KEYWORDS: Brain, Neuroimaging, Photoacoustic tomography, Calcium, Acquisition tracking and pointing, Neurons, Luminescence, Tissue optics, In vivo imaging, Signal detection
Significance: Optical imaging of responses in fluorescently labeled neurons has progressed significantly in recent years. However, there is still a need to monitor neural activities at divergent spatial scales and at depths beyond the optical diffusion limit.Aim: To meet these needs, we aim to develop multiscale photoacoustic tomography (PAT) to image neural activities across spatial scales with a genetically encoded calcium indicator GCaMP.Approach: First, using photoacoustic microscopy, we show that depth-resolved GCaMP signals can be monitored in vivo from a fly brain in response to odor stimulation without depth scanning and even with the cuticle intact. In vivo monitoring of GCaMP signals was also demonstrated in mouse brains. Next, using photoacoustic computed tomography, we imaged neural responses of a mouse brain slice at depths beyond the optical diffusion limit.Results: We provide the first unambiguous demonstration that multiscale PAT can be used to record neural activities in transgenic flies and mice with select neurons expressing GCaMP.Conclusions: Our results indicate that the combination of multiscale PAT and fluorescent neural activity indicators provides a methodology for imaging targeted neurons at various scales.
We report a handheld multi-parametric photoacoustic microscopy system for functional and oxygen-metabolic imaging of the human skin microvasculature. With the high spatiotemporal resolution (3 μm and 10-Hz frame rate), the traverse of single red blood cells through the capillary can be visualized, from which the flow speed can be quantified. Furthermore, the multi-parametric measurement enables comprehensive quantification of the oxygen saturation and release in individual microvessels. The utility of this new technique has been demonstrated by studying the microvascular reactivity in humans.
We report a new multi-parametric photoacoustic microscopy (PAM) system, which enables high-resolution imaging of blood perfusion, oxygenation and flow at 0.2-Hz frame rate over an area of 4.5×4.5 mm2. Extending the laser scanning range by using a cylindrically focused transducer (focus: 50 µm by 4.5 mm), it increases the speed of our previous hybrid-scan system with a weakly focused transducer (focal diameter: 250 µm) by 18-fold without compromising the sensitivity. We have demonstrated the feasibility of this technique in the transmission mode in vivo. Further development of a reflection-mode system will enable real-time cortex-wide imaging of cerebral hemodynamics and metabolism.
Multi-parametric photoacoustic microscopy (PAM) is uniquely capable of quantifying the cerebral hemodynamics and oxygen metabolism at the microscopic level. However, the limited depth of focus of conventional PAM is insufficient to encompass the depth variation of the mouse brain when imaging a large area. For instance, the surface contour of the mouse cortex is dome-shaped and spans several hundred microns along the depth direction. When out of focus, the resolution and sensitivity of PAM quickly degrades. Moreover, quantitative measurements (e.g., blood oxygenation and flow) are no longer accurate with the compromised resolution and sensitivity. Here, we report automated contour-scan multi-parametric PAM, which enables simultaneous imaging of blood perfusion, oxygenation and flow with high resolution and sensitivity over the entire mouse cortex. Different from the traditional contour-scan method that requires three steps (pre-scan, off-line calculation of the contour map, and contour scan), our technique can perform high-resolution wide-field contour scan without the first two steps, thereby significantly reducing the acquisition time. We first tested the feasibility of this technique by imaging a plastic ball coated with black ink. Then, we quantitatively analyzed the influence of out-of-focus on the measurement of blood flow in a vessel-mimicking phantom. Finally, we demonstrated cortex-wide multi-parametric PAM in the live mouse brain with high resolution and sensitivity.
Surgery for rectal cancer is associated with significant side effects including wound infections, incontinence, sexual and bladder dysfunction, and long-term ostomies. Though studies have shown that patients who completely respond to preoperative treatment can safely avoid surgery, nonoperative options remain limited by the poor performance of MRI and endorectal ultrasound after initial therapy. Therefore, new imaging modalities are needed to improve posttreatment tumor assessment and enable the widespread adoption of nonoperative management in rectal cancer. An acoustic resolution photoacoustic microscope (AR-PAM) was constructed with high frequency ultrasonic transducer and near infrared laser. We performed initial phantom, and then imaged ex vivo human colorectal specimens to evaluate different AR-PAM characteristics in each tissue type (normal, untreated tumor, and treated tumor). Our data suggest that photoacoustic imaging can differentiate the distorted vasculature of rectal tumors from normal vascular patterns. However, the vascular distribution of rectal tissue in pathological complete responders showed similar distribution as the normal colorectal tissue; mucosa, submucosa and muscle layer are clearly presented in ultrasound images, while photoacoustic images have revealed that most vasculatures distribute in submucosa. Encouraged by these initial results, we are developed a high-speed scanning (1 second for 20mm B-scan) AR-PAM with laser pulse repetition rate of 1kHz for large field 3D imaging. Lateral resolution of 65μm, axial resolution of 45μm, and 8mm tissue imaging depth can be achieved.
