution photoacoustic and fluorescence microscopy.

A novel combined photoacoustic tomography (PAT), optical resolution photoacoustic microscopy (ORPAM) and optical coherence tomography (OCT) instrument has been developed for imaging biological tissues. The system is based on the use of a Fabry-Perot (FP) polymer film ultrasound sensor. This is designed to be transparent to wavelengths between 590nm and 1200nm so that photoacoustic excitation laser pulses in this spectral range can be transmitted through the sensor into the underlying tissue to allow backward mode operation. The dual PAT-ORPAM capability of the system was demonstrated by imaging a tissue phantom composed of 7μm diameter carbon fibres immersed in an optically scattering liquid. The lateral and vertical spatial resolutions in ORPAM mode are approximately 7μm and 10μm respectively for sub-mm depths. In PAT mode, the lateral spatial resolution is less than 50μm for depths up to 5mm and the vertical resolution is approximately 10μm. The transparent nature of the FP polymer film ultrasound sensor offers a convenient platform for combining other optical imaging modalities with PAT and ORPAM. To illustrate this, a frequency-domain OCT system operating at 1060nm was integrated into the system and combined PAT/OCT images of the skin of a mouse were obtained in vivo.

Photoacoustic microscopy for cells in a 3D microenvironment: Systems and applications

Optical resolution photoacoustic microscopy (OR-PAM) provides functional and molecular information at the cellular scale. OR-PAM typically employs ultrasonic transducers, which require coupling with tissue, to detect the thermo-elastic waves generated in tissues irradiated by nanosecond laser. Images are acquired by scanning the excitation beam, which is usually focused to a spot size of a few microns, i.e., lateral resolution. However, the axial resolution is constrained to an order of magnitude lower by the bandwidth of the transducer used. Additionally, imaging depth of OR-PAM is constrained to the depth that allows ballistic photons, which is usually less than 1 mm. We present an All Optical Frequency Domain Photoacoustic Microscope where the photoacoustic signal is generated by an amplitude modulated CW laser and detected using a two wave mixing (interferometer) in a photorefractive crystal. Sweeping the amplitude-modulation frequency and using an ultra-broadband optical detector can potentially prov...

In this Letter, reflection-mode optical resolution photoacoustic microscopy (OR-PAM) using glancing angle-deposited (GLAD) nanostructured Fabry-Perot interferometers (FPI) for in vivo applications is reported. GLAD is a single-step physical vapor deposition (PVD) technique used to fabricate porous nanostructured thin films. Using titanium dioxide, a transparent semiconductor with a high refractive index (n=2.4), the GLAD technique can be employed to fabricate samples with tailored nano-porosity, refractive index periodicities, and high Q-factor reflectance spectra. The OR-PAM in vivo images of chorioallantoic membrane (CAM) of 5-day chicken embryo model are demonstrated. The phantom study shows lateral resolution and signal-to-noise ratio better than 7μm and 35dB, respectively. The sensitive GLAD FPI allows photoacoustic imaging down to a few-nJ pulse energy. To the best of our knowledge, this is the first time that a FPI-based reflection mode optical resolution photoacoustic imaging technique is demonstrated for in vivo applications.

Optical resolution photoacoustic microscopy (ORPAM) is a high-resolution hybrid imaging modality having potential for microscale in vivo imaging. Optical diffraction limits the lateral resolution of ORPAM. A photonic nanojet (PNJ) was used to break this diffraction limit. A single round microsphere can generate a PNJ with subwavelength waist, but its short axial length limits its applications to surface imaging only. We investigate different sphere designs to achieve ultralong nanojets that will make the nanojet more viable in far-field applications, such as photoacoustic imaging. The PNJ properties, including effective length, waist size, working distance, and peak intensity, can be tuned and controlled by changing the sphere design and its refractive index. A truncated multilayer microsphere design could generate an ultraelongated PNJ with length larger than ∼172λ (∼138 μm) while retaining a large working distance ∼32λ (∼26 μm). Through simulation study, we observed ∼11-fold enhancement in lateral resolution with 5 μm round sphere (refractive index 2.2) when used in a conventional ORPAM setup with NA=0.1 and λ=800 nm.

