Tapered catheter-based transurethral photoacoustic and ultrasonic endoscopy of the urinary system.

Early diagnosis is critical for treating bladder cancer, as this cancer is very aggressive and lethal if detected too late. To address this important clinical issue, a photoacoustic tomography (PAT)-based transabdominal imaging approach was suggested in previous reports, in which its in vivo feasibility was also demonstrated based on a small animal model. However, successful translation of this approach to real clinical settings would be challenging because the human bladder is located at a depth that far exceeds the typical penetration depth of PAT (∼3 cm for in vivo cases). In this study, we developed a tapered catheter-based, transurethral photoacoustic and ultrasonic endoscopic probe with a 2.8 mm outer diameter to investigate whether the well-known benefits of PAT can be harnessed to resolve unmet urological issues, including early diagnosis of bladder cancer. To demonstrate the in vivo imaging capability of the proposed imaging probe, we performed a rabbit model-based urinary system imaging experiment and acquired a 3D microvasculature map distributed in the wall of the urinary system, which is a first in PAT, to the best of our knowledge. We believe that the results strongly support the use of this transurethral imaging approach as a feasible strategy for addressing urological diagnosis issues.

Deep-Learning-Based Algorithm for the Removal of Electromagnetic Interference Noise in Photoacoustic Endoscopic Image Processing.

Despite all the expectations for photoacoustic endoscopy (PAE), there are still several technical issues that must be resolved before the technique can be successfully translated into clinics. Among these, electromagnetic interference (EMI) noise, in addition to the limited signal-to-noise ratio (SNR), have hindered the rapid development of related technologies. Unlike endoscopic ultrasound, in which the SNR can be increased by simply applying a higher pulsing voltage, there is a fundamental limitation in leveraging the SNR of PAE signals because they are mostly determined by the optical pulse energy applied, which must be within the safety limits. Moreover, a typical PAE hardware situation requires a wide separation between the ultrasonic sensor and the amplifier, meaning that it is not easy to build an ideal PAE system that would be unaffected by EMI noise. With the intention of expediting the progress of related research, in this study, we investigated the feasibility of deep-learning-based EMI noise removal involved in PAE image processing. In particular, we selected four fully convolutional neural network architectures, U-Net, Segnet, FCN-16s, and FCN-8s, and observed that a modified U-Net architecture outperformed the other architectures in the EMI noise removal. Classical filter methods were also compared to confirm the superiority of the deep-learning-based approach. Still, it was by the U-Net architecture that we were able to successfully produce a denoised 3D vasculature map that could even depict the mesh-like capillary networks distributed in the wall of a rat colorectum. As the development of a low-cost laser diode or LED-based photoacoustic tomography (PAT) system is now emerging as one of the important topics in PAT, we expect that the presented AI strategy for the removal of EMI noise could be broadly applicable to many areas of PAT, in which the ability to apply a hardware-based prevention method is limited and thus EMI noise appears more prominently due to poor SNR.

Photoacoustic Tomography Opening New Paradigms in Biomedical Imaging.

After the emergence of the ultrasound, X-ray CT, PET, and MRI, photoacoustic tomography (PAT) is now in the phase of its exponential growth, with its expected full maturation being another form of mainstream clinical imaging modality. By combining the high contrast benefit of optical imaging and the high-resolution deep imaging capability of ultrasound, PAT can provide unprecedented anatomical image contrasts at clinically relevant depths as well as enable the use of a variety of functional and molecular imaging information, which is not possible with conventional imaging modalities. With these strengths, PAT has achieved numerous breakthroughs in various biomedical applications and also provided new technical platforms that may be able to resolve unmet issues in clinics. In this chapter, we provide an overview of the development of PAT technology for several major biomedical applications and provide an approximate projection of the future of PAT.

High-speed label-free functional photoacoustic microscopy of mouse brain in action.

We present fast functional photoacoustic microscopy (PAM) for three-dimensional high-resolution, high-speed imaging of the mouse brain, complementary to other imaging modalities. We implemented a single-wavelength pulse-width-based method with a one-dimensional imaging rate of 100 kHz to image blood oxygenation with capillary-level resolution. We applied PAM to image the vascular morphology, blood oxygenation, blood flow and oxygen metabolism in both resting and stimulated states in the mouse brain.

