Handheld Autofluorescence Imaging System for Intraoperative Parathyroid Detection.

Introduction: Hypoparathyroidism and hypocalcemia are common complications after thyroid surgery. Parathyroids may be incidentally damaged or removed because they are difficult to distinguish from surrounding tissue. Intraoperative optical technologies such as near infrared autofluorescence (NIRAF) are becoming increasingly popular to help identify parathyroids during thyroid surgery. The objective of this video is to introduce a developing NIRAF device called hANDY-i and compare the device with existing Food and Drug Administration approved technology. Materials and Methods: hANDY-i is developed by Optosurgical, LLC. The device consists of a coaxial 785 nm laser excitation module and coregistred red-green-blue and near-infrared cameras. Operation of the device and output from preliminary intraoperative use are shown. Results: hANDY-i performs well, producing intuitive side-by-side NIRAF and RGB images of the operating field. The device demonstrates high contrast between suspected parathyroid glands and surrounding tissue. Operating theater, overhead lamps, and surgical headlights can all be used with the device. The device is also shown to be effective in both in vivo and ex vivo applications. Conclusions: The prototype described advance NIRAF technology by reducing light sensitivity and improving output representation. In doing so, hANDY-i makes NIRAF more accessible and less obstructive to the surgical workflow. Sources of Funding: This study was supported by the National Institute of Biomedical Imaging and Bioengineering of the National Institutes of Health under Award Number R43EB030874. Disclaimer: The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.Yoseph Kim is an employee of Optosurgical LLC. Jaepyeong Cha has ownership stake in Optosurgical LLC. For all other authors, no competing financial interests exist.Runtime of video: 7 mins 14 secs.

Multicore fiber optic imaging reveals that astrocyte calcium activity in the cerebral cortex is modulated by internal motivational state.

Astrocytes are a direct target of neuromodulators and can influence neuronal activity on broad spatial and temporal scales through their close proximity to synapses. However, our knowledge about how astrocytes are functionally recruited during different animal behaviors and their diverse effects on the CNS remains limited. To enable measurement of astrocyte activity patterns in vivo during normative behaviors, we developed a high-resolution, long working distance, multi-core fiber optic imaging platform that allows visualization of cortical astrocyte calcium transients through a cranial window in freely moving mice. Using this platform, we defined the spatiotemporal dynamics of astrocytes during diverse behaviors, ranging from circadian fluctuations to novelty exploration, showing that astrocyte activity patterns are more variable and less synchronous than apparent in head-immobilized imaging conditions. Although the activity of astrocytes in visual cortex was highly synchronized during quiescence to arousal transitions, individual astrocytes often exhibited distinct thresholds and activity patterns during explorative behaviors, in accordance with their molecular diversity, allowing temporal sequencing across the astrocyte network. Imaging astrocyte activity during self-initiated behaviors revealed that noradrenergic and cholinergic systems act synergistically to recruit astrocytes during state transitions associated with arousal and attention, which was profoundly modulated by internal state. The distinct activity patterns exhibited by astrocytes in the cerebral cortex may provide a means to vary their neuromodulatory influence in response to different behaviors and internal states.

Emerging Imaging Technologies for Parathyroid Gland Identification and Vascular Assessment in Thyroid Surgery: A Review From the American Head and Neck Society Endocrine Surgery Section.

Importance: Identification and preservation of parathyroid glands (PGs) remain challenging despite advances in surgical techniques. Considerable morbidity and even mortality result from hypoparathyroidism caused by devascularization or inadvertent removal of PGs. Emerging imaging technologies hold promise to improve identification and preservation of PGs during thyroid surgery. Observation: This narrative review (1) comprehensively reviews PG identification and vascular assessment using near-infrared autofluorescence (NIRAF)-both label free and in combination with indocyanine green-based on a comprehensive literature review and (2) offers a manual for possible implementation these emerging technologies in thyroid surgery. Conclusions and Relevance: Emerging technologies hold promise to improve PG identification and preservation during thyroidectomy. Future research should address variables affecting the degree of fluorescence in NIRAF, standardization of signal quantification, definitions and standardization of parameters of indocyanine green injection that correlate with postoperative PG function, the financial effect of these emerging technologies on near-term and longer-term costs, the adoption learning curve and effect on surgical training, and long-term outcomes of key quality metrics in adequately powered randomized clinical trials evaluating PG preservation.

