Multi-focus image fusion with enhancement filtering for robust vascular quantification using photoacoustic microscopy.

Optical-resolution photoacoustic microscopy (OR-PAM) can achieve submicron lateral resolution by tightly focusing the excitation light, while the axial resolution is still limited by the frequency bandwidth of the ultrasonic transducer. The Grueneisen relaxation effect, in which the Grueneisen parameter changes within the thermal relaxation time following a laser impulse heating, can provide excellent axial resolution due to its optical sectioning property. Based on this effect, Grueneisen relaxation photoacoustic microscopy (GR-PAM) was developed and demonstrated ex vivo. Here, we present for the first time in vivo imaging of mouse brains with improved axial resolution based on GR-PAM. An intensity-modulated continuous-wave (CW) 532 nm laser thermally heated the in-focus absorber. Another 532 nm pulsed laser, which is aligned confocally with the CW laser, generated the photoacoustic (PA) signal from the absorber. The difference between the amplitudes of the photoacoustic signals with and without heating was used for image reconstruction. The achieved axial resolution is ~12.5 µm, which is fivefold better than the acoustically determined value for a 20 MHz-bandwidth ultrasound transducer. The system was demonstrated by imaging a blood-filled tube ex vivo and blood vessels of mouse brains in vivo. The blood-filled tube diameter obtained from the PA image by GR-PAM is 105 µm, which is much closer to its actual diameter (100 µm) than the value from conventional OR-PAM (160 µm). This axial resolution improvement was further validated in imaging mouse brains in vivo, and yielded significantly narrower axial profiles of the vessels. This in vivo demonstration of imaging by GR-PAM might inspire more applications in PA biomedical imaging and sensing.

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.

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

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.

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) 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.

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.

For visual surveillance and other applications, there is a one big challenge to identify the information of defocused images. This challenge can be resolved using multifocused image fusion approaches using similar images of same object. Visual sensor networks (VSN) make use of these image fusion methods effectively for effective surveillance and motion tracking. The fusion algorithms are then compared using a multi-focus image dataset and established objective fusion parameters. Both the subjective and objective evaluation criteria are given, along with a detailed discussion of a few common objective assessment indicators. Here we found that deep learning provides best outcomes in compare to traditional methods. Multi-focus optical image fusion has come a long way in the past few years, but it still has a long way to go. Therefore in this paper, recent advancements on intelligent multi-focus image fusion have been reviewed. In this review paper, we review existing studies on non-conventional multi focus image fusion approaches, highlighting the most recent progresses and future directions.

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.

Freehand scanning photoacoustic microscopy with simultaneous localization and mapping

Multi-focus image fusion is an effective way to overcome the shallow depth of field of ordinary cameras and to achieve all-in-focus imaging across the entire depth range. However, existing multi-focus image fusion methods are usually designed for single-view and static scenes, mainly because the current focal stack imaging technology for capturing multi-focus images can only capture one partially focused image at each moment. To simultaneously capture multi-focus images for all-in-focus imaging in dynamic scenes, we propose a computational imaging scheme for all-in-focus dynamic image fusion. Specifically, a multi-view multi-focus (MVMF) imaging technology that integrates the characteristics of both multi-view imaging and focal stack imaging is designed to simultaneously capture MVMF images. For the fusion of MVMF images, we design an MVMF image fusion method based on focus clarity evaluation. This method consists of three modules: image alignment, focus clarity evaluation, and image fusion, which can effectively fuse MVMF images into a high-quality all-in-focus image. In addition, to fill the gap of lacking dynamic multi-focus image datasets in the existing multi-focus image fusion field, we render an MVMF image dataset (i.e., DYN-MVMF) capturing dynamic scenes using the Blender software. On the DYN-MVMF dataset, we conduct extensive comparative experiments with state-of-the-art multi-focus image fusion methods. The experimental results validate the effectiveness of the proposed method in fusing MVMF images into a high-quality all-in-focus image. Moreover, we also conduct comparative experiments with the conventional focal stack imaging scheme on dynamic scene imaging and salient object detection tasks. The results demonstrate that our proposed computational imaging scheme can achieve all-in-focus imaging for each frame of dynamic scenes, thereby enabling more accurate salient object detection results. Therefore, the proposed scheme is expected to contribute to the development of high-performance imaging systems.

Optical-resolution photoacoustic microscopy (OR-PAM) has demonstrated high-spatial-resolution imaging of optical absorption in biological tissue. To date, most OR-PAM systems rely on mechanical scanning with confocally aligned optical excitation and ultrasonic detection, limiting the wide-field imaging speed of these systems. Although several multifocal OR-PA (MFOR-PA) systems have attempted to address this limitation, they are hindered by the complex design in a constrained physical space. Here, we present a two-dimensional (2D) MFOR-PAM system that utilizes a 2D microlens array and an acoustic ergodic relay. Using a single-element ultrasonic transducer, this system can detect PA signals generated from 400 optical foci in parallel and then raster scan the optical foci patterns to form an MFOR-PAM image. This system improves the imaging resolution of an acoustic ergodic relay system from 220 to 13 μm and enables 400-folds shorter scanning time than that of a conventional OR-PAM system at the same resolution and laser repetition rate. We demonstrated the imaging ability of the system with both in vitro and in vivo experiments.

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.

Multi-scale convolutional neural network for multi-focus image fusion