Use of Photoacoustic Imaging to Study the Effects of Anemia on Placental Oxygen Saturation in Normoxic and Hypoxic Conditions.

We aimed to evaluate fetal and placental oxygen saturation (sO2) in anemic and non-anemic pregnant rats throughout gestation using photoacoustic imaging (PAI). Female Sprague-Dawley rats were fed an iron-restricted or iron-replete diet before and during pregnancy. On gestational days 13, 18, and 21, PAI was coupled with high resolution ultrasound to measure oxygenation of the fetus, whole placenta, mesometrial triangle, as well as the maternal and fetal faces of the placenta. PAI was performed in 3D, which allowed sO2 to be measured within an entire region, as well as in 2D, which enabled sO2 measurements in response to a hypoxic event in real time. Both 3D and 2D PAI were performed at varying levels of FiO2 (fraction of inspired oxygen). Iron restriction caused anemia in dams and fetuses, a reduction in fetal body weight, and an increase in placental weight, but overall had minimal effects on sO2. Reductions in FiO2 caused corresponding reductions in sO2 which correlated to the severity of the hypoxic challenge. Regional differences in sO2 were evident within the placenta and between the placenta and fetus. In conclusion, PAI enables non-invasive measurement of sO2 both rapidly and with a high degree of sensitivity. The lack of overt changes in sO2 levels between control and anemic fetuses may suggest reduced oxygen extraction and utilization in the latter group, which could be attributed to compensatory changes in growth and developmental trajectories.

Deep learning-enabled realistic virtual histology with ultraviolet photoacoustic remote sensing microscopy.

The goal of oncologic surgeries is complete tumor resection, yet positive margins are frequently found postoperatively using gold standard H&E-stained histology methods. Frozen section analysis is sometimes performed for rapid intraoperative margin evaluation, albeit with known inaccuracies. Here, we introduce a label-free histological imaging method based on an ultraviolet photoacoustic remote sensing and scattering microscope, combined with unsupervised deep learning using a cycle-consistent generative adversarial network for realistic virtual staining. Unstained tissues are scanned at rates of up to 7 mins/cm2, at resolution equivalent to 400x digital histopathology. Quantitative validation suggests strong concordance with conventional histology in benign and malignant prostate and breast tissues. In diagnostic utility studies we demonstrate a mean sensitivity and specificity of 0.96 and 0.91 in breast specimens, and respectively 0.87 and 0.94 in prostate specimens. We also find virtual stain quality is preferred (P = 0.03) compared to frozen section analysis in a blinded survey of pathologists.

Rapid ultraviolet photoacoustic remote sensing microscopy using voice-coil stage scanning.

There is an unmet need for fast virtual histology technologies that exhibit histological realism and can scan large sections of fresh tissue within intraoperative time-frames. Ultraviolet photoacoustic remote sensing microscopy (UV-PARS) is an emerging imaging modality capable of producing virtual histology images that show good concordance to conventional histology stains. However, a UV-PARS scanning system that can perform rapid intraoperative imaging over mm-scale fields-of-view at fine resolution (<500 nm) has yet to be demonstrated. In this work, we present a UV-PARS system which utilizes voice-coil stage scanning to demonstrate finely resolved images for 2×2 mm2 areas at 500 nm sampling resolution in 1.33 minutes and coarsely resolved images for 4×4 mm2 areas at 900 nm sampling resolution in 2.5 minutes. The results of this work demonstrate the speed and resolution capabilities of the UV-PARS voice-coil system and further develop the potential for UV-PARS microscopy to be employed in a clinical setting.

High-Performance Electrode-Post CMUTs: Fabrication Details and Best Practices.

Capacitive micromachined ultrasound transducers (CMUTs) have been investigated for over 25 years due to their promise for mass manufacturing and electronic co-integration. Previously, CMUTs were fabricated with many small membranes comprising a single transducer element. This, however, resulted in suboptimal electromechanical efficiency and transmit performance, such that resulting devices were not necessarily competitive with piezoelectric transducers. Moreover, many previous CMUT devices were subject to dielectric charging and operational hysteresis that limited long-term reliability. Recently, we demonstrated a CMUT architecture using a single long rectangular membrane per transducer element and novel electrode-post (EP) structures. This architecture not only offers long-term reliability, but also provides performance advantages over previously published CMUT and piezoelectric arrays. The purpose of this article is to highlight these performance advantages and provide details of the fabrication process, including the best practices to avoid common pitfalls. The objective is to provide sufficient detail to inspire a new generation of microfabricated transducers, which could lead to performance gains of future ultrasound systems.