Unsupervised and quantitative intestinal ischemia detection using conditional adversarial network in multimodal optical imaging.

Purpose: Intraoperative evaluation of bowel perfusion is currently dependent upon subjective assessment. Thus, quantitative and objective methods of bowel viability in intestinal anastomosis are scarce. To address this clinical need, a conditional adversarial network is used to analyze the data from laser speckle contrast imaging (LSCI) paired with a visible-light camera to identify abnormal tissue perfusion regions. Approach: Our vision platform was based on a dual-modality bench-top imaging system with red-green-blue (RGB) and dye-free LSCI channels. Swine model studies were conducted to collect data on bowel mesenteric vascular structures with normal/abnormal microvascular perfusion to construct the control or experimental group. Subsequently, a deep-learning model based on a conditional generative adversarial network (cGAN) was utilized to perform dual-modality image alignment and learn the distribution of normal datasets for training. Thereafter, abnormal datasets were fed into the predictive model for testing. Ischemic bowel regions could be detected by monitoring the erroneous reconstruction from the latent space. The main advantage is that it is unsupervised and does not require subjective manual annotations. Compared with the conventional qualitative LSCI technique, it provides well-defined segmentation results for different levels of ischemia. Results: We demonstrated that our model could accurately segment the ischemic intestine images, with a Dice coefficient and accuracy of 90.77% and 93.06%, respectively, in 2560 RGB/LSCI image pairs. The ground truth was labeled by multiple and independent estimations, combining the surgeons' annotations with fastest gradient descent in suspicious areas of vascular images. The total processing time was 0.05 s for an image size of 256 × 256 . Conclusions: The proposed cGAN can provide pixel-wise and dye-free quantitative analysis of intestinal perfusion, which is an ideal supplement to the traditional LSCI technique. It has potential to help surgeons increase the accuracy of intraoperative diagnosis and improve clinical outcomes of mesenteric ischemia and other gastrointestinal surgeries.

DXM-TransFuse U-net: Dual cross-modal transformer fusion U-net for automated nerve identification.

Accurate nerve identification is critical during surgical procedures to prevent damage to nerve tissues. Nerve injury can cause long-term adverse effects for patients, as well as financial overburden. Birefringence imaging is a noninvasive technique derived from polarized images that have successfully identified nerves that can assist during intraoperative surgery. Furthermore, birefringence images can be processed under 20 ms with a GPGPU implementation, making it a viable image modality option for real-time processing. In this study, we first comprehensively investigate the usage of birefringence images combined with deep learning, which can automatically detect nerves with gains upwards of 14% over its color image-based (RGB) counterparts on the F2 score. Additionally, we develop a deep learning network framework using the U-Net architecture with a Transformer based fusion module at the bottleneck that leverages both birefringence and RGB modalities. The dual-modality framework achieves 76.12 on the F2 score, a gain of 19.6 % over single-modality networks using only RGB images. By leveraging and extracting the feature maps of each modality independently and using each modality's information for cross-modal interactions, we aim to provide a solution that would further increase the effectiveness of imaging systems for enabling noninvasive intraoperative nerve identification.

A coaxial excitation, dual-red-green-blue/near-infrared paired imaging system toward computer-aided detection of parathyroid glands in situ and ex vivo.

Early and precise detection of parathyroid glands (PGs) is a challenging problem in thyroidectomy due to their small size and similar appearance to surrounding tissues. Near-infrared autofluorescence (NIRAF) has stimulated interest as a method to localize PGs. However, high incidence of false positives for PGs has been reported with this technique. We introduce a prototype equipped with a coaxial excitation light (785 nm) and a dual-sensor to address the issue of false positives with the NIRAF technique. We test the clinical feasibility of our prototype in situ and ex vivo using sterile drapes on 10 human subjects. Video data (1287 images) of detected PGs were collected to train, validate and compare the performance for PG detection. We achieved a mean average precision of 94.7% and a 19.5-millisecond processing time/detection. This feasibility study supports the effectiveness of the optical design and may open new doors for a deep learning-based PG detection method.

A pilot feasibility study to assess vascularity and perfusion of parathyroid glands using a portable hand-held imager.