Urogenital photoacoustic endoscope.

Photoacoustic (PA) endoscopy for human urogenital imaging has the potential to diagnose many important diseases, such as endometrial and prostate cancers. We have specifically developed a 12.7 mm diameter, rigid, side-scanning PA endoscopic probe for such applications. The key features of this endoscope are the streamlined structure for smooth cavity introduction and the proximal actuation mechanism for fast scanning. Here we describe the probe's composition and scanning mechanism and present in vivo experimental results suggesting its potential for comprehensive clinical applications.

A 2.5-mm diameter probe for photoacoustic and ultrasonic endoscopy.

We have created a 2.5-mm outer diameter integrated photo-acoustic and ultrasonic mini-probe which can be inserted into a standard video endoscope's instrument channel. A small-diameter focused ultrasonic transducer made of PMN-PT provides adequate signal sensitivity, and enables miniaturization of the probe. Additionally, this new endoscopic probe utilizes the same scanning mirror and micromotor-based built-in actuator described in our previous reports; however, the length of the rigid distal section of the new probe has been further reduced to ~35 mm. This paper describes the technical details of the mini-probe and presents experimental results that both quantify the imaging performance and demonstrate its in vivo imaging capability, which suggests that it could work as a mini-probe for certain clinical applications.

Intra-instrument channel workable, optical-resolution photoacoustic and ultrasonic mini-probe system for gastrointestinal endoscopy.

There has been a long-standing expectation that the optical-resolution embodiment of photoacoustic tomography could have a substantial impact on gastrointestinal endoscopy by enabling microscopic visualization of the vasculature based on the endogenous contrast mechanism. Although multiple studies have demonstrated the in vivo imaging capability of a developed imaging device over the last decade, the implementation of such an endoscopic system that can be applied immediately when necessary via the instrument channel of a video endoscope has been a challenge. In this study, we developed a 3.38-mm diameter catheter-based, integrated optical-resolution photoacoustic and ultrasonic mini-probe system and successfully demonstrated its intra-instrument channel workability for the standard 3.7-mm diameter instrument channel of a clinical video endoscope based on a swine model. Through the instrument channel, we acquired the first in vivo dual-mode photoacoustic and ultrasonic endoscopic images from the esophagogastric junction of a swine. Further, in a rat colorectum in vivo imaging experiment, we visualized hierarchically developed mesh-like capillary networks with a hole size as small as ~50 µm, which suggests the potential level of image details that could be photoacoustically provided in clinical settings in the future.

Microfluidic sorting of mammalian cells by optical force switching.

Microfluidic-based devices have allowed miniaturization and increased parallelism of many common functions in biological assays; however, development of a practical technology for microfluidic-based fluorescence-activated cell sorting has proved challenging. Although a variety of different physical on-chip switch mechanisms have been proposed, none has satisfied simultaneously the requirements of high throughput, purity, and recovery of live, unstressed mammalian cells. Here we show that optical forces can be used for the rapid (2-4 ms), active control of cell routing on a microfluidic chip. Optical switch controls reduce the complexity of the chip and simplify connectivity. Using all-optical switching, we have implemented a fluorescence-activated microfluidic cell sorter and evaluated its performance on live, stably transfected HeLa cells expressing a fused histone-green fluorescent protein. Recovered populations were verified to be both viable and unstressed by evaluation of the transcriptional expression of two genes, HSPA6 and FOS, known indicators of cellular stress.

Year-long biophoton measurements: normalized frequency count analysis and seasonal dependency.

Biophoton emissions from three healthy human subjects were measured for 52 weeks. The active nature of dorsal hands and personal discernable patterns in palmar hands were investigated through frequency count analysis of biophoton emission rates. Also, the seasonal dependency of biophoton emission rates from human hands was studied and we found that emission rates were lowest in autumn. There was a reversion of relative emission rates from the palms and the dorsa depending upon the season. The emission rates from the palms remain rather stable throughout the year, but those from the dorsa vary widely depending upon the season. These features of biophoton emission rates were considered in light of the diagnostic view of traditional Chinese medicine.

