Quantification and visualization of metastatic lung tumors in mice.

Histopathological examination is important for the diagnosis of various diseases. Conventional histopathology provides a two-dimensional view of the tissues, and requires the tissue to be extracted, fixed, and processed using histotechnology techniques. However, there is an increasing need for three-dimensional (3D) images of structures in biomedical research. The objective of this study was to develop reliable, objective tools for visualizing and quantifying metastatic tumors in mouse lung using micro-computed tomography (micro-CT), optical coherence tomography (OCT), and field emission-scanning electron microscopy (FE-SEM). Melanoma cells were intravenously injected into the tail vein of 8-week-old C57BL/6 mice. The mice were euthanized at 2 or 4 weeks after injection. Lungs were fixed and examined by micro-CT, OCT, FE-SEM, and histopathological observation. Micro-CT clearly distinguished between tumor and normal cells in surface and deep lesions, thereby allowing 3D quantification of the tumor volume. OCT showed a clear difference between the tumor and surrounding normal tissues. FE-SEM clearly showed round tumor cells, mainly located in the alveolar wall and growing inside the alveoli. Therefore, whole-tumor 3D imaging successfully visualized the metastatic tumor and quantified its volume. This promising approach will allow for fast and label-free 3D phenotyping of diverse tissue structures.

Encoder-Weighted W-Net for Unsupervised Segmentation of Cervix Region in Colposcopy Images.

Cervical cancer can be prevented and treated better if it is diagnosed early. Colposcopy, a way of clinically looking at the cervix region, is an efficient method for cervical cancer screening and its early detection. The cervix region segmentation significantly affects the performance of computer-aided diagnostics using a colposcopy, particularly cervical intraepithelial neoplasia (CIN) classification. However, there are few studies of cervix segmentation in colposcopy, and no studies of fully unsupervised cervix region detection without image pre- and post-processing. In this study, we propose a deep learning-based unsupervised method to identify cervix regions without pre- and post-processing. A new loss function and a novel scheduling scheme for the baseline W-Net are proposed for fully unsupervised cervix region segmentation in colposcopy. The experimental results showed that the proposed method achieved the best performance in the cervix segmentation with a Dice coefficient of 0.71 with less computational cost. The proposed method produced cervix segmentation masks with more reduction in outliers and can be applied before CIN detection or other diagnoses to improve diagnostic performance. Our results demonstrate that the proposed method not only assists medical specialists in diagnosis in practical situations but also shows the potential of an unsupervised segmentation approach in colposcopy.

Compartmentalized Arrays of Matrix Droplets for Quantitative Mass Spectrometry Imaging of Adsorbed Peptides.

Mass spectrometry imaging (MSI) based on matrix-assisted laser desorption/ionization (MALDI) provides information on the identification and spatial distribution of biomolecules. Quantitative analysis, however, has been challenging largely due to heterogeneity in both the size of the matrix crystals and the extraction area. In this work, we present a compartmentalized elastomeric stamp for quantitative MALDI-MSI of adsorbed peptides. Filling the compartments with matrix solution and stamping onto a planar substrate extract and concentrate analytes adsorbed in each compartment into a single analyte-matrix cocrystal over the entire stamped area. Walls between compartments help preserve spatial information on the adsorbates. The mass intensity of the cocrystals directly correlates with the surface coverage of analytes, which enables not only quantitative analysis but estimation of an equilibrium constant for the adsorption. We demonstrate via MALDI-MSI relative quantitation of peptides adsorbed along a microchannel with varying surface coverages.

Quantitative Screening of Cervical Cancers for Low-Resource Settings: Pilot Study of Smartphone-Based Endoscopic Visual Inspection After Acetic Acid Using Machine Learning Techniques.