OBJECTIVES: Intraoperative localization and preservation of parathyroid glands (PGs) are challenging during thyroid surgery. A new noninvasive technique of combined near-infrared PG autofluorescence detection and dye-free imaging angiography that allows intraoperative feedback has recently been introduced. The objective of this study was to evaluate this technique in real-time. MATERIALS AND METHODS: A pilot feasibility study of a portable imaging device in four patients who underwent either thyroid lobectomy or total thyroidectomy is presented. PG autofluorescence and vascularity/tissue perfusion were monitored using a real-time screen display during the surgical procedure. RESULTS: Three lobectomies and one total thyroidectomy were performed. Among the nine PGs identified by the operating surgeon, eight PGs were confirmed using the autofluorescence device. Each PG was successfully determined to be either well-perfused or devascularized, and devascularized PGs were autotransplanted. CONCLUSIONS: The preliminary results suggest that the combination of PG autofluorescence detection and dye-free angiography can potentially be used to assess PG function. With further validation studies, the effectiveness of this technique in clinical practice can be further delineated.

Laparoscopic cholecystectomy in a swine model using a novel near-infrared fluorescent IV dye (BL-760).

BACKGROUND AND OBJECTIVES: Bile duct injury during laparoscopic cholecystectomy has an incidence rate of 1%-2% and commonly appears under conditions of severe inflammation, adhesion, or unexpected anatomical variations. Despite the difficulties and rising concerns of identifying bile duct during surgeries, surgeons do not have a specific modality to identify bile duct except intraoperative cholangiography. While no biliary-specific fluorescent dye exists for clinical use, our team has previously described the development of a preclinical biliary-specific dye, BL-760. Here, we present our study of laparoscopic cholecystectomy using the fluorescent dye in a swine model. STUDY DESIGN/MATERIALS AND METHODS: With an approval from Institutional Animal Care and Use Committee, two 20-25 kg swine underwent laparoscopic abdominal surgery using a Food and Drug Administration-cleared fluorescent laparoscopic system. Images of the liver and gallbladder were taken both before and after intravenous injection of the novel fluorescent dye. The dye was dosed at 60 µg/kg and injected via the ear vein. The amount of time taken to visualize fluorescence in the biliary tract was measured. Fluorescent signal was observed after injection, and target-to-background ratio (TBR) of the biliary tract to surrounding cystic artery and liver parenchyma was measured. RESULTS: Biliary tract visualization under fluorescent laparoscopy was achieved within 5 min after the dye injection without any adverse effects. Cystic duct and extrahepatic duct were clearly visualized and identified with TBR values of 2.19 and 2.32, respectively, whereas no fluorescent signal was detected in liver. Cystic duct and artery were successfully ligated by an endoscopic clip applier with the visual assistance of highlighted biliary tract images. Laparoscopic cholecystectomy was completed within 30 min in each case without any complications. CONCLUSIONS: BL-760 is a novel preclinical fluorescent dye useful for intraoperative identification and visualization of biliary tract. Such fluorescent dye that is exclusively metabolized by liver and rapidly excreted into biliary tract would be beneficial for all types of hepato-biliary surgeries. With the validation of additional preclinical data, this novel dye has potential to be a valuable tool to prevent any iatrogenic biliary injuries and/or bile leaks during laparoscopic abdominal and liver surgeries.

Multimodal Imaging of Laser Speckle Contrast Imaging Combined With Mosaic Filter-Based Hyperspectral Imaging for Precise Surgical Guidance.

OBJECTIVE: To enable a real-time surgical guidance system that simultaneously monitors blood vessel perfusion, oxygen saturation, thrombosis, and tissue recovery by combining multiple optical imaging techniques into a single system: visible imaging, mosaic filter-based snapshot hyperspectral imaging (HSI), and laser speckle contrast imaging (LSCI). METHODS: The multimodal optical imaging system was demonstrated by clamping blood vessels in the small intestines of rats to create areas of restricted blood flow. Subsequent tissue damage and regeneration were monitored during procedures. Using LSCI, vessel perfusion was measured, revealing the biological activity and survival of organ tissues. Blood oxygen saturation was monitored using HSI in the near-infrared region. Principal component analysis was used over the spectral dimension to identify an HSI wavelength combination optimized for hemodynamic biomarker visualization. HSI and LSCI were complimentary, identifying thrombus generation and tissue recovery, which was not possible in either modalityalone. RESULTS AND CONCLUSION: By analyzing multimodal tissue information from visible imaging, LSCI perfusion imaging, and HSI, a recovery prognosis could be determined based on the blood supply to the organ. The unique combination of the complementary imaging techniques into a single surgical microscope holds promise for improving the real-time determination of blood supply and tissue prognosis during surgery. SIGNIFICANCE: Precise real-time monitoring for vascular anomalies promises to reduce the risk of organ damage in precise surgical operations such as tissue resection and transplantation. In addition, the convergence of label-free imaging technologies removes delays associated with the injection and diffusion of vascular monitoring dyes.