Three-dimensional photoacoustic endoscopic imaging of the rabbit esophagus.

We report photoacoustic and ultrasonic endoscopic images of two intact rabbit esophagi. To investigate the esophageal lumen structure and microvasculature, we performed in vivo and ex vivo imaging studies using a 3.8-mm diameter photoacoustic endoscope and correlated the images with histology. Several interesting anatomic structures were newly found in both the in vivo and ex vivo images, which demonstrates the potential clinical utility of this endoscopic imaging modality. In the ex vivo imaging experiment, we acquired high-resolution motion-artifact-free three-dimensional photoacoustic images of the vasculatures distributed in the walls of the esophagi and extending to the neighboring mediastinal regions. Blood vessels with apparent diameters as small as 190 µm were resolved. Moreover, by taking advantage of the dual-mode high-resolution photoacoustic and ultrasound endoscopy, we could better identify and characterize the anatomic structures of the esophageal lumen, such as the mucosal and submucosal layers in the esophageal wall, and an esophageal branch of the thoracic aorta. In this paper, we present the first photoacoustic images showing the vasculature of a vertebrate esophagus and discuss the potential clinical applications and future development of photoacoustic endoscopy.

Optical-resolution photoacoustic endomicroscopy in vivo.

Optical-resolution photoacoustic microscopy (OR-PAM) has become a major experimental tool of photoacoustic tomography, with unique imaging capabilities for various biological applications. However, conventional imaging systems are all table-top embodiments, which preclude their use in internal organs. In this study, by applying the OR-PAM concept to our recently developed endoscopic technique, called photoacoustic endoscopy (PAE), we created an optical-resolution photoacoustic endomicroscopy (OR-PAEM) system, which enables internal organ imaging with a much finer resolution than conventional acoustic-resolution PAE systems. OR-PAEM has potential preclinical and clinical applications using either endogenous or exogenous contrast agents.

An integrated, stacked microlaboratory for biological agent detection with DNA and immunoassays.

An integrated, stacked microlaboratory for performing automated electric-field-driven immunoassays and DNA hybridization assays was developed. The stacked microlaboratory was fabricated by orderly laminating several different functional layers (all 76 x 76 mm(2)) including a patterned polyimide layer with a flip-chip bonded CMOS chip, a pressure sensitive acrylic adhesive (PSA) layer with a fluidic cutout, an optically transparent polymethyl methacrylate (PMMA) film, a PSA layer with a via, a patterned polyimide layer with a flip-chip bonded silicon chip, a PSA layer with a fluidic cutout, and a glass cover plate layer. Versatility of the stacked microlaboratory was demonstrated by various automated assays. Escherichia coli bacteria and Alexa-labeled protein toxin staphylococcal enterotoxin B (SEB) were detected by electric-field-driven immunoassays on a single chip with a specific-to-nonspecific signal ratios of 4.2:1 and 3.0:1, respectively. Furthermore, by integrating the microlaboratory with a module for strand displacement amplification (SDA), the identification of the Shiga-like toxin gene (SLT1) from E. coli was accomplished within 2.5 h starting from a dielectrophoretic concentration of intact E. coli bacteria and finishing with an electric-field-driven DNA hybridization assay, detected by fluorescently labeled DNA reporter probes. The integrated microlaboratory can be potentially used in a wide range of applications including detection of bacteria and biowarfare agents, and genetic identification.

Photon counting statistics analysis of biophotons from hands.

The photon counting statistics of biophotons emitted from hands is studied with a view to test its agreement with the Poisson distribution. The moments of observed probability up to seventh order have been evaluated. The moments of biophoton emission from hands are in good agreement while those of dark counts of photomultiplier tube show large deviations from the theoretical values of Poisson distribution. The present results are consistent with the conventional delta-value analysis of the second moment of probability.

Left-right asymmetry of biophoton emission from hemiparesis patients.

