Wide‐field monitoring and real‐time local recording of microvascular networks on small animals with a dual‐raster‐scanned photoacoustic microscope

Photoacoustic microscopy (PAM) provides a new method for the imaging of small‐animals with high‐contrast and deep‐penetration. However, the established PAM systems have suffered from a limited field‐of‐view or imaging speed, which are difficult to both monitor wide‐field activity of organ and record real‐time change of local tissue. Here, we reported a dual‐raster‐scanned photoacoustic microscope (DRS‐PAM) that integrates a two‐dimensional motorized translation stage for large field‐of‐view imaging and a two‐axis fast galvanometer scanner for real‐time imaging. The DRS‐PAM provides a flexible transition from wide‐field monitoring the vasculature of organs to real‐time imaging of local dynamics. To test the performance of DRS‐PAM, clear characterization of angiogenesis and functional detail was illustrated, hemodynamic activities of vasculature in cerebral cortex of a mouse were investigated. Furthermore, response of tumor to treatment were successfully monitored during treatment. The experimental results demonstrate the DRS‐PAM holds the great potential for biomedical research of basic biology.     

Front Cover: Wide‐field monitoring and real‐time local recording of microvascular networks on small animals with a dual‐raster‐scanned photoacoustic microscope

A dual‐raster‐scanned photoacoustic microscope (DRS‐PAM) was reported, which integrates a two‐dimensional motorized translation stage for large field‐of‐view imaging and a two‐axis fast galvanometer scanner for real‐time imaging. The DRS‐PAM provides a flexible transition from wide‐field monitoring the vasculature of organs to real‐time imaging of local dynamics. Further details can be found in the article by Fei Yang, Zhiyang Wang, Wuyu Zhang, et al. ( e202000022 ).

     

Miniature probe for dual‐modality photoacoustic microscopy and white‐light microscopy for image guidance: A prototype toward an endoscope

In this study, a novel photoacoustic microscopy (PAM) probe integrating white‐light microscopy (WLM) modality that provides guidance for PAM imaging and complementary information is implemented. One single core of an imaging fiber bundle is employed to deliver a pulsed laser for photoacoustic excitation for PAM mode, which provides high resolution with deep penetration. Meanwhile, for WLM mode, the imaging fiber bundle is used to transmit two‐dimensional superficial images. Lateral resolution of 7.2 μm for PAM is achieved. Since miniature components are used, the probe diameter is only 1.7 mm. Imaging of phantom and animals in vivo is conducted to show the imaging capability of the probe. The probe has several advantages by introducing the WLM mode, such as being able to conveniently identify regions of interest and align the focus for PAM mode. The prototype of an endoscope shows potential to facilitate clinical photoacoustic endoscopic applications.     

A new deep learning method for image deblurring in optical microscopic systems

Deconvolution is the most commonly used image processing method in optical imaging systems to remove the blur caused by the point‐spread function (PSF). While this method has been successful in deblurring, it suffers from several disadvantages, such as slow processing time due to multiple iterations required to deblur and suboptimal in cases where the experimental operator chosen to represent PSF is not optimal. In this paper, we present a deep‐learning‐based deblurring method that is fast and applicable to optical microscopic imaging systems. We tested the robustness of proposed deblurring method on the publicly available data, simulated data and experimental data (including 2D optical microscopic data and 3D photoacoustic microscopic data), which all showed much improved deblurred results compared to deconvolution. We compared our results against several existing deconvolution methods. Our results are better than conventional techniques and do not require multiple iterations or pre‐determined experimental operator. Our method has several advantages including simple operation, short time to compute, good deblur results and wide application in all types of optical microscopic imaging systems. The deep learning approach opens up a new path for deblurring and can be applied in various biomedical imaging fields.     