Development of a non-invasive, dual-sensor handheld imager for intraoperative preservation of parathyroid glands.

Intraoperative localization and preservation of parathyroid glands (PTGs) are challenging during thyroid surgery. Using a technique of combined near-infrared PTG autofluorescence detection and dye-free imaging angiography, this study developed a portable device for localization of PTGs and assessment of viability by confirming tissue perfusion. The imager's performance was evaluated through a pilot clinical study (N=10).

Improved Nerve Visualization in Head and Neck Surgery Using Mueller Polarimetric Imaging: Preclinical Feasibility Study in a Swine Model.

BACKGROUND AND OBJECTIVES: Meticulous dissection and identification of nerves during head and neck surgery are crucial for preventing nerve damage. At present, nerve identification relies heavily on the surgeon's knowledge of anatomy, optionally combined with intraoperative neuromonitoring. Recently, optical techniques such as Mueller polarimetric imaging (MPI) have shown potential to improve nerve identification. STUDY DESIGN/MATERIALS AND METHODS: With institutional approval, seven 25-35 kg Yorkshire pigs underwent cervical incision in the central neck. Intraoperative images were obtained using our in-house MPI system. Birefringence maps from the MPI system were processed to quantify the values between 0 and 255 from different tissue types; an active contour model was applied to further improve nerve visualization on the corresponding color images. RESULTS: Among the seven pigs, the vagus nerves and recurrent laryngeal nerves were successfully differentiated with a mean intensity of 130.954 ± 20.611, which was significantly different (P < 0.05) from those of arteries (78.512 ± 27.78) and other surrounding tissues (82.583 ± 35.547). There were no imaging-related complications during the procedure. © 2021 Wiley Periodicals LLC. CONCLUSIONS: MPI is a potentially complementary intraoperative tool for nerve identification in adjacent tissues.

Intraoperative Laser Speckle Contrast Imaging For Real-Time Visualization of Cerebral Blood Flow in Cerebrovascular Surgery: Results From Pre-Clinical Studies.

Cerebrovascular surgery can benefit from an intraoperative system that conducts continuous monitoring of cerebral blood flow (CBF). Such a system must be handy, non-invasive, and directly integrated into the surgical workflow. None of the currently available techniques, considered alone, meets all these criteria. Here, we introduce the SurgeON™ system: a newly developed non-invasive modular tool which transmits high-resolution Laser Speckle Contrast Imaging (LSCI) directly onto the eyepiece of the surgical microscope. In preclinical rodent and rabbit models, we show that this system enabled the detection of acute perfusion changes as well as the recording of temporal response patterns and degrees of flow changes in various microvascular settings, such as middle cerebral artery occlusion, femoral artery clipping, and complete or incomplete cortical vessel cautery. During these procedures, a real-time visualization of vasculature and CBF was available in high spatial resolution through the eyepiece as a direct overlay on the live morphological view of the surgical field. Upon comparison with indocyanine green angiography videoangiography (ICG-VA) imaging, also operable via SurgeON, we found that direct-LSCI can produce greater information than ICG-VA and that continuous display of data is advantageous for performing immediate LSCI-guided adjustments in real time.

Ureter Identification In an Inflammatory Pig Model Using a Novel Near-Infrared Fluorescent Dye.