Investigating mechanisms of laser pulse-induced reflectivity modulations in photoacoustic remote sensing with a 10 million frames-per-second camera.

Photoacoustic remote sensing has been recently developed as an all-optical imaging modality capable of imaging a variety of endogenous contrast agents label-free. Initially predicted laser pulse-induced refractive index perturbation-based interrogation beam reflectivity modulations have been found to be orders of magnitude smaller than those typically observed experimentally. In this report we utilize a 10 million frames-per-second camera to further investigate these predicted reflectivity modulations, while also exploring other potential mechanisms of laser pulse-induced reflectivity modulations. Laser-induced motion is demonstrated both laterally for gold wires suspended and submerged in air and water, respectively, and carbon fibers submerged in water, and axial motion is observed in gold wires submerged in a depth gradient of intralipid solution. This laser-induced sample motion is anticipated to cause reflectivity modulations local to the interrogation beam profile in microscopy set-ups. Non-motion-based maximum intensity modulations of 3% are also observed in gold wires submerged in water, indicating the presence of the originally predicted reflectivity modulations. Overall, these observations are important as they provide a widefield view of laser-pulse interactions unavailable in previous point scanning-based photoacoustic remote sensing microscopy configurations, where observed mechanisms occur on time-scales orders of magnitude faster than equivalent field of view point scanning capabilities.

Outperforming piezoelectric ultrasonics with high-reliability single-membrane CMUT array elements.

It has long been hypothesized that capacitive micromachined ultrasound transducers (CMUTs) could potentially outperform piezoelectric technologies. However, challenges with dielectric charging, operational hysteresis, and transmit sensitivity have stood as obstacles to these performance outcomes. In this paper, we introduce key architectural features to enable high-reliability CMUTs with enhanced performance. Typically, a CMUT element in an array is designed with an ensemble of smaller membranes oscillating together to transmit or detect ultrasound waves. However, this approach can lead to unreliable behavior and suboptimal transmit performance if these smaller membranes oscillate out of phase or collapse at different voltages. In this work, we designed CMUT array elements composed of a single long rectangular membrane, with the aim of improving the output pressure and electromechanical efficiency. We compare the performance of three different modifications of this architecture: traditional contiguous dielectric, isolated isolation post (IIP), and insulated electrode-post (EP) CMUTs. EPs were designed to improve performance while also imparting robustness to charging and minimization of hysteresis. To fabricate these devices, a wafer-bonding process was developed with near-100% bonding yield. EP CMUT elements achieved electromechanical efficiency values as high as 0.95, higher than values reported with either piezoelectric transducers or previous CMUT architectures. Moreover, all investigated CMUT architectures exhibited transmit efficiency 2-3 times greater than published CMUT or piezoelectric transducer elements in the 1.5-2.0 MHz range. The EP and IIP CMUTs demonstrated considerable charging robustness, demonstrating minimal charging over 500,000 collapse-snap-back actuation cycles while also mitigating hysteresis. Our proposed approach offers significant promise for future ultrasonic applications.

Fast hybrid optomechanical scanning photoacoustic remote sensing microscopy for virtual histology.

A rapid scanning microscopy method for hematoxylin and eosin (H&E) like images is sought after for interoperative diagnosis of solid tumor margins. The rapid observation and diagnosis of histological samples can greatly lower surgical risk and improve patient outcomes from solid tumor resection surgeries. Photoacoustic remote sensing (PARS) has recently been demonstrated to provide images of virtual H&E stains with excellent concordance with true H&E staining of formalin-fixed, paraffin embedded tissues. By using PARS with constant velocity and 1D galvanometer mirror scanning we acquire large virtual H&E images (10mm x 5mm) of prostate tissue in less than 3.5 minutes without staining, and over two orders of magnitude faster data acquisition than the current PARS imaging speed.

Virtual histopathology with ultraviolet scattering and photoacoustic remote sensing microscopy.

Realistic label-free virtual histopathology has been a long sought-after goal not yet achieved with current methods. Here, we introduce high-resolution hematoxylin and eosin (H&E)-like virtual histology of unstained human breast lumpectomy specimen sections using ultraviolet scattering-augmented photoacoustic remote sensing microscopy. Together with a colormap-matching algorithm based on blind stain separation from a reference true H&E image, we are able to produce virtual H&E images of unstained tissues with close concordance to true H&E-stained sections, with promising diagnostic utility.