Tissue clearing‐based method for unobstructed three‐dimensional imaging of mouse penis with subcellular resolution

Although mice are widely used to elucidate factors contributing to penile disorders and develop treatment options, quantification of tissue changes upon intervention is either limited to minuscule tissue volume (histology) or acquired with limited spatial resolution (MRI/CT). Thus, imaging method suitable for expeditious acquisition of the entire mouse penis with subcellular resolution is described that relies on both aqueous‐ (clear, unobstructed brain imaging cocktails and computational analysis) and solvent‐based (fluorescence‐preserving capability imaging of solvent‐cleared organs) tissue optical clearing (TOC). The combined TOC approach allows to image mouse penis innervation and vasculature with unprecedented detail and, for the first time, reveals the three‐dimensional structure of murine penis fibrocartilage.     

Biophotonic technologies for assessment of breast tumor surgical margins—A review

Breast conserving surgery (BCS) offering similar surgical outcomes as mastectomy while retaining breast cosmesis is becoming increasingly popular for the management of early stage breast cancers. However, its association with reoperation rates of 20% to 40% following incomplete tumor removal warrants the need for a fast and accurate intraoperative surgical margin assessment tool that offers cellular, structural and molecular information of the whole specimen surface to a clinically relevant depth. Biophotonic technologies are evolving to qualify as such an intraoperative tool for clinical assessment of breast cancer surgical margins at the microscopic and macroscopic scale. Herein, we review the current research in the application of biophotonic technologies such as photoacoustic imaging, Raman spectroscopy, multimodal multiphoton imaging, diffuse optical imaging and fluorescence imaging using medically approved dyes for breast cancer detection and/or tumor subtype differentiation toward intraoperative assessment of surgical margins in BCS specimens, and possible challenges in their route to clinical translation.     

Structured illumination imaging with quasi‐periodic patterns

Structured illumination microscopy (SIM) is a well‐established method for optical sectioning and super‐resolution. The core of structured illumination is using a periodic pattern to excite image signals. This work reports a method for estimating minor pattern distortions from the raw image data and correcting these distortions during SIM image processing. The method was tested with both simulated and experimental image data from two‐photon Bessel light‐sheet SIM. The results proves the method is effective in challenging situations, where strong scattering background exists, signal‐to‐noise ratio (SNR) is low and the sample structure is sparse. Experimental results demonstrate restoring synaptic structures in deep brain tissue, despite the presence of strong light scattering and tissue‐induced SIM pattern distortion.     

Non‐invasive sentinel lymph node mapping and needle guidance using clinical handheld photoacoustic imaging system in small animal

Translating photoacoustic imaging (PAI) into clinical setup is a challenge. Handheld clinical real‐time PAI systems are not common. In this work, we report an integrated photoacoustic (PA) and clinical ultrasound imaging system by combining light delivery with the ultrasound probe for sentinel lymph node imaging and needle guidance in small animal. The open access clinical ultrasound platform allows seamless integration of PAI resulting in the development of handheld real‐time PAI probe. Both methylene blue and indocyanine green were used for mapping the sentinel lymph node using 675 and 690 nm wavelength illuminations, respectively. Additionally, needle guidance with combined ultrasound and PAI was demonstrated using this imaging system. Up to 1.5 cm imaging depth was observed with a 10 Hz laser at an imaging frame rate of 5 frames per second, which is sufficient for future translation into human sentinel lymph node imaging and needle guidance for fine needle aspiration biopsy.      

Fast and sensitive delineation of brain tumor with clinically compatible moxifloxacin labeling and confocal microscopy

Delineation of brain tumor margins during surgery is critical to maximize tumor removal while preserving normal brain tissue to obtain optimal clinical outcomes. Although various imaging methods have been developed, they have limitations to be used in clinical practice. We developed a high‐speed cellular imaging method by using clinically compatible moxifloxacin and confocal microscopy for sensitive brain tumor detection and delineation. Moxifloxacin is a Food and Drug Administration (FDA) approved antibiotic and was used as a cell labeling agent through topical administration. Its strong fluorescence at short visible excitation wavelengths allowed video‐rate cellular imaging. Moxifloxacin‐based confocal microscopy (MBCM) was characterized in normal mouse brain specimens and visualized their cytoarchitecture clearly. Then, MBCM was applied to both brain tumor murine models and two malignant human brain tumors of glioblastoma and metastatic cancer. MBCM detected tumors in all the specimens by visualizing dense and irregular cell distributions, and tumor margins were easily delineated based on the cytoarchitecture. An image analysis method was developed for automated detection and delineation. MBCM demonstrated sensitive delineation of brain tumors through cytoarchitecture visualization and would have potentials for human applications, such as a surgery‐guiding method for tumor removal.      