BACKGROUND AND OBJECTIVES: Ureters are at risk of injury in settings of inflammation and distorted anatomy. The use of a fluorescent dye can improve intraoperative ureteral identification without the need for any additional invasive procedures. Our team has previously described the development of a preclinical ureter-specific dye, UL-766, tested in a rat model. Here, we present the use of the fluorescent dye during laparoscopy to assist in ureteral identification in a swine model with an inflamed abdomen; the results of this study serve as proof of feasibility for use in the setting of tissue edema and erythema. STUDY DESIGN/MATERIALS AND METHODS: With institutional approval, two 20-25 kg pigs underwent abdominal surgery with the use of a Food and Drug Administration-approved fluorescence laparoscopic system. Using standard laparoscopy, inflammation was induced with sharp and blunt dissection and irritation was induced with gauze. The animals were allowed to recover and returned to the operating room after 7 days. Images of the inflamed right retroperitoneum, with fluorescence imaging, turned on, were taken before and after intravenous injection of the novel fluorescent dye at 120 µg/kg. The time until fluorescence visualization of the ureters was measured, and the fluorescent signal was measured for up to 4 hours from the time of the initial dye injection. Partial and complete transection of ureteral injuries was made by scissors and monitored under both standard video and fluorescence laparoscopy. RESULTS: Inflammation reduced the certainty of ureter identification by white light alone. Despite surrounding tissue erythema and edema, ureteral visualization under fluorescence laparoscopy was achieved within 5-10 minutes after dye injection. The fluorescent signal remained visible for at least 4 hours after injection, and the fluorescent dye showed a partial ureteral injury that would not have been observed under standard laparoscopy. CONCLUSIONS: UL-766 is a preclinical fluorescent dye useful for the intraoperative identification of the ureters and ureteral injuries in an inflamed abdomen. With the acquisition of additional preclinical data, this novel dye can be a valuable tool during laparoscopic abdominal and pelvic surgeries. Lasers Surg. Med. © 2019 Wiley Periodicals, Inc.

Deep Neural Network Regression for Automated Retinal Layer Segmentation in Optical Coherence Tomography Images.

Segmenting the retinal layers in optical coherence tomography (OCT) images helps to quantify the layer information in early diagnosis of retinal diseases, which are the main cause of permanent blindness. Thus, the segmentation process plays a critical role in preventing vision impairment. However, because there is a lack of practical automated techniques, expert ophthalmologists still have to manually segment the retinal layers. In this study, we propose an automated segmentation method for OCT images based on a feature-learning regression network without human bias. The proposed deep neural network regression takes the intensity, gradient, and adaptive normalized intensity score (ANIS) of an image segment as features for learning, and then predicts the corresponding retinal boundary pixel. Reformulating the segmentation as a regression problem obviates the need for a huge dataset and reduces the complexity significantly, as shown in the analysis of computational complexity given here. In addition, assisted by ANIS, the method operates robustly on OCT images containing intensity variances, low-contrast regions, speckle noise, and blood vessels, yet remains accurate and time-efficient. In evaluation of the method conducted using 114 images, the processing time was approximately 10.596 s per image for identifying eight boundaries, and the training phase for each boundary line took only 30 s. Further, the Dice similarity coefficient used for assessing accuracy gave a computed value of approximately 0.966. The absolute pixel distance of manual and automatic segmentation using the proposed scheme was 0.612, which is less than a one-pixel difference, on average.

A Biliary Tract-Specific Near-Infrared Fluorescent Dye for Image-Guided Hepatobiliary Surgery.

Despite advances, visual inspection, palpation, and intraoperative ultrasound remain the most utilized tools during surgery today. A particularly challenging issue is the identification of the biliary system due to its complex architecture partially embedded within the liver. Fluorescence guided surgical interventions, particularly using near-infrared (NIR) wavelengths, are an emerging approach for the real-time assessment of the hepatobiliary system. However, existing fluorophores, such as the FDA-approved indocyanine green (ICG), have significant limitations for rapid and selective visualization of bile duct anatomy. Here we report a novel NIR fluorophore, BL (Bile Label)-760, which is exclusively metabolized by the liver providing high signal in the biliary system shortly after intravenous administration. This molecule was identified by first screening a small set of known heptamethine cyanines including clinically utilized agents. After finding that none of these were well-suited, we then designed and tested a small series of novel dyes within a prescribed polarity range. We validated the molecule that emerged from these efforts, BL-760, through animal studies using both rodent and swine models employing a clinically applicable imaging system. In contrast to ICG, BL-760 fluorescence revealed a high target-to-background ratio (TBR) of the cystic duct relative to liver parenchyma 5 min after intravenous injection. During hepatic resection surgery, intrahepatic ducts were clearly highlighted, and bile leakage was easily detected. In conclusion, BL-760 has highly promising properties for intraoperative navigation during hepatobiliary surgery.