Photoacoustic endoscopy offers in vivo examination of the visceral tissue using endogenous contrast, but its typical B-scan rate is ∼10 Hz, restricted by the speed of the scanning unit and the laser pulse repetition rate. Here, we present a transvaginal fast-scanning optical-resolution photoacoustic endoscope with a 250-Hz B-scan rate over a 3-mm scanning range. Using this modality, we not only illustrated the morphological differences of vasculatures among the human ectocervix, uterine body, and sublingual mucosa but also showed the longitudinal and cross-sectional differences of cervical vasculatures in pregnant women. This technology is promising for screening the visceral pathological changes associated with angiogenesis.
Optical-resolution photoacoustic microscopy (OR-PAM) has demonstrated fast, label-free volumetric imaging of optical-absorption contrast within the quasiballistic regime of photon scattering. However, the limited numerical aperture of the ultrasonic transducer restricts the detectability of the photoacoustic waves, thus resulting in incomplete reconstructed features. To tackle the limited-view problem, we added an oblique illumination beam to the original coaxial optical-acoustic scheme to provide a complementary detection view. The virtual augmentation of the detection view was validated through numerical simulations and tissue-phantom experiments. More importantly, the combination of top and oblique illumination successfully imaged a mouse brain in vivo down to 1 mm in depth, showing detailed brain vasculature. Of special note, it clearly revealed the diving vessels that were long missing in images from original OR-PAM.
In biomedical imaging, all optical techniques face a fundamental trade-off between spatial resolution and tissue penetration. Therefore, obtaining an organelle-level resolution image of a whole organ has remained a challenging and yet appealing scientific pursuit. Over the past decade, optical microscopy assisted by mechanical sectioning or chemical clearing of tissue has been demonstrated as a powerful technique to overcome this dilemma, one of particular use in imaging the neural network. However, this type of techniques needs lengthy special preparation of the tissue specimen, which hinders broad application in life sciences. Here, we propose a new label-free three-dimensional imaging technique, named microtomy-assisted photoacoustic microscopy (mPAM), for potentially imaging all biomolecules with 100% endogenous natural staining in whole organs with high fidelity. We demonstrate the first label-free mPAM, using UV light for label-free histology-like imaging, in whole organs (e.g., mouse brains), most of them formalin-fixed and paraffin- or agarose-embedded for minimal morphological deformation. Furthermore, mPAM with dual wavelength illuminations is also employed to image a mouse brain slice, demonstrating the potential for imaging of multiple biomolecules without staining. With visible light illumination, mPAM also shows its deep tissue imaging capability, which enables less slicing and hence reduces sectioning artifacts. mPAM could potentially provide a new insight for understanding complex biological organs.
We report a photoacoustic thermal flowmetry based on optical-resolution photoacoustic microscopy (OR-PAM) using a single laser source for both thermal tagging and photoacoustic excitation. When an optically absorbing medium is flowing across the optical focal zone of OR-PAM, a small volume of the medium within the optical focus is repeatedly illuminated and heated by a train of laser pulses with a high repetition rate. The average temperature of the heated volume at each laser pulse is indicated by the photoacoustic signal excited by the same laser pulse due to the well-established linear relationship between the Grueneisen coefficient and the local temperature. The thermal dynamics of the heated medium volume, which are closely related to the flow speed, can therefore be measured from the time course of the detected photoacoustic signals. Here, we have developed a lumped mathematical model to describe the time course of the photoacoustic signals as a function of the medium’s flow speed. We conclude that the rising time constant of the photoacoustic signals is linearly dependent on the flow speed. Thus, the flow speed can be quantified by fitting the measured photoacoustic signals using the derived mathematical model. We first performed proof-of-concept experiments using defibrinated bovine blood flowing in a plastic tube. The experiment results have demonstrated that the proposed method has high accuracy (∼±6%) and a wide range of measurable flow speeds. We further validated the method by measuring the blood flow speeds of the microvasculature in a mouse ear in vivo.