BACKGROUND: Approximately 90% of global cervical cancer (CC) is mostly found in low- and middle-income countries. In most cases, CC can be detected early through routine screening programs, including a cytology-based test. However, it is logistically difficult to offer this program in low-resource settings due to limited resources and infrastructure, and few trained experts. A visual inspection following the application of acetic acid (VIA) has been widely promoted and is routinely recommended as a viable form of CC screening in resource-constrained countries. Digital images of the cervix have been acquired during VIA procedure with better quality assurance and visualization, leading to higher diagnostic accuracy and reduction of the variability of detection rate. However, a colposcope is bulky, expensive, electricity-dependent, and needs routine maintenance, and to confirm the grade of abnormality through its images, a specialist must be present. Recently, smartphone-based imaging systems have made a significant impact on the practice of medicine by offering a cost-effective, rapid, and noninvasive method of evaluation. Furthermore, computer-aided analyses, including image processing-based methods and machine learning techniques, have also shown great potential for a high impact on medicinal evaluations. OBJECTIVE: In this study, we demonstrate a new quantitative CC screening technique and implement a machine learning algorithm for smartphone-based endoscopic VIA. We also evaluated the diagnostic performance and practicability of the approach based on the results compared to the gold standard and from physicians' interpretation. METHODS: A smartphone-based endoscope system was developed and applied to the VIA screening. A total of 20 patients were recruited for this study to evaluate the system. Overall, five were healthy, and 15 were patients who had shown a low to high grade of cervical intraepithelial neoplasia (CIN) from both colposcopy and cytology tests. Endoscopic VIA images were obtained before a loop electrosurgical excision procedure for patients with abnormal tissues, and their histology tissues were collected. Endoscopic VIA images were assessed by four expert physicians relative to the gold standard of histopathology. Also, VIA features were extracted from multiple steps of image processing techniques to find the differences between abnormal (CIN2+) and normal (≤CIN1). By using the extracted features, the performance of different machine learning classifiers, such as k-nearest neighbors (KNN), support vector machine, and decision tree (DT), were compared to find the best algorithm for VIA. After determining the best performing classifying model, it was used to evaluate the screening performance of VIA. RESULTS: An average accuracy of 78%, with a Cohen kappa of 0.571, was observed for the evaluation of the system by four physicians. Through image processing, 240 sliced images were obtained from the cervicogram at each clock position, and five features of VIA were extracted. Among the three models, KNN showed the best performance for finding VIA within holdout 10-fold cross-validation, with an accuracy of 78.3%, area under the curve of 0.807, a specificity of 80.3%, and a sensitivity of 75.0%, respectively. The trained model performed using an unprovided data set resulted in an accuracy of 80.8%, specificity of 84.1%, and sensitivity of 71.9%. Predictions were visualized with intuitive color labels, indicating the normal/abnormal tissue using a circular clock-type segmentation. Calculating the overlapped abnormal tissues between the gold standard and predicted value, the KNN model overperformed the average assessments of physicians for finding VIA. CONCLUSIONS: We explored the potential of the smartphone-based endoscopic VIA as an evaluation technique and used the cervicogram to evaluate normal/abnormal tissue using machine learning techniques. The results of this study demonstrate its potential as a screening tool in low-resource settings.

Substrate curvature affects the shape, orientation, and polarization of renal epithelial cells.

The unique structure of kidney tubules is representative of their specialized function. Because maintaining tubular structure and controlled diameter is critical for kidney function, it is critical to understand how topographical cues, such as curvature, might alter cell morphology and biological characteristics. Here, we examined the effect of substrate curvature on the shape and phenotype of two kinds of renal epithelial cells (MDCK and HK-2) cultured on a microchannel with a broad range of principal curvature. We found that cellular architecture on curved substrates was closely related to the cell type-specific characteristics (stiffness, cell-cell adherence) of the cells and their density, as well as the sign and degree of curvature. As the curvature increased on convex channels, HK-2 cells, having lower cell stiffness and monolayer integrity than those of MDCK cells, aligned their in-plane axis perpendicular to the channel but did not significantly change in morphology. By contrast, MDCK cells showed minimal change in both morphology and alignment. However, on concave channels, both cell types were elongated and showed longitudinal directionality, although the changes in MDCK cells were more conservative. Moreover, substrate curvature contributed to cell polarization by enhancing the expression of apical and basolateral cell markers with height increase of the cells. Our study suggests curvature to be an important guiding principle for advanced tissue model developments, and that curved and geometrically ambiguous substrates can modulate the cellular morphology and phenotype. STATEMENT OF SIGNIFICANCE: In many tissues, such as renal tubules or intestinal villi, epithelial layers exist in naturally curved forms, a geometry that is not reproduced by flat cultures. Because maintaining tubular structure is critical for kidney function, it is important to understand how topographical cues, such as curvature, might alter cell morphology and biological characteristics. We found that cellular architecture on curved substrates was closely related to cell type and density, as well as the sign and degree of the curvature. Moreover, substrate curvature contributed to cell polarization by enhancing the expression of apical and basolateral cell markers with height increase. Our results suggested that substrate curvature might contribute to cellular architecture and enhance the polarization of kidney tubule cells.