Left-right biophoton asymmetry from the palm and the dorsum of hands from 7 Korean hemiparesis patients were studied. There is a strong tendency that the left-hemiparesis patients emit more biophotons from the right than the left hands, while the right-hemiparesis patient emits more from the left hand. Acupuncture treatment reduces dramatically the left-right asymmetry of biophoton emission rates. However there is no systematic difference for the patients in the emission rates from the palm and the dorsum of hands.

Left-right and Yin-Yang balance of biophoton emission from hands.

Yearlong measurements of biophotons from palm and back of hand of three healthy people were performed. The detection of biophoton was done with two photomultiplier tubes whose spectral range was from 300 nm to 650 nm. The measurement was done on a circular area of diameter 46 mm whose centers were at the acupuncture point Laogong (PC8) of a palm and the mid-point of a back, respectively. The emission rates from the dorsa showed strong seasonal dependence which is consistent with the active nature of Yang meridians, while the palm sides show less seasonal dependence as they belong to the passive Yin meridians. This could be quantified simply by the standard deviations from the yearly average: They were 47.6 counts per second (cps), 66.1 cps and 66.0 cps from the dorsa, and 23.8 cps, 29.7 cps and 30.4 cps from the palms of subject 1, 2 and 3, respectively. Biophoton emission revealed qualitative individual tendencies: The subjects 1 and 2 emitted more strongly from the left dorsa, while the subject 3 from the right dorsum. The left-right balance of biophoton emission was well kept for normal people, which is in contrast with the severe imbalance for some stroke patients. Thus biophoton measurement has a diagnostic potential that encourages more studies.

Catheter-based photoacoustic endoscope.

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.

Wide-field fast-scanning photoacoustic microscopy based on a water-immersible MEMS scanning mirror.

By offering images with high spatial resolution and unique optical absorption contrast, optical-resolution photoacoustic microscopy (OR-PAM) has gained increasing attention in biomedical research. Recent developments in OR-PAM have improved its imaging speed, but have to sacrifice either the detection sensitivity or field of view or both. We have developed a wide-field fast-scanning OR-PAM by using a water-immersible microelectromechanical systems (MEMS) scanning mirror (MEMS-OR-PAM). In MEMS-OR-PAM, the optical and acoustic beams are confocally configured and simultaneously steered, which ensures the uniform detection sensitivity. A B-scan imaging speed as high as 400 Hz can be achieved over a 3 mm scanning range. Using the system, we imaged the flow dynamics of both red blood cells and carbon particles in a mouse ear in vivo. Presented results show that MEMS-OR-PAM could be a powerful tool for studying highly dynamic and time-sensitive biological phenomena.

Simultaneous functional photoacoustic and ultrasonic endoscopy of internal organs in vivo.

At present, clinicians routinely apply ultrasound endoscopy in a variety of interventional procedures that provide treatment solutions for diseased organs. Ultrasound endoscopy not only produces high-resolution images, but also is safe for clinical use and broadly applicable. However, for soft tissue imaging, its mechanical wave-based image contrast fundamentally limits its ability to provide physiologically specific functional information. By contrast, photoacoustic endoscopy possesses a unique combination of functional optical contrast and high spatial resolution at clinically relevant depths, ideal for imaging soft tissues. With these attributes, photoacoustic endoscopy can overcome the current limitations of ultrasound endoscopy. Moreover, the benefits of photoacoustic imaging do not come at the expense of existing ultrasound functions; photoacoustic endoscopy systems are inherently compatible with ultrasound imaging, thereby enabling multimodality imaging with complementary contrast. Here we present simultaneous photoacoustic and ultrasonic dual-mode endoscopy and show its ability to image internal organs in vivo, thus illustrating its potential clinical application.

Photoacoustic endoscopy.

We have developed photoacoustic endoscopy with a miniaturized imaging probe. A light-guiding optical fiber, an ultrasonic sensor, and a mechanical scanning unit are integrated to enable circumferential sector scanning, which produces B-scan images. Biological tissues, including the gastrointestinal tract of a rat, have been imaged ex vivo or in situ.