Enabling simultaneous high-resolution imaging of the total concentration of hemoglobin (CHb), oxygen saturation of hemoglobin (sO2), and cerebral blood flow (CBF), multiparametric photoacoustic microscopy (PAM) holds the potential to quantify the cerebral metabolic rate of oxygen at the microscopic level. However, its imaging speed has been severely limited by the pulse repetition rate of the dual-wavelength photoacoustic excitation and the scanning mechanism. To address these limitations, we have developed a new generation of multiparametric PAM. Capitalizing on a self-developed high-repetition dual-wavelength pulsed laser and an optical–mechanical hybrid-scan configuration, this innovative technique has achieved an unprecedented A-line rate of 300 kHz, leading to a 20-fold increase in the imaging speed over our previously reported multiparametric PAM that is based on pure mechanical scanning. The performance of the high-speed multiparametric PAM has been examined both in vitro and in vivo. Simultaneous PAM of microvascular CHb, sO2, and CBF in absolute values over a ∼3-mm-diameter brain region of interest can be accomplished within 10 min.
Intravital microscopy techniques have become increasingly important in biomedical research because they can provide unique microscopic views of various biological or disease developmental processes in situ. Here we present an optical-resolution photoacoustic endomicroscopy (OR-PAEM) system that visualizes internal organs with a much finer resolution than conventional acoustic-resolution photoacoustic endoscopy systems. By combining gradient index (GRIN) lens-based optical focusing and ultrasonic ring transducer-based acoustic focusing, we achieved a transverse resolution as fine as ~10 μm at an optical working distance of 6.5 mm. The OR-PAEM system’s high-resolution intravital imaging capability is demonstrated through animal experiments.
The addition of photoacoustic endoscopy to conventional endoscopic ultrasound offers imaging capabilities that may improve diagnosis and clinical care of gastrointestinal tract diseases. In this study, using a 3.8-mm diameter dual-mode photoacoustic and ultrasonic endoscopic probe, we investigated photoacoustic and ultrasonic image features of rabbit esophagi. Specifically, we performed ex vivo imaging of intact rabbit esophagi and correlated the acquired images with histology. Without motion artifact-based limitations, we were able to utilize the full resolving power of the endoscopic device and acquire the first three-dimensional vasculature map of the esophagus and mediastinum, along with coregistered tissue density information. Here, we present the experimental results and discuss potential clinical applications of the technique.
We have successfully developed a fully-sheathed, flexible shaft-based, mechanical scanning photoacoustic endoscopy (PAE) system for imaging the human gastrointestinal tract via the instrument channel of a clinical video endoscope. The endoscopic system uses a single element ultrasonic transducer and flexible shaft-based proximal actuation mechanism, and it has a 2.5 m long and 3.2 mm diameter catheter section, which can be accommodated in the 3.7 mm diameter instrument channel of a clinical video endoscope. Here, we demonstrate the intra-instrument channel workability and in vivo imaging capability of the PAE system.
We report a flexible shaft-based mechanical scanning photoacoustic endoscopy (PAE) system that can be potentially used for imaging the human gastrointestinal tract via the instrument channel of a clinical video endoscope. The development of such a catheter endoscope has been an important challenge to realize the technique’s benefits in clinical settings. We successfully implemented a prototype PAE system that has a 3.2-mm diameter and 2.5-m long catheter section. As the instrument’s flexible shaft and scanning tip are fully encapsulated in a plastic catheter, it easily fits within the 3.7-mm diameter instrument channel of a clinical video endoscope. Here, we demonstrate the intra-instrument channel workability and in vivo animal imaging capability of the PAE system.
KEYWORDS: In vivo imaging, Tumors, Ear, Photoacoustic microscopy, Acoustics, Biomedical optics, Optical scanning, 3D image processing, 3D scanning, Transducers
Combined optical and mechanical scanning (COMS) in optical-resolution photoacoustic microscopy (OR-PAM) has provided five scanning modes with fast imaging speed and wide field of view (FOV). With two-dimensional (2D) galvanometer-based optical scanning, we have achieved a 2 KHz B-scan rate and 50 Hz volumetric-scan rate, which enables real-time tracking of cell activities in vivo. With optical-mechanical hybrid 2D scanning, we are able to image a wide FOV (10×8 mm2) within 150 seconds, which is 20 times faster than the conventional mechanical scan in our second-generation OR-PAM. With three-dimensional mechanical-based contour scanning, we can maintain the optimal signal-to-noise ratio and spatial resolution of OR-PAM while imaging objects with uneven surfaces, which is ideal for fast and quantitative studies of tumors and the brain.