Lamellar keratoplasty using position-guided surgical needle and M-mode optical coherence tomography.

Deep anterior lamellar keratoplasty (DALK) is an emerging surgical technique for the restoration of corneal clarity and vision acuity. The big-bubble technique in DALK surgery is the most essential procedure that includes the air injection through a thin syringe needle to separate the dysfunctional region of the cornea. Even though DALK is a well-known transplant method, it is still challenged to manipulate the needle inside the cornea under the surgical microscope, which varies its surgical yield. Here, we introduce the DALK protocol based on the position-guided needle and M-mode optical coherence tomography (OCT). Depth-resolved 26-gage needle was specially designed, fabricated by the stepwise transitional core fiber, and integrated with the swept source OCT system. Since our device is feasible to provide both the position information inside the cornea as well as air injection, it enables the accurate management of bubble formation during DALK. Our results show that real-time feedback of needle end position was intuitionally visualized and fast enough to adjust the location of the needle. Through our research, we realized that position-guided needle combined with M-mode OCT is a very efficient and promising surgical tool, which also to enhance the accuracy and stability of DALK.

Quantitative phase imaging for medical diagnosis.

Optical microscopy is an indispensable diagnostic tool in modern healthcare. As a prime example, pathologists rely exclusively on light microscopy to investigate tissue morphology in order to make a diagnosis. While advances in light microscopy and contrast markers allow pathologists to visualize cells and tissues in unprecedented detail, the interpretation of these images remains largely subjective, leading to inter- and intra-observer discrepancy. Furthermore, conventional microscopy images capture qualitative information which makes it difficult to automate the process, reducing the throughput achievable in the diagnostic workflow. Quantitative Phase Imaging (QPI) techniques have been advanced in recent years to address these two challenges. By quantifying physical parameters of cells and tissues, these systems remove subjectivity from the disease diagnosis process and allow for easier automation to increase throughput. In addition to providing quantitative information, QPI systems are also label-free and can be easily assimilated into the current diagnostic workflow in the clinic. In this paper we review the advances made in disease diagnosis by QPI techniques. We focus on the areas of hematological diagnosis and cancer pathology, which are the areas where most significant advances have been made to date. [Image adapted from Y. Park, M. Diez-Silva, G. Popescu, G. Lykotrafitis, W. Choi, M. S. Feld, and S. Suresh, Proc. Natl. Acad. Sci. 105, 13730-13735 (2008).].

Optical coherence tomography for advanced screening in the primary care office.

Optical coherence tomography (OCT) has long been used as a diagnostic tool in the field of ophthalmology. The ability to observe microstructural changes in the tissues of the eye has proved very effective in diagnosing ocular disease. However, this technology has yet to be introduced into the primary care office, where indications of disease are first encountered. We have developed a portable, handheld imaging probe for use in the primary care setting and evaluated its tissue site accessibility, ability to observe diseased tissue, and screening capabilities in in vivo human patients, particularly for pathologies related to the eye, ear and skin. Various stages of diabetic retinopathy were investigated using the handheld probe and early-stage diabetic retinopathy was flagged as abnormal from the OCT images. At such early stages of disease, it is difficult to observe abnormalities with the limited tools that are currently available to primary care physicians. These results indicate that OCT shows promise to transform from being a diagnostic technology in the medical and surgical specialities to a screening technology in the primary care office and at the front-line of healthcare.

Optical coherence tomography for rapid tissue screening and directed histological sectioning.

In pathology, histological examination of the tissue is the "gold standard" to diagnose various diseases. It has contributed significantly toward identifying the abnormalities in tissues and cells, but has inherent drawbacks when used for fast and accurate diagnosis. These limitations include the lack of in vivo observation in real time and sampling errors due to limited number and area coverage of tissue sections. Its diagnostic yield also varies depending on the ability of the physician and the effectiveness of any image guidance technique that may be used for tissue screening during excisional biopsy. In order to overcome these current limitations of histology-based diagnostics, there are significant needs for either complementary or alternative imaging techniques which perform non-destructive, high resolution, and rapid tissue screening. Optical coherence tomography (OCT) is an emerging imaging modality which allows real-time cross-sectional imaging with high resolutions that approach those of histology. OCT could be a very promising technique which has the potential to be used as an adjunct to histological tissue observation when it is not practical to take specimens for histological processing, when large areas of tissue need investigating, or when rapid microscopic imaging is needed. This review will describe the use of OCT as an image guidance tool for fast tissue screening and directed histological tissue sectioning in pathology.