In order to understand the pathophysiology of glaucoma, Lamina cribrosa (LC) perfusion needs to be the subject of thorough investigation. It is currently difficult to obtain high resolution images of the embedded microcapillary network of the LC using conventional imaging techniques. In this study, an optical resolution photoacoustic microscopy (OR-PAM) system was used for imaging lamina cribrosa of an ex vivo porcine eye. Extrinsic contrast agent was used to perfuse the eye via its ciliary arteries. The OR-PAM system have a lateral resolution of 4 μm and an axial resolution of 30 μm. The high resolution system could able resolve a perfused LC microcapillary network to show vascular structure within the LC thickness. OR-PAM could be a promising imaging modality to study the LC perfusion and hence could be used to elucidate the hemodynamic aspect of glaucoma.

The penetration depth of ballistic optical imaging technologies is limited by light scattering. To study the effect of scattering on optical-resolution photoacoustic microscopy (OR-PAM), we divided the signals in OR-PAM into two classes: one is from the target volume defined by the optical resolution cell (Class I); the other is from the rest of the acoustic resolution cell (Class II). We developed a way to simulate the point spread function (PSF) of our OR-PAM system considering both optical illumination and acoustic detection, then used the PSF to calculate the contributions of each class of signal to the total signal at different focal depths. Our simulation results showed that: 1) The Class II signal decays much more slowly than the Class I signal; 2) The full width at half maximum (FWHM) of the PSF for the focal depth of 0.9 transport mean free path (TMFP) is not broadened much (~10%) compared with that for a clear medium; 3) Image contrast is degraded with increasing depth when there is a uniform absorption background.

We show that optical properties of dense biological tissues can be determined from backscattered power curves measured by a low-coherence reflectometer. Our measurement approach is based on a first-order scattering theory that relates the backscattered power to the total and backscattering cross sections of scatterers in a turbid medium. As a validation of the technique, measurements were made with a commercially available reflectometer on suspensions of polystyrene microspheres having known optical properties. With this reflectometer, which employs a 1300-nm LED source that emits less than 20 µW, we found that skin tissues could be probed to a depth of nearly 1 mm. Estimates of optical coefficients of human dermis and of a variety of excised animal tissues are given.

Tissue optical properties can be determined with optical coherence tomography (OCT) by fitting a model to the OCT signal. Using calibrated samples in the fixed focus geometry, the validity of the single-scattering and multiple-scattering models for both highly scattering and weakly scattering media (scattering coefficients mu(s) ranging from 1.25 to 25.11mm(-1)) has been investigated. The results show that, with a proper correction for the confocal properties of the sample arm, both models are appropriate to extract the scattering coefficients of weakly scattering media. For highly scattering media, the multiple scattering should be taken into account, and the multiple-scattering model can provide higher accuracy. Finally, the scattering properties of in vitro rat liver and in vivo human skin are determined. The results are useful for quantitatively characterizing tissue scattering in biomedical research and clinical diagnosis.

Optical-resolution photoacoustic microscopy (OR-PAM) can visualize wavelength-dependent optical absorption at the cellular level. However, OR-PAM suffers from a limited depth of field (DOF) due to the tight focus of the optical excitation beam, making it challenging to acquire high-resolution images of samples with uneven surfaces or high-quality volumetric images without z-scanning. To overcome this limitation, we propose needle-shaped beam photoacoustic microscopy (NB-PAM), which can extend the DOF to up to ~28-fold Rayleigh lengths via customized diffractive optical elements (DOEs). The DOE generate a needle beam with a well-maintained beam diameter, a uniform axial intensity distribution, and negligible sidelobes. The advantage of using NB-PAM is demonstrated by both histology-like imaging of fresh slide-free organs using a 266 nm laser and in vivo mouse brain vasculature imaging using a 532 nm laser. The approach provides new perspectives for slide-free intraoperative pathological imaging and in-vivo organ-level imaging.