Real-time, label-free, intraoperative visualization of peripheral nerves and micro-vasculatures using multimodal optical imaging techniques.

Accurate, real-time identification and display of critical anatomic structures, such as the nerve and vasculature structures, are critical for reducing complications and improving surgical outcomes. Human vision is frequently limited in clearly distinguishing and contrasting these structures. We present a novel imaging system, which enables noninvasive visualization of critical anatomic structures during surgical dissection. Peripheral nerves are visualized by a snapshot polarimetry that calculates the anisotropic optical properties. Vascular structures, both venous and arterial, are identified and monitored in real-time using a near-infrared laser-speckle-contrast imaging. We evaluate the system by performing in vivo animal studies with qualitative comparison by contrast-agent-aided fluorescence imaging.

A chemically stable fluorescent marker of the ureter.

Surgical methods guided by exogenous fluorescent markers have the potential to define tissue types in real time. Small molecule dyes with efficient and selective renal clearance could enable visualization of the ureter during surgical procedures involving the abdomen and pelvis. These studies report the design and synthesis of a water soluble, net neutral C4'-O-alkyl heptamethine cyanine, Ureter-Label (UL)-766, with excellent properties for ureter visualization. This compound is accessed through a concise synthetic sequence involving an N- to O-transposition reaction that provides other inaccessible C4'-O-alkyl heptamethine cyanines. Unlike molecules containing a C4'-O-aryl substituent, which have also been used for ureter visualization, UL-766 is not reactive towards glutathione and the cellular proteome. In addition, rat models of abdominal surgery reveal that UL-766 undergoes efficient and nearly exclusive renal clearance in vivo. In total, this molecule represents a promising candidate for visualizing the ureter during a variety of surgical interventions.

Dual-display laparoscopic laser speckle contrast imaging for real-time surgical assistance.

Laser speckle contrast imaging (LSCI) utilizes the speckle pattern of a laser to determine the blood flow in tissues. The current approaches for its use in a clinical setting require a camera system with a laser source on a separate optical axis making it unsuitable for minimally invasive surgery (MIS). With blood flow visualization, bowel viability, for example, can be determined. Thus, LSCI can be a valuable tool in gastrointestinal surgery. In this work, we develop the first-of-its-kind dual-display laparoscopic vision system integrating LSCI with a commercially available 10mm rigid laparoscope where the laser has the same optical axis as the laparoscope. Designed for MIS, our system permits standard color RGB, label-free vasculature imaging, and fused display modes. A graphics processing unit accelerated algorithm enables the real-time display of three different modes at the surgical site. We demonstrate the capability of our system for imaging relative flow rates in a microfluidic phantom with channels as small as 200 µm at a working distance of 1-5 cm from the laparoscope tip to the phantom surface. Using our system, we reveal early changes in bowel perfusion, which are invisible to standard color vision using a rat bowel occlusion model. Furthermore, we apply our system for the first time for imaging intestinal vasculature during MIS in a swine.

Multispectral tissue characterization for intestinal anastomosis optimization.

Intestinal anastomosis is a surgical procedure that restores bowel continuity after surgical resection to treat intestinal malignancy, inflammation, or obstruction. Despite the routine nature of intestinal anastomosis procedures, the rate of complications is high. Standard visual inspection cannot distinguish the tissue subsurface and small changes in spectral characteristics of the tissue, so existing tissue anastomosis techniques that rely on human vision to guide suturing could lead to problems such as bleeding and leakage from suturing sites. We present a proof-of-concept study using a portable multispectral imaging (MSI) platform for tissue characterization and preoperative surgical planning in intestinal anastomosis. The platform is composed of a fiber ring light-guided MSI system coupled with polarizers and image analysis software. The system is tested on ex vivo porcine intestine tissue, and we demonstrate the feasibility of identifying optimal regions for suture placement.

Random transverse motion-induced spatial compounding for fiber bundle imaging.

We propose and demonstrate a novel fiber bundle imaging based on spatial compounding induced by random transverse motion to remove the pixelation effect, to improve resolution, and to increase image quality. The experimental results using a USAF target and pyramidal neuron cell showed that 20-frame compounding improved image quality (contrast-to-noise ratio by >9 dB, global SNR by >6 dB, equivalent number of looks by >1.8 times, and 1/ß by >1.5 times), resolution by better than 2 µm, and completely eliminated pixelation artifact.