In‐vivo hemodynamic imaging of acute prenatal ethanol exposure in fetal brain by photoacoustic tomography

Prenatal ethanol exposure (PEE) can lead to structural and functional abnormalities in fetal brain. Although neural developmental deficits due to PEE have been recognized, the immediate effects of PEE on fetal brain vasculature and hemodynamics remain poorly understood. One of the major obstacles that preclude the rapid advancement of studies on fetal vascular dynamics is the limitation of the imaging techniques. Thus, a technique for noninvasive in‐vivo imaging of fetal vasculature and hemodynamics is desirable. In this study, we explored the dynamic changes of the vessel dimeter, density and oxygen saturation in fetal brain after acute maternal ethanol exposure in the second‐trimester equivalent murine model using a real‐time photoacoustic tomography system we developed for imaging embryo of small animals. The results indicate a significant decrease in fetal brain vessel diameter, perfusion and oxygen saturation. This work demonstrated that PAT can provide high‐resolution noninvasive imaging ability to monitor fetal vascular dynamics.     

Deep learning of diffraction image patterns for accurate classification of five cell types

Development of label‐free methods for accurate classification of cells with high throughput can yield powerful tools for biological research and clinical applications. We have developed a deep neural network of DINet for extracting features from cross‐polarized diffraction image (p‐DI) pairs on multiple pixel scales to accurately classify cells in five types. A total of 6185 cells were measured by a polarization diffraction imaging flow cytometry (p‐DIFC) method followed by cell classification with DINet on p‐DI data. The averaged value and SD of classification accuracy were found to be 98.9% ± 1.00% on test data sets for 5‐fold training and test. The invariance of DINet to image translation, rotation, and blurring has been verified with an expanded p‐DI data set. To study feature‐based classification by DINet, two sets of correctly and incorrectly classified cells were selected and compared for each of two prostate cell types. It has been found that the signature features of large dissimilarities between p‐DI data of correctly and incorrectly classified cell sets increase markedly from convolutional layers 1 and 2 to layers 3 and 4. These results clearly demonstrate the importance of high‐order correlations extracted at the deep layers for accurate cell classification.      

Photoacoustic and ultrasound (PAUS) dermoscope with high sensitivity and penetration depth by using a bimorph transducer

A bimorph transducer was proposed to improve the detection sensitivity and imaging depth of photoacoustic and ultrasound (PAUS) dermoscope. By applying the bimorph transducer, the imaging depth and sensitivity of PAUS dermoscope were enhanced by simultaneously improving excitation efficiency and reception bandwidth. The integrated design of the imaging head of the dermoscope makes it highly convenient for detecting human skin. The PAUS imaging performance was demonstrated via visualizing subcutaneous tumor and depicting full structures of different skin layers from epidermis to subcutaneous tissue. The results confirm that the dermoscope with the bimorph transducer is well suited for PA and US dual‐modality imaging, which can provide multi‐information for skin disease.     

Label-free high frame rate imaging of circulating blood clots using a dual modal ultrasound and photoacoustic system

Deep vein thrombosis (DVT) is a disorder when a blood clot (thrombus) is formed in one of the deep veins. These clots detach from the original sites and circulate in the blood stream at high velocities. Diagnosing these blood clots at an early stage is necessary to decide the treatment strategy. For label-free, in vivo, and real-time detection, high framerate photoacoustic imaging can be used. In this work, a dual modal clinical ultrasound and photoacoustic (PA) system is used for the high framerate PA imaging of circulating blood clots in blood at linear velocities up to 107 cm/sec. Blood clot had 1.4 times higher signal-to-noise ratio (SNR) in the static mode and 1.3 times higher SNR compared to blood PA signal in the flow experiments. This work demonstrates that fast-moving circulating blood clots are easy to recognize against the background PA signal and may aid in early diagnosis.     