Optical coherence tomography for rapid tissue screening and directed histological sectioning.

In pathology, histological examination of the "gold standard" to diagnose various diseases. It has contributed significantly toward identifying the abnormalities in tissues and cells, but has inherent drawbacks when used for fast and accurate diagnosis. These limitations include the lack of in vivo observation in real time and sampling errors due to limited number and area coverage of tissue sections. Its diagnostic yield also varies depending on the ability of the physician and the effectiveness of any image guidance technique that may be used for tissue screening during excisional biopsy. In order to overcome these current limitations of histology-based diagnostics, there are significant needs for either complementary or alternative imaging techniques which perform non-destructive, high resolution, and rapid tissue screening. Optical coherence tomography (OCT) is an emerging imaging modality which allows real-time cross-sectional imaging with high resolutions that approach those of histology. OCT could be a very promising technique which has the potential to be used as an adjunct to histological tissue observation when it is not practical to take specimens for histological processing, when large areas of tissue need investigating, or when rapid microscopic imaging is needed. This review will describe the use of OCT as an image guidance tool for fast tissue screening and directed histological tissue sectioning in pathology.

Preliminary investigation on use of high-resolution optical coherence tomography to monitor injury and repair in the rat sciatic nerve.

BACKGROUND AND OBJECTIVE: Optical coherence tomography (OCT) has been used in limited settings to study peripheral nerve injury. The purpose of the study is to determine whether high-resolution OCT can be used to monitor nerve injury and regeneration in the rat sciatic nerve following crush injury, ligation, and transection with microsurgical repair. STUDY DESIGN/MATERIALS AND METHODS: Forty-five rats were segregated into three groups. The right sciatic nerve was suture ligated (n = 15), cut then microsurgically repaired (n = 15), or crushed (n = 15). The left sciatic nerve served as the control; only surgical exposure and skin closure were performed. Each group was further divided into three subgroups where they were assigned survival durations of 4, 15, or 24 weeks. Following euthanasia, nerves were harvested, fixed in formalin, and imaged at the injury site, as well as proximal and distal ends. The OCT system resolution was approximately 7 microm in tissue with a 1,060 nm central wavelength. RESULTS: Control (uninjured) nerve tissue showed homogenous signal distribution to a relatively uniform depth; in contrast, damaged nerves showed irregular signal distribution and intensity. Changes in signal distribution were most significant at the injury site and distal regions. Increases in signal irregularity were evident during longer recovery times. Histological analysis determined that OCT imaging was limited to the surrounding perineurium and scar tissue. CONCLUSION: OCT has the potential to be a valuable tool for monitoring nerve injury and repair, and the changes that accompany wound healing, providing clinicians with a non-invasive tool to treat nerve injuries.

Detection and monitoring of early airway injury effects of half-mustard (2-chloroethylethylsulfide) exposure using high-resolution optical coherence tomography.

Optical coherence tomography (OCT) is a noninvasive, high-resolution imaging technology capable of delivering real-time, near-histologic images of tissues. Mustard gas is a vesicant-blistering agent that can cause severe and lethal damage to airway and lungs. The ability to detect and assess airway injury in the clinical setting of mustard exposure is currently limited. The purpose of this study is to assess the ability to detect and monitor progression of half-mustard [2-chloroethylethylsulfide (CEES)] airway injuries with OCT techniques. A ventilated rabbit mustard exposure airway injury model is developed. A flexible fiber optic OCT probe is introduced into the distal trachea to image airway epithelium and mucosa in vivo. Progression of airway injury is observed over eight hours with OCT using a prototype time-domain superluminescent diode OCT system. OCT tracheal images from CEES exposed animals are compared to control rabbits for airway mucosal thickening and other changes. OCT detects the early occurrence and progression of dramatic changes in the experimental group after exposure to CEES. Histology and immunofluorescence staining confirms this finding. OCT has the potential to be a high resolution imaging modality capable of detecting, assessing, and monitoring treatment for airway injury following mustard vesicant agent exposures.