Photoacoustic ophthalmoscopy (PAOM) is a high-resolution in vivo imaging modality that is capable of providing specific optical absorption information for the retina. A high-frequency ultrasonic transducer is one of the key components in PAOM, which is in contact with the eyelid through coupling gel during imaging. The ultrasonic transducer plays a crucial role in determining the image quality affected by parameters such as spatial resolution, signal-to-noise ratio, and field of view. In this paper, we present the results of a systematic study on a high-frequency ultrasonic transducer design for PAOM. The design includes piezoelectric material selection, frequency selection, and the fabrication process. Transducers of various designs were successfully applied for capturing images of biological samples in vivo. The performances of these designs are compared and evaluated.
We have developed a new photoacoustic endoscopic probe specifically designed for human urogenital imaging. The
endoscopic probe uses a parabolic mirror-based mechanical scanning mechanism, providing an angular field of view of 270°. And it has a rigid, dome shaped end section for smooth cavity introduction. Here we introduce the new
endoscope’s design and imaging principle, and present experimental results validating its in vivo imaging ability.
We performed a photoacoustic endoscopic imaging study of melanoma tumor growth in a nude rat in vivo. After
inducing the tumor at the colorectal wall of the animal, we monitored the tumor development in situ by using a
photoacoustic endoscopic system. This paper introduces our experimental method for tumor inoculation and presents
imaging results showing the morphological changes of the blood vasculature near the tumor region according to the
tumor progress. Our study could provide insights for future studies on tumor development in small animals.
Like ultrasound endoscopy, photoacoustic endoscopy (PAE) could become a valuable addition to clinical practice due
to its deep imaging capability. Results from our recent in vivo transesophageal endoscopic imaging study on rabbits
demonstrate the technique’s capability to image major organs in the mediastinal region, such as the lung, trachea, and
cardiovascular systems. Here, we present various features from photoacoustic images from the mediastinal region of
several rabbits and discuss possible clinical contributions of this technique and directions of future technology
development.
Many diseases involve changes in the biomechanical properties of tissue, and there is a close correlation between tissue elasticity and pathology. We report on the development of a phase-resolved acoustic radiation force optical coherence elastography method (ARF-OCE) to evaluate the elastic properties of tissue. This method utilizes chirped acoustic radiation force to produce excitation along the sample's axial direction, and it uses phase-resolved optical coherence tomography (OCT) to measure the vibration of the sample. Under 500-Hz square wave modulated ARF signal excitation, phase change maps of tissue mimicking phantoms are generated by the ARF-OCE method, and the resulting Young's modulus ratio is correlated with a standard compression test. The results verify that this technique could efficiently measure sample elastic properties accurately and quantitatively. Furthermore, a three-dimensional ARF-OCE image of the human atherosclerotic coronary artery is obtained. The result indicates that our dynamic phase-resolved ARF-OCE method can delineate tissues with different mechanical properties.
In order to image noninvasively cell nuclei in vivo without staining, we have developed ultraviolet photoacoustic microscopy (UV-PAM), in which ultraviolet light excites nucleic acids in cell nuclei to produce photoacoustic waves. Equipped with a tunable laser system, the UV-PAM was applied to in vivo imaging of cell nuclei in small animals. We found that 250 nm was the optimal wavelength for in vivo photoacoustic imaging of cell nuclei. The optimal wavelength enables UV-PAM to image cell nuclei using as little as 2 nJ laser pulse energy. Besides the optimal wavelength, application of a wavelength between 245 and 275 nm can produce in vivo images of cell nuclei with specific, positive, and high optical contrast.
We have developed a 2.5-mm outer diameter photoacoustic endoscopic mini-probe to use in the instrument channel
(typically 2.8 or 3.7 mm in diameter) of standard video endoscopes. To achieve adequate signal sensitivity, we
fabricated a focused ultrasonic transducer using a highly-sensitive PMN-PT piezoelectric material. We quantified the
PMN-PT transducer's operating parameters and validated the
mini-probe's endoscopic imaging capability through an ex
vivo imaging experiment on a rat colon.
KEYWORDS: In vivo imaging, Ultraviolet radiation, Photoacoustic spectroscopy, Skin, Photoacoustic microscopy, Ultrasonography, Ear, Signal to noise ratio, Cancer, Ultrasonics
Ultraviolet photoacoustic microscopy (UVPAM) can image cell nuclei in vivo with high contrast and resolution
noninvasively without staining. Here, we used UV light at wavelengths of 210-310 nm for excitation of DNA and RNA
to produce photoacoustic waves. We applied the UVPAM to in vivo imaging of cell nuclei in mouse skin, and obtained
UVPAM images of the unstained cell nuclei at wavelengths of 245-282 nm as ultrasound gel was used for acoustic
coupling. The largest ratio of contrast to noise was found for the images of cell nuclei at a 250 nm wavelength.