Optical‐resolution photoacoustic microscopy with ultrafast dual‐wavelength excitation

Fast functional and molecular photoacoustic microscopy requires pulsed laser excitations at multiple wavelengths with enough pulse energy and short wavelength‐switching time. Recent development of stimulated Raman scattering in optical fiber offers a low‐cost laser source for multiwavelength photoacoustic imaging. In this approach, long fibers temporally separate different wavelengths via optical delay. The time delay between adjacent wavelengths may eventually limits the highest A‐line rate. In addition, a long‐time delay in fiber may limit the highest pulse energy, leading to poor image quality. In order to achieve high pulse energy and ultrafast dual‐wavelength excitation, we present optical‐resolution photoacoustic microscopy with ultrafast dual‐wavelength excitation and a signal separation method. The signal separation method is validated in numerical simulation and phantom experiments. We show that when two photoacoustic signals are partially overlapped with a 50‐ns delay, they can be recovered with 98% accuracy. We apply this ultrafast dual‐wavelength excitation technique to in vivo OR‐PAM. Results demonstrate that A‐lines at two wavelengths can be successfully separated, and sO₂ values can be reliably computed from the separated data. The ultrafast dual‐wavelength excitation enables fast functional photoacoustic microscopy with negligible misalignment among different wavelengths and high pulse energy, which is important for in vivo imaging of microvascular dynamics.     

Transmission in near‐infrared optical windows for deep brain imaging

Near‐infrared (NIR) radiation has been employed using one‐ and two‐photon excitation of fluorescence imaging at wavelengths 650–950 nm (optical window I) for deep brain imaging; however, longer wavelengths in NIR have been overlooked due to a lack of suitable NIR‐low band gap semiconductor imaging detectors and/or femtosecond laser sources. This research introduces three new optical windows in NIR and demonstrates their potential for deep brain tissue imaging. The transmittances are measured in rat brain tissue in the second (II, 1,100–1,350 nm), third (III, 1,600–1,870 nm), and fourth (IV, centered at 2,200 nm) NIR optical tissue windows. The relationship between transmission and tissue thickness is measured and compared with the theory. Due to a reduction in scattering and minimal absorption, window III is shown to be the best for deep brain imaging, and windows II and IV show similar but better potential for deep imaging than window I.

     

Towards quantitative assessment of burn based on photoacoustic and optical coherence tomography

Accurate and timely assessment of the severity of burn is essential for the treatment of burns. Currently, although most first‐degree and third‐degree burns are easily diagnosed through visual inspection or auxiliary diagnostic methods, the second‐degree burn is still difficult to distinguish due to the ambiguity boundaries of second‐degree with first‐degree and third‐degree burns. In this study, we proposed a non‐invasive technique by combing photoacoustic imaging (PAI) and optical coherence tomography (OCT) to multi‐parameter quantitatively assess the burns. The feasibility and capacity of the dual‐mode PAT/OCT for assessing the burns was first testified by tissue‐mimicking phantom and burn wounds in mouse pinna in vivo. The further experiments conducted on the back of rats showed that the changes in skin scattering structure, vascular morphology and blood flow provided by the dual‐mode PAI/OCT system can determine distinct boundaries and depth of the burns. The experimental results prove that combined PAI/OCT as a novel method can be used to assess the severity of burn, which has the potential to diagnose the burns in clinic.     

Inside Cover: In‐vivo hemodynamic imaging of acute prenatal ethanol exposure in fetal brain by photoacoustic tomography (J. Biophotonics 5/2020)

The figure shows the detailed morphology of vasculature and dynamic changes of the blood vessel diameter and density and the oxygen saturation in the blood vessels in fetal brain after acute prenatal ethanol exposure in the second‐trimester equivalent murine model obtained using a real‐time photoacoustic tomography (PAT) system. Further details can be found in the article by Tianqi Shan, Yuan Zhao, Shixie Jiang, Huabei Jiang ( e201960161 ).