In vivo three-dimensional spectral domain endoscopic optical coherence tomography using a microelectromechanical system mirror.

A biopsy is a well-known medical test used to evaluate tissue abnormality. Biopsy specimens are invasively taken from part of a lesion and visualized by microscope after chemical treatment. However, diagnosis by means of biopsy is not only variable due to depth and location of specimen but may also damage the specimen. In addition, only a limited number of specimens can be obtained, thus, the entire tissue morphology cannot be observed. We introduce a three-dimensional (3-D) endoscopic optical biopsy via optical coherence tomography employing a dual-axis microelectromechanical system scanning mirror. Since this technique provides high-resolution, noninvasive, direct, and multiple visualization of tissue, it could function as a clinical biopsy with advanced performance. The device was integrated with a conventional endoscope and utilized to generate in vivo 3-D clinical images in humans and animals.

Use of polar decomposition for the diagnosis of oral precancer.

The Mueller matrix describes all the polarizing properties of a sample and, therefore, the optical differences between noncancerous and precancerous tissue that may be present within the matrix elements. A high-speed polarimetry system that generates 16 (4x4) full Mueller matrices to characterize tissues is presented. Feature extraction is done on the Mueller matrix elements resulting in depolarization and retardance images by polar decomposition. These are used to detect and classify early oral cancers and precancerous changes in epithelium such as dysplasia. These images are compared with orthogonal polarization images and analyzed in an attempt to identity useful factors for the differentiation between cancerous lesions and their benign counterparts. Our results indicate that polarimetry has potential as a method for the in vivo early detection and diagnosis of oral premalignancy.

Evaluation of rabbit tracheal inflammation using optical coherence tomography.

BACKGROUND: Optical coherence tomography (OCT) is an evolving technology that is capable of delivering real-time, high-resolution images of tissues. The purpose of this study was to evaluate the feasibility of using OCT for detecting airway pathology in a septic animal model. METHODS: The tracheas of New Zealand white rabbits were inoculated endobronchially with various concentrations of live Streptococcus pneumoniae bacteria. After the development of pneumonia/sepsis, the animals were killed. OCT tracheal images and corresponding histologic specimens from these experimental animals were compared to control rabbit tracheas for morphologic features and quantitative tracheal mucosal thickness measurements. RESULTS: The results revealed significant airway mucosal thickening in the experimental group that was consistent with tracheal edema. Morphologic changes, including epithelial denuding and mucosal sloughing, were evident in regions of the experimental tracheas. CONCLUSION: This study suggests that OCT is a potentially valuable imaging modality that is capable of evaluating superficial airway pathology with high-resolution in vivo images. Numerous applications of OCT can be envisioned in the realm of pulmonary medicine and thoracic surgery that may substantially increase the precision and accuracy of current bronchoscopic diagnostic and surgical techniques.

Feasibility of three-dimensional optical coherence tomography and optical Doppler tomography of malignancy in hamster cheek pouches.

OBJECTIVE: Hamster cheek pouches (HCP) with various degrees of 9,10-dimethyl-1,2-benzanthracene (DMBA)-induced dysplasia and malignancies were imaged with OCT/ODT in vivo and in vitro to assess the potential for three-dimensional high-resolution optical localization of airway malignancy. BACKGROUND DATA: Optical coherence tomography (OCT)/optical doppler tomography (ODT) provide potential capability for real-time in vivo high-resolution (2-20 microm) cross-sectional imaging of tissues and spatially resolved blood flow in microvasculature for pathology diagnostics. METHODS: DMBA was applied to the right side of the cheek pouch (HCP), and mineral oil (control) to the left side three times weekly for 10-18 weeks in Syrian Golden Hamsters using a standard protocol for malignancy induction. HCP were imaged in vivo with OCT/ODT as well as in vitro post-excision, using a prototype 1310-nm broadband superluminescent diode-based OCT/ODT device constructed in our laboratory. Three-dimensional images were constructed, and compared to standard and three-dimensional histology hematoxylin and eosin staining. RESULTS AND CONCLUSION: OCT imaging offered exceptional resolution of the HCP to depths of 1-2 mm and confirmed ability to detect dysplasia and malignancy. Three-dimensional OCT images were readily constructed, allowing visualization of extent and localization of tumor margins. ODT demonstrated increased vascularity in the area of neoplasia. OCT/ODT is a promising new technology for oral airway diagnostics.