Multimodal 3D photoacoustic remote sensing and confocal fluorescence microscopy imaging.

SIGNIFICANCE: Complementary absorption and fluorescence contrast could prove useful for a wide range of biomedical applications. However, current absorption-based photoacoustic microscopy systems require the ultrasound transducers to physically touch the samples, thereby increasing contamination and limiting strong optical focusing in reflection mode. AIM: We sought to develop an all-optical system for imaging cells and tissues using the three combined imaging modalities: photoacoustic remote sensing (PARS), epifluorescence, and confocal laser scanning microscopy (CLSM). APPROACH: A PARS subsystem with ultraviolet excitation was used to obtain label-free absorption-contrast images of nucleic acids in ex vivo tissue samples. Co-integrated epifluorescence and CLSM subsystems were used to verify the 2D and 3D nuclei distribution. RESULTS: Complementary absorption and fluorescence contrast were demonstrated in phantom imaging experiments and subsequent cell and tissue imaging experiments. Lateral and axial resolution of ultraviolet-PARS (UV-PARS) is shown to be 0.39 and 1.6 µm, respectively, with 266-nm light. CLSM lateral and axial resolution was measured as 0.97 and 2.0 µm, respectively. This resolution is sufficient to image individual cell layers with fine optical sectioning. UV-PARS images of cell nuclei are validated in thick tissue using CLSM. CONCLUSIONS: Multimodal absorption and fluorescence contrast are obtained with a non-contact all-optical microscopy system for the first time and utilized to obtain images of cells and tissues with subcellular resolution.

F-mode ultraviolet photoacoustic remote sensing for label-free virtual H&E histopathology using a single excitation wavelength.

Photoacoustic remote sensing (PARS) is a novel all-optical imaging modality that allows for non-contact detection of initial photoacoustic pressures. Using 266-nm excitation pulses, ultraviolet PARS (UV-PARS) has previously demonstrated imaging contrast for cell nuclei in histological samples with <400nm resolution. In prior PARS-based imaging schemes, the signal amplitude at an interrogation point was determined by the maximum deflection from the DC scattering signal in response to a pulsed excitation. This method, however, does not take into consideration additional information encoded in the frequency domain of the recorded PARS signals. Here, we present a frequency domain technique called F-mode PARS that can be used to generate images with nuclear and cytoplasmic enhanced contrast, enabling label-free virtual hematoxylin-and-eosin-like microscopy, using only a single excitation wavelength. With F-mode processing, we have been able to demonstrate contrast-to-noise ratios of up to 38 dB between cell nuclei and surrounding cytoplasm, which represents up to a 25-dB improvement over previous implementations of UV-PARS systems.

Virtual hematoxylin and eosin histopathology using simultaneous photoacoustic remote sensing and scattering microscopy.

Hematoxylin and eosin (H&E) staining is the gold standard for most histopathological diagnostics but requires lengthy processing times not suitable for point-of-care diagnosis. Here we demonstrate a 266-nm excitation ultraviolet photoacoustic remote sensing (UV-PARS) and 1310-nm microscopy system capable of virtual H&E 3D imaging of tissues. Virtual hematoxylin staining of nuclei is achieved with UV-PARS, while virtual eosin staining is achieved using the already implemented interrogation laser from UV-PARS for scattering contrast. We demonstrate the capabilities of this dual-contrast system for en-face planar and depth-resolved imaging of human tissue samples exhibiting high concordance with H&E staining procedures and confocal fluorescence microscopy. To our knowledge, this is the first microscopy approach capable of depth-resolved imaging of unstained thick tissues with virtual H&E contrast.

Dual-Frequency CMUT Arrays for Multiband Ultrasound Imaging Applications.