Optical resolution photoacoustic microscopy (OR-PAM) can achieve an optical-dependent high lateral resolution (<; 1 μm) and visualize photoacoustic (PA) property at single-cell level. The spatial resolvability of the PA imaging is expected to precisely characterize biological activities of cells such as their interactions with drugs and contrast agents by comparing the acquired PA characteristics with the cell's morphology. However, since the conventional OR-PAM systems only visualize objects that generate PA signals, it was difficult to acquire a PA image and an optical morphological image simultaneously. Here, we report a novel system of the hybrid optical / PA microscopy that can acquire morphological and PA characteristics of cells at the same time. The performance of the developed system was validated by measuring a USAF1951 target and bovine red blood cells.

Optical-resolution photoacoustic microscopy (OR-PAM) offers high lateral resolution for imaging fine tissue structures, such as tissue vasculature and cancerous tumour margins. However, its volumetric imaging capability is limited by optical scattering, which degrades resolution with increasing imaging depth. While deconvolution methods have been proposed to enhance resolution, they typically assume a static system point spread function (PSF) and fail to account for depthdependent degradation caused by tissue scattering. To address this, we propose a physics-informed deconvolution framework that incorporates depth-varying PSFs based on tissue optical properties. We use Monte Carlo eXtreme (MCX) to model optical fluence and the MATLAB k-Wave toolbox to simulate the response function of an ultrasound transducer to produce depth-dependent PSFs. These are then incorporated as prior knowledge in a Richardson-Lucy algorithm applied to three-dimensional PAM images. This framework leads to almost two-fold resolution recovery in a three-dimensional OR-PAM image, demonstrating its potential for extending high-resolution OR-PAM imaging beyond the ballistic regime.

Optical resolution photoacoustic microscopy (OR-PAM) is a high-resolution imaging configuration of photoacoustic Imaging. However, due to limited active detection area of ultrasound transducer, the field of view of an OR-PAM is limited to only few millimeter. Researchers have tried to improve the field of view of OR-PAM by implementing the mechanical scanning of the ultrasound transducer, but this has slowed down the speed of imaging significantly. We have developed a very fast, ultra-wide laser scanning photoacoustic system that provides a very large active detection area. The system utilizes a novel spiral scanning mechanism, using traditional 2D galvo mirrors to scan an area of ~4cm in less than 5 seconds. To characterize and demonstrate the imaging capability of the system, we image rat brain in vivo.

Photoacoustic (PA) imaging is rapidly progressing imaging modality in which very short pulsed laser causes thermal expansion to generate PA signal. In previous PA microscope systems, relatively long time was required for image acquisition because they required mechanical scan of the PA transducer. The objective of the present study is to develop fast laser scanning optical resolution photoacoustic microscopy (OR-PAM) with fixed PA signal detector to realize very high frame rate PA imaging. Q-switched Nd:YAG laser with the wavelength of 532 nm, pulse width of 5.5 ns and pulse repetition rate of up to 50 kHz was equipped in the system. Low frequency PA detector was comprised of a glass prism configuring acoustic focusing and a polymethyl methacrylate (PMMA) prism with 5 MHz PZT (lead zirconate titanate) transducer. High frequency PA detector was comprised of the same glass prism and a glass prism with ZnO (zinc oxide) thin film transducer. Galvano scanner operating in the air was controlled by a microcomputer to scan laser beam. PA signal was detected with the fixed PA detector thus realized the frame rate of 5 fps for C-mode equivalent to 500 fps for B-mode. The lateral resolution of the low frequency system was found to be 11.6 μm and the blood vessel on the surface of cod roe was clearly visualized. The system may be applicable for imaging blood flow and vascular dynamics of micro vessel.