Noninvasive imaging of oral premalignancy and malignancy.

Early detection of cancer and its precursors remains the best way to ensure patient survival and quality of life. Our specific aim is to test a multimodality approach to noninvasive diagnostics of oral premalignancy and malignancy. In the hamster cheek pouch model (120 hamsters), in vivo optical coherence tomography (OCT) and optical Doppler tomography (ODT) map epithelial, subepithelial, and vascular change throughout carcinogenesis. In vivo multiwavelength multiphoton (MPM) and second-harmonic generated (SHG) fluorescence techniques provided parallel data on surface and subsurface tissue structure, specifically collagen presence and structure, cellular presence, and vasculature. Images are diagnosed by two blinded, prestandardized investigators using a scale from 0 to 6 for all modalities. After sacrifice, histopathology is evaluated on a scale of 0 to 6. Imaging data are reproducibly obtained with good accuracy. Carcinogenesis-related structural and vascular changes are clearly visible to tissue depths of 2 mm. Sensitivity (OCT/ODT alone, 71 to 88%; OCT+MPMSHG, 79 to 91%) and specificity (OCT alone, 62 to 83%; OCT+MPMSHG, 67 to 90%) compare well with conventional techniques. Our conclusions are that OCT/ODT and MPM/SHG are promising noninvasive in vivo diagnostic modalities for oral dysplasia and malignancy.

In vivo optical coherence tomography for the diagnosis of oral malignancy.

BACKGROUND AND OBJECTIVE: Oral cancer results in 10,000 U.S. deaths annually. Improved highly sensitive diagnostics allowing early detection of oral cancer would benefit patient survival and quality of life. Objective was to investigate in vivo non-invasive optical coherence tomography (OCT) techniques for imaging and diagnosing neoplasia-related epithelial, sub-epithelial changes throughout carcinogenesis. STUDY DESIGN/MATERIALS AND METHODS: In the standard hamster cheek pouch model for oral carcinogenesis (n = 36), in vivo OCT was used to image epithelial and sub-epithelial change. OCT- and histopathology-based diagnoses on a scale of 0 (healthy) to 6 (squamous cell carcinoma, SCC) were performed at all stages throughout carcinogenesis by two blinded investigators. RESULTS: Epithelial, sub-epithelial structures were clearly discernible using OCT. OCT diagnosis agreed with the histopathological gold standard in 80% of readings. CONCLUSION: In vivo OCT demonstrates excellent potential as a diagnostic tool in the oral cavity.

Feasibility study of normal and septic tracheal imaging using optical coherence tomography.

BACKGROUND AND OBJECTIVES: Optical coherence tomography (OCT) is an imaging technology that may be adapted for use with flexible fiberoptic bronchoscopy, potentially allowing it to play an important role in pulmonary diagnostics. The goal of this study was to evaluate the feasibility of OCT to image tracheal pathology. STUDY DESIGN/MATERIALS AND METHODS: Tracheas were harvested from normal and septic New Zealand White rabbits and imaged using OCT. Two delivery devices were employed. One was a moving stage with an objective lens and collimator, the other a linear scanning flexible fiberoptic catheter using a GRIN lens and prism for endoscopic OCT. After OCT images were obtained from normal and septic tracheas, the excised tissues were prepared for standard histologic examination. Areas imaged by OCT were compared with corresponding histology slides. RESULTS: OCT images demonstrated in detail tracheal sub-surface structures such as the epithelium, lamina propria, submucosa, and cartilage. The appearance of structures imaged by OCT corresponded very well with histologic pictures obtained by light microscopy. The OCT images from septic tracheas showed marked swelling of the mucosal and submucosal layers. Such pathology was equally imaged by either the moving stage or fiberoptic catheter for endoscopic OCT. CONCLUSIONS: OCT images of the trachea can distinguish many sub-surface structural features usually requiring biopsy and light microscopy for visualization. Marked differences between normal and septic trachea were apparent in OCT images. In the future, OCT may be a valuable tool for evaluating tracheal pathology in situ with high image resolution.