     

Photoacoustic imaging of the human placental vasculature

Minimally invasive fetal interventions require accurate imaging from inside the uterine cavity. Twin‐to‐twin transfusion syndrome (TTTS), a condition considered in this study, occurs from abnormal vascular anastomoses in the placenta that allow blood to flow unevenly between the fetuses. Currently, TTTS is treated fetoscopically by identifying the anastomosing vessels, and then performing laser photocoagulation. However, white light fetoscopy provides limited visibility of placental vasculature, which can lead to missed anastomoses or incomplete photocoagulation. Photoacoustic (PA) imaging is an alternative imaging method that provides contrast for hemoglobin, and in this study, two PA systems were used to visualize chorionic (fetal) superficial and subsurface vasculature in human placentas. The first system comprised an optical parametric oscillator for PA excitation and a 2D Fabry‐Pérot cavity ultrasound sensor; the second, light emitting diode arrays and a 1D clinical linear‐array ultrasound imaging probe. Volumetric photoacoustic images were acquired from ex vivo normal term and TTTS‐treated placentas. It was shown that superficial and subsurface branching blood vessels could be visualized to depths of approximately 7 mm, and that ablated tissue yielded negative image contrast. This study demonstrated the strong potential of PA imaging to guide minimally invasive fetal therapies.      

Quantitative phase‐filtered wavelength‐modulated differential photoacoustic radar tumor hypoxia imaging toward early cancer detection

Overcoming the limitations of conventional linear spectroscopy used in multispectral photoacoustic imaging, wherein a linear relationship is assumed between the absorbed optical energy and the absorption spectra of the chromophore at a specific location, is crucial for obtaining accurate spatially‐resolved quantitative functional information by exploiting known chromophore‐specific spectral characteristics. This study introduces a non‐invasive phase‐filtered differential photoacoustic technique, wavelength‐modulated differential photoacoustic radar (WM‐DPAR) imaging that addresses this issue by eliminating the effect of the unknown wavelength‐dependent fluence. It employs two laser wavelengths modulated out‐of‐phase to significantly suppress background absorption while amplifying the difference between the two photoacoustic signals. This facilitates pre‐malignant tumor identification and hypoxia monitoring, as minute changes in total hemoglobin concentration and hemoglobin oxygenation are detectable. The system can be tuned for specific applications such as cancer screening and SO₂ quantification by regulating the amplitude ratio and phase shift of the signal. The WM‐DPAR imaging of a head and neck carcinoma tumor grown in the thigh of a nude rat demonstrates the functional PA imaging of small animals in vivo. The PA appearance of the tumor in relation to tumor vascularity is investigated by immunohistochemistry. Phase‐filtered WM‐DPAR imaging is also illustrated, maximizing quantitative SO₂ imaging fidelity of tissues.

Oxygenation levels within a tumor grown in the thigh of a nude rat using the two‐wavelength phase‐filtered differential PAR method.     

Noninvasive photoacoustic and fluorescent tracking of optical dye labeled T cellular activities of diseased sites at new depth

The migration of immune cells is crucial to the immune response. Visualization of these processes has previously been limited because of the imaging depth. We developed a deep‐penetrating, sensitive and high‐resolution method to use fast photoacoustic tomography (PAT) to image the dynamic changes of T cells in lymph node and diseases at new depth (up to 9.5 mm). T cells labeled with NIR‐797‐isothiocyanate, an excellent near‐infrared photoacoustic and fluorescent agent, were intravenously injected to the mice. We used fluorescence imaging to determine the location of T cells roughly and photoacoustic imaging is used to observe T‐cell responses in diseased sites deeply and carefully. The dynamic changes of T cells in lymph node, acute disease (bacterial infection) and chronic disease (tumor) were observed noninvasively by photoacoustic and fluorescence imaging at different time points. T cells accumulated gradually and reached a maximum at 4 hours and declined afterwards in lymph node and bacterial infection site. At tumor model, T cells immigrated to the tumor with a maximum at 12 hours. Our study can not only provide a new observing method for immune activities tracking, but also enable continuous monitoring for therapeutic interventions.