Optical resolution photoacoustic microscopy and fluorescence imaging with a multimode fiber

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

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

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.

Accurate identification and quantification of microvascular patterns are important for clinical diagnosis and therapeutic monitoring using optical-resolution photoacoustic microscopy (OR-PAM). Due to its limited depth of field, conventional OR-PAM may not fully reveal microvascular patterns with enough details in depth range, which affects the segmentation and quantification. Here, we propose a robust vascular quantification approach via combining multi-focus image fusion with enhancement filtering (MIFEF). The multi-focus image fusion is constructed based on multi-scale gradients and image matting to improve image fusion quality by considerably achieving accurate focus measurement for initial segmentation as well as decision map refinement. The enhancement filtering identifies the vessels and handles noise without deforming microvasculature. The performance of the MIFEF were evaluated employing a leaf phantom, mouse livers and brains. The proposed method for OR-PAM can significantly facilitate the clinical provision of optical biopsy of vascular-related diseases.

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.

Freehand scanning photoacoustic microscopy with simultaneous localization and mapping

Studying oxygen metabolism during brain ischemia has been limited by imaging modalities that have either low resolution but good tissue penetration, or high resolution requiring invasive preparations (open-skull windows). Here, we report a study of cerebrovascular oxygen metabolism in mouse focal ischemia using a novel minimally invasive, high-resolution technique known as optical-resolution photoacoustic microscopy (OR-PAM). Using OR-PAM, we serially imaged cortical vessels through the intact skull of Swiss Webster mice (n=3) before, during, and up to 72 hrs after a 1-hr intraluminal transient middle cerebral artery occlusion (MCAO). Vessels were imaged at 2 different wavelengths (532 & 563 nm) to quantify oxy-, deoxy-hemoglobin and oxygen saturation (sO 2 , Fig , A-C). Oxygen extraction fraction (OEF) was calculated from venous sO 2 values (assuming a constant arterial sO 2 of 95%). Seventy-two hrs after ischemia, animals were sacrificed and whole brains were stained with TTC to delineate infarct at the cortical surface (D). Arterioles were easily distinguished from venules in baseline images (A, mean arterial sO 2 , 87%; mean venous sO 2 , 76%). During MCAO, mean arteriolar sO 2 values did not change; however, venular sO 2 decreased precipitously (B, E), greater in regions that went on to infarct (45±2%) compared to surrounding regions (52±2%, p<0.001). Three and 7 hrs after reperfusion, venous sO 2 slowly recovered but not back to baseline levels. Twenty-four (C) and 72 hrs after reperfusion, venous sO 2 in regions of infarction increased above baseline (83%), approaching arterial sO 2 values of 90%, while venous sO2 in peri-infarct regions (71%) returned near baseline levels of 76% (E). OEF increased from 18% to 55% in regions of eventual infarction, during MCAO; following reperfusion, OEF progressively dropped below baseline levels (12%). In surrounding surviving tissue, OEF returned close to baseline levels (24%) following reperfusion (F). OR-PAM is capable of measuring oxygen metabolism at high resolution during focal ischemia. Consistent with previous PET studies in human stroke, we found OEF increased during acute ischemia; however, OEF remained elevated (above baseline values) after reperfusion, suggesting that injured tissue may have increased oxygen metabolism. OEF gradually decreased below baseline values in infarcted tissue, but returned to near-baseline values in viable peri-infarct regions.

Photoacoustic microscopy (PAM) has remained one of the fastest developing biomedical imaging modalities in the past decade. The confocal strategy of optical illumination and acoustic detection is a way to boost the sensitivity of PAM. To achieve confocal PAM, current PAM systems utilize separate acoustic and optical converging devices, making the systems bulky and complicated. In this Letter, we demonstrate the use of a single-liquid lens to successfully achieve acoustic and optical confocal configuration for optical-resolution PAM (ORPAM). Using the lens with a numerical aperture of 0.43, we show that the resolution of the ORPAM system is 4.8μm with a significantly improved sensitivity of acoustic detection. We also apply this compact ORPAM system to in vivo imaging of the vasculature of a rat ear.

The increasing use of mouse models for human brain disease studies, coupled with the fact that existing high-resolution functional imaging modalities cannot be easily applied to mice, presents an emerging need for a new functional imaging modality. Utilizing both mechanical and optical scanning in the photoacoustic microscopy, we can image spontaneous cerebral hemodynamic fluctuations and their associated functional connections in the mouse brain. The images is going to be acquired noninvasively with a fast frame rate, a large field of view, and a high spatial resolution. We developed an optical resolution photoacoustic microscopy (OR-PAM) with diode laser. Laser light was raster scanned due to XY-stage movement. Images from ultra-high OR-PAM can then be used to study brain disorders such as stroke, Alzheimer's, schizophrenia, multiple sclerosis, autism, and epilepsy.

The use of an optical resolution photoacoustic microscopy (OR-PAM) system to evaluate the vascular disruptive effect of combretastatin A4 Phosphate (CA4P) on a murine orthotopic glioma with intact skull is described here. Second generation optical-resolution photoacoustic microscopy scanner with a 532 nm pulsed diode-pumped solid-state laser that specifically matches the absorption maximum of hemoglobin in tissues was used to image orthotopic glioma inoculated in mouse brain. Two-dimensional maps of brain vasculature with a lateral resolution of 5 μm and a depth of 700 μm at a field of view 5 × 4 mm were acquired on normal brain and glioma brain. Longitudinal imaging of the brain pre- and post-administration of CA4P, a FDA approved drug for solid tumors, enabled the monitoring of hemodynamic changes in tumor vasculature revealing the well documented vascular shutdown and recovery associated with this drug. Our study marks the beginning of potential prospects of this technology as an imaging tool for preclinical and clinical study of pathologies characterized by changes in the vasculature.

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.

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.

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 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.

As an important part of optical-resolution photoacoustic microscopy, the acoustic lens is responsible for efficient collection of photoacoustic signals. The spherical focused acoustic lens is commonly used in photoacoustic microscopy because of its efficient detection of the photoacoustic signal in the focus area. However, the narrow depth of field of the spherical focused acoustic lens limits the expansion of the depth of field of the photoacoustic microscopy. To solve this problem, a Bessel acoustic-beam acoustic lens is proposed. The Bessel acoustic-beam acoustic lens replaces the spherical concave surface with a conical concave surface to generate a Bessel acoustic beam with non-diffraction. Using the simulation model of Bessel acoustic-beam acoustic lens constructed by COMSOL Multiphysics, it is verified theoretically that the Bessel acoustic-beam acoustic lens can improve the depth of field of detection by ∼2 times. The Bessel acoustic-beam acoustic lens can further promote the capability of high-speed and large volumetric imaging of optical-resolution photoacoustic microscopy and will be helpful in the acquisition of physiological and pathological processes.