Dual-frequency capacitive micromachined ultrasonic transducers (CMUTs) are introduced for multiscale imaging applications, where a single array transducer can be used for both deep low-resolution imaging and shallow high-resolution imaging. These transducers consist of low- and high-frequency membranes interlaced within each subarray element. They are fabricated using a modified sacrificial release process. Successful performance is demonstrated using wafer-level vibrometer testing, as well as acoustic testing on wirebonded dies consisting of arrays of 2- and 9-MHz elements of up to 64 elements for each subarray. The arrays are demonstrated to provide multiscale, multiresolution imaging using wire phantoms and can span frequencies from 2 MHz up to as high as 17 MHz. Peak transmit sensitivities of 27 and 7.5 kPa/V are achieved with the low- and high-frequency subarrays, respectively. At 16-mm imaging depth, lateral spatial resolution achieved is 0.84 and 0.33 mm for low- and high-frequency subarrays, respectively. The signal-to-noise ratio of the low-frequency subarray is significantly higher for deep targets compared to the high-frequency subarray. The array achieves multiband imaging capabilities difficult to achieve with current transducer technologies and may have applications to multipurpose probes and novel contrast agent imaging schemes.

Enrichment and ratiometric detection of circulating tumor cells using PSMA- and folate receptor-targeted magnetic and surface-enhanced Raman scattering nanoparticles.

The presence of circulating tumor cells (CTCs) in a patient's bloodstream is a hallmark of metastatic cancer. The detection and analysis of CTCs is a promising diagnostic and prognostic strategy as they may carry useful genetic information from their derived primary tumor, and the enumeration of CTCs in the bloodstream has been known to scale with disease progression. However, the detection of CTCs is a highly challenging task owing to their sparse numbers in a background of billions of background blood cells. To effectively utilize CTCs, there is a need for an assay that can detect CTCs with high specificity and can locally enrich CTCs from a liquid biopsy. We demonstrate a versatile methodology that addresses these needs by utilizing a combination of nanoparticles. Enrichment is achieved using targeted magnetic nanoparticles and high specificity detection is achieved using a ratiometric detection approach utilizing multiplexed targeted and non-targeted surface-enhanced Raman Scattering Nanoparticles (SERS-NPs). We demonstrate this approach with model prostate and cervical circulating tumor cells and show the ex vivo utility of our methodology for the detection of PSMA or folate receptor over-expressing CTCs. Our approach allows for the mitigation of interference caused by the non-specific uptake of nanoparticles by other cells present in the bloodstream and our results from magnetically trapped CTCs reveal over a 2000% increase in targeted SERS-NP signal over non-specifically bound SERS-NPs.

Multimodal imaging with spectral-domain optical coherence tomography and photoacoustic remote sensing microscopy.

We develop a multimodal imaging platform, combining depth-resolved scattering contrast from spectral-domain optical coherence tomography (SD-OCT) with complementary, non-contact absorption contrast using photoacoustic remote sensing (PARS) microscopy. The system provides a widefield OCT mode using a telecentric scan lens, and a high-resolution, dual-contrast mode using a 0.26 numerical aperture apochromatic objective. An interlaced acquisition approach is used to achieve simultaneous, co-registered imaging. The SD-OCT modality provides a 9.7 µm axial resolution. Comprehensive in vivo imaging of a nude mouse ear is demonstrated, with the SD-OCT scattering intensity revealing dermal morphology, and PARS microscopy providing a map of microvasculature.

Label-free lipid contrast imaging using non-contact near-infrared photoacoustic remote sensing microscopy.

Histopathology of lipid-rich tissues is often a difficult endeavor, owing to the limited tissue processing workflows that can appropriately preserve tissue while keeping fatty deposits intact. Here, we present the first usage of near-infrared (NIR) photoacoustic remote sensing (PARS) to achieve imaging contrast from lipids without the need for exogenous stains or labels. In our system, the facile production of 1225 nm excitation pulses is achieved by the stimulated Raman scattering of a 1064 nm source propagating through an optical fiber. PARS-based detection is achieved by monitoring the change in the scattering profile of a co-aligned 1550 nm continuous-wave interrogation beam in response to absorption of the 1225 nm light by lipids. Our non-contact, reflection-mode approach can achieve a FWHM resolution of up to 0.96 µm and signal-to-noise ratios as high as 45 dB from carbon fibers and 9.7 dB from a lipid phantom. NIR-PARS offers a promising approach to image lipid-rich samples with a simplified workflow.

Real-time functional photoacoustic remote sensing microscopy.

A fiber-tetherable non-contact photoacoustic remote sensing microscopy system capable of multiplex functional imaging is reported. By utilizing stimulated Raman scattering within an over-pumped polarization-maintaining single-mode optical fiber, rapid pulse-to-pulse switching (500 kHz) of excitation spectral content is demonstrated and utilized as a photoacoustic excitation source. These rapid acquisitions aim to reduce motion artifacts and facilitate high frame rates appropriate for real-time feedback to users. The system is characterized by estimating blood oxygen saturation in blood-flow phantoms and within a mouse ear in vivo.

