Simultaneous deep ultraviolet transmission and scattering microscopy for virtual histology.

In recent years, the emergence of a variety of novel optical microscopy techniques has enabled the generation of virtual optical stains of unlabeled tissue specimens, which have the potential to transform existing clinical histopathology workflows. In this work, we present a simultaneous deep ultraviolet transmission and scattering microscopy system that can produce virtual histology images that show concordance to conventional gold-standard histological processing techniques. The results of this work demonstrate the system's diagnostic potential for characterizing unlabeled thin tissue sections and streamlining histological workflows.

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.

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.

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.

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.