Submicron-resolution photoacoustic microscopy (PAM) currently exists only in transmission mode, due to the technical difficulties of combining high numerical-aperture (NA) optical illumination with high NA acoustic detection. The lateral resolution of reflection-mode PAM has not reached <2 μm in the visible light range. Here we develop the first reflection-mode submicron-resolution PAM system with a new compact design. By using a parabolic mirror to focus and reflect the photoacoustic waves, sufficient signals were collected for good sensitivity without distorting the optical focusing. By imaging nanospheres and a resolution test chart, the lateral resolution was measured to be ∼ 0.5 μm with an optical wavelength of 532 nm, an optical NA of 0.63. The axial resolution was measured at 15 μm. Here the axial resolution was measured by a different experiment with the lateral resolution measurement. But we didn't describe the details of axial resolution measurement due to space limit. The maximum penetration was measured at ∼ 0.42 mm in optical-scattering soft tissue. As a comparison, both the submicron-resolution PAM and a 2.4 μm-resolution PAM were used to image a mouse ear in vivo with the same optical wavelength and similar pulse energy. Capillaries were resolved better by the submicron-resolution PAM. Therefore, the submicron-resolution PAM is suitable for in vivo high-resolution imaging, or even subcellular imaging, of optical absorption.
Photoacoustic endoscopy (PAE) provides unique functional information with high spatial resolution at super depths.
The provision of functional information is predicated on its strong spectroscopic imaging ability, and its deep imaging
capability is derived from its ultrasonic detection of diffused photon absorption. To accurately image functional
physiological parameters, it is necessary to rapidly alternate laser pulses of sufficient energy and different wavelengths.
In this study, we developed peripheral optical systems for PAE based on two identical pulsed-laser systems to achieve
the fast laser wavelength switching that is essential for accurate functional imaging. Each laser system was comprised of
a tunable dye laser pumped by a solid-state, diode-pumped Nd:YLF laser. Both systems deliver adequate energy at the
scanning head of the endoscope for imaging biological tissue in the optically diffusive regime (~0.3-0.6 mJ per pulse
with a repetition rate of ~1 kHz). In this paper, we introduce the employed laser systems and design of the light delivery
optics, and present results from an ex vivo animal imaging experiment that validates the system's multi-wavelength
functional imaging capability.
Photoacoustic microscopy (PAM) offers label-free, optical absorption contrast. A high-speed,
high-resolution PAM system in an inverted microscope configuration with a laser pulse repetition
rate of 100,000 Hz and a stationary ultrasonic transducer was built. Four-dimensional in vivo
imaging of microcirculation in mouse skin was achieved at 18 three-dimensional volumes per
second with repeated two-dimensional raster scans of 100 by 50 points. The corresponding twodimensional
B-scan (50 A-lines) frame rate was 1800 Hz, and the one-dimensional A-scan rate
was 90,000 Hz. The lateral resolution is 0.23±0.03 μm for Au nano-wire imaging, which is 2.0
times below the diffraction limit.
We have successfully implemented a focused ultrasonic transducer for photoacoustic endoscopy. The photoacoustic
endoscopic probe's ultrasound transducer determines the lateral resolution of the system. By using a focused ultrasonic
transducer, we significantly improved the endoscope's spatial resolution and signal-to-noise ratio. This paper describes
the technical details of the ultrasonic transducer incorporated into the photoacoustic endoscopic probe and the
experimental results from which the transducer's resolution is quantified and the image improvement is validated.
We investigate the saturation effect, which describes the violation of the linearity between the measured photoacoustic amplitude and the object's optical absorption coefficient in functional photoacoustic imaging when the optical absorption in the object increases. We model the optical energy deposition and photoacoustic signal generation and detection in a semi-infinite optical absorbing object. Experiments are carried out by measuring photoacoustic signals generated from an ink-filled plastic tube. The saturation effect is studied by varying the optical absorption coefficient in the model and the ink concentration in the photoacoustic experiments. By changing the center frequency of the ultrasonic detector, the requirement to minimize the saturation effect in functional photoacoustic imaging is established.
In this study, we further developed our photoacoustic endoscopic system to produce three-dimensional images of
internal organs by performing pullback C-scans. Employing the side-scanning photoacoustic endoscopic probe
discussed in the Optical Society of America's journal Optics Letters, we could acquire successive B-scan images by
pulling back the probe with a motorized linear stage. We demonstrate the endoscopic system's volumetric imaging
ability through imaging of a metal wire phantom and an in situ rat rectum.
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