Ultraviolet photoacoustic remote sensing microscopy.

Traditional histopathology involves fixing, sectioning, and staining protocols that are time consuming and subject to staining variability. In this Letter, we present ultraviolet photoacoustic remote sensing microscopy, capable of imaging cell nuclei without the need for exogenous stains or labelling. Our reflection mode approach is non-contact and has the potential to provide useful histological information without laborious sample preparation steps. Tumor cell cultures and excised tissue samples were imaged with the 0.7 µm resolution and signal-to-noise ratios as high as 53 dB, with close agreement to traditional hematoxylin and eosin staining.

A Nonlinear Lumped Equivalent Circuit Model for a Single Uncollapsed Square CMUT Cell.

An accurate nonlinear lumped equivalent circuit model is used for modeling of capacitive micromachined ultrasonic transducers (CMUTs). Finite-element analysis (FEA) is a powerful tool for the analysis of CMUT arrays with a small number of cells while with the harmonic balance (HB) analysis of the lumped equivalent circuit model, the entire behavior of a large-scale arbitrary CMUT array can be modeled in a very short time. Recently, an accurate nonlinear equivalent circuit model for uncollapsed single circular CMUT cells has been developed. However, the need for an accurate large-signal circuit model for CMUT cells with square membranes motivated us to produce a new nonlinear large-signal equivalent circuit model for uncollapsed CMUT cells. In this paper, using analytical calculations and FEA as the tuning tool, a precise large signal equivalent circuit model of square CMUT dynamics was developed and showed excellent agreement with finite-element modeling (FEM) results. Then, different CMUT single cells with square and circular membranes were fabricated using a standard sacrificial release process. Model predictions of resonance frequencies and displacements closely matched experimental vibrometer measurements. The framework presented here may prove valuable for future design and modeling of CMUT arrays with square membranes for ultrasound imaging and therapy applications.

In vivo photoacoustic difference-spectra imaging of bacteria using photoswitchable chromoproteins.

Photoacoustic (PA) imaging offers great promise for deep molecular imaging of optical reporters but has difficulties in imaging multiple molecular probes simultaneously in a strong blood background. Photoswitchable chromoproteins like BphP1 have recently allowed for sensitive PA detection by reducing high-blood background signals but lack multiplexing capabilities. We propose a method known as difference-spectra demixing for multiplexing multiple photoswitchable chromoproteins and introduce a second photoswitchable chromoprotein, sGPC2. sGPC2 has a far-red and orange state with peaks at 700 and 630 nm, respectively. It is roughly one-tenth the size of BphP1 and photoswitches four times as fast (2.4% per mJ / cm2). We simultaneously image Escherichia coli expressing sGPC2 and BphP1 injected in mice in vivo. Difference-spectra demixing obtained successful multiplexed images of photoswitchable molecular probes, resulting in a 21.6-fold increase in contrast-to-noise ratio in vivo over traditional PA imaging and an 8% to 40% reduction in erroneously demixed signals in comparison with traditional spectral demixing. PA imaging and characterization were conducted using a custom-built photoswitching PA imaging system.

Coherence-gated photoacoustic remote sensing microscopy.

Photoacoustic remote sensing microscopy (PARS) represents a new paradigm within the optical imaging community by providing high sensitivity (>50 dB in vivo) non-contact optical absorption contrast in scattering media with a reflection-mode configuration. Unlike contact-based photoacoustic modalities which can acquire complete A-scans with a single excitation pulse due to slow acoustic propagation facilitating the use of time-gated collection of returning acoustic signals, PARS provides depth resolution only through optical sectioning. Here we introduce a new approach for providing coherence-gated depth-resolved PARS imaging using a difference between pulsed-interrogation optical coherence tomography scan-lines with and without excitation pulses. Proposed methods are validated using simulations which account for pulsed-laser induced initial-pressures and accompanying refractive index changes. The changes in refractive index are shown to be proportional to optical absorption. It is demonstrated that to achieve optimal image quality, several key parameters must be selected including interrogation pulse duration and delay. The proposed approach offers the promise of non-contact depth-resolved optical absorption contrast at optical-resolution scales and may complement the scattering contrast offered by optical coherence tomography.