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Confocal laser endoscopy is a new method of real-time optical biopsy. Fluorescent images of histological quality can be obtained instantly from the epithelium of hollow organs. Currently, scanning is performed proximally with probe-based instruments that are commonly used in clinical practice, with limited flexibility in focus control. We demonstrate the use of a parametric resonant scanner mounted at the distal end of an endoscope to perform high-speed lateral deflection. A hole has been etched into the center of the reflector to roll up the light path. This design reduces the size of the instrument to 2.4 mm in diameter and 10 mm in length, allowing it to be passed forward through the working channel of standard medical endoscopes. The compact lens provides lateral and axial resolutions of 1.1 and 13.6 µm, respectively. A working distance of 0 µm and a field of view of 250 µm × 250 µm are achieved at frame rates up to 20 Hz. Excitation at 488 nm excites fluorescein, an FDA approved dye for high tissue contrast. Endoscopes have been reprocessed for 18 cycles without failure using clinically approved sterilization methods. Fluorescent images were obtained from normal colonic mucosa, tubular adenomas, hyperplastic polyps, ulcerative colitis, and Crohn’s colitis during routine colonoscopy. Single cells can be identified, including colonocytes, goblet cells, and inflammatory cells. Mucosal features such as crypt structures, crypt cavities, and lamina propria can be distinguished. The instrument can be used as an adjunct to conventional endoscopy.
Confocal laser endoscopy is a novel imaging modality being developed for clinical use as an adjunct to routine endoscopy1,2,3. These flexible, fiber-optic-connected instruments can be used to detect diseases in the epithelial cells that line hollow organs, such as the colon. This thin layer of tissue is highly metabolically active and is the source of many disease processes such as cancer, infection, and inflammation. Endoscopy can achieve subcellular resolution, providing real-time, near-histological quality in vivo images to help clinicians make clinical decisions. Physical tissue biopsy carries the risk of bleeding and perforation. Too many or too few biopsy specimens are often collected. Each sample removed increases the surgical cost. It takes several days for the sample to be evaluated by a pathologist. During the days of waiting for pathology results, patients often experience anxiety. In contrast, other clinical imaging modalities such as MRI, CT, PET, SPECT, and ultrasound lack the spatial resolution and temporal speed required to visualize epithelial processes in vivo with real-time, subcellular resolution.
A probe-based instrument (Cellvizio) is currently commonly used in clinics to perform “optical biopsy”. The design is based on a spatially coherent fiber optic bundle4 that collects and transmits fluorescent images. The single fiber core acts as a “hole” to spatially filter defocused light for subcellular resolution. Scanning is performed proximally using a large, bulky galvanometer. This provision limits the ability of the focus control tool. Proper staging of early epithelial carcinoma requires visualization below the tissue surface to assess invasion and determine appropriate therapy. Fluorescein, an FDA-approved contrast agent, is administered intravenously to highlight structural features of the epithelium. These endomicroscopes have dimensions <2.4 mm in diameter, and can be passed forward easily through the biopsy channel of standard medical endoscopes. These endomicroscopes have dimensions <2.4 mm in diameter, and can be passed forward easily through the biopsy channel of standard medical endoscopes. Эти эндомикроскопы имеют размеры <2,4 мм в диаметре и могут быть легко проведены через биопсийный канал стандартных медицинских эндоскопов. These endomicroscopes are <2.4 mm in diameter and can be easily passed through the biopsy channel of standard medical endoscopes. These borescopes are less than 2.4 mm in diameter and easily pass through the biopsy channel of standard medical borescopes. This flexibility allows for a wide range of clinical applications and is independent of endoscope manufacturers. Numerous clinical studies have been performed using this imaging device, including the early detection of cancers of the esophagus, stomach, colon, and oral cavity. Imaging protocols have been developed and the safety of the procedure has been established.
Microelectromechanical systems (MEMS) is a powerful technology for designing and manufacturing tiny scanning mechanisms used in the distal end of endoscopes. This position (relative to proximal) allows for greater flexibility in controlling the focus position5,6. In addition to lateral deflection, the distal mechanism can also perform axial scans, post-objective scans, and random access scans. These capabilities enable more comprehensive epithelial cell interrogation, including vertical cross-sectional imaging7, large field of view (FOV)8 aberration-free scanning, and improved performance in user-defined sub-regions9. MEMS solves the serious problem of packaging the scanning engine with the limited space available at the far end of the instrument. Compared to bulky galvanometers, MEMS provide superior performance at a small size, high speed, and low power consumption. A simple manufacturing process can be scaled up for mass production at low cost. Many MEMS designs have been previously reported10,11,12. None of the technologies has yet been sufficiently developed to enable the widespread clinical use of real-time in vivo imaging through the working channel of a medical endoscope. Here, we aim to demonstrate the use of a MEMS scanner at the distal end of an endoscope for in vivo human image acquisition during routine clinical endoscopy.
A fiber optic instrument was developed using a MEMS scanner at the distal end to collect real-time in vivo fluorescent images with similar histological characteristics. A single-mode fiber (SMF) is enclosed in a flexible polymer tube and excited at λex = 488 nm. This configuration shortens the length of the distal tip and allows it to be passed forward through the working channel of standard medical endoscopes. Use the tip to center the optic. These lenses are designed to achieve nearly diffractive axial resolution with a numerical aperture (NA) = 0.41 and working distance = 0 µm13. Precision shims are made to precisely align the optics 14. The scanner is packaged in an endoscope with a rigid distal tip 2.4 mm in diameter and 10 mm long (Fig. 1a). These dimensions allow it to be used in clinical practice as an accessory during endoscopy (Fig. 1b). The maximum power of the laser incident on the tissue was 2 mW.
Confocal laser endoscopy (CLE) and MEMS scanners. Photograph showing (a) a packaged instrument with rigid distal tip dimensions of 2.4 mm diameter and 10 mm length and (b) straight passage through the working channel of a standard medical endoscope (Olympus CF-HQ190L). (c) Front view of the scanner showing a reflector with a central aperture of 50 µm through which the excitation beam passes. The scanner is mounted on a gimbal driven by a set of quadrature comb drive drives. The resonant frequency of the device is determined by the size of the torsion spring. (d) Side view of the scanner showing the scanner mounted on a stand with wires connected to electrode anchors that provide connection points for drive and power signals.
The scanning mechanism consists of a gimbal-mounted reflector driven by a set of comb-driven quadrature actuators to deflect the beam laterally (XY plane) in a Lissajous pattern (Fig. 1c). A hole 50 µm in diameter was etched in the center through which the excitation beam passed. The scanner is driven at the resonant frequency of the design, which can be tuned by changing the dimensions of the torsion spring. Electrode anchors were engraved on the periphery of the device to provide connection points for power and control signals (Fig. 1d).
The imaging system is mounted on a portable cart that can be rolled into the operating room. The graphical user interface has been designed to support users with minimal technical knowledge, such as doctors and nurses. Manually check the scanner drive frequency, beamform mode, and image FOV.
The overall length of the endoscope is approximately 4m to allow full passage of instruments through the working channel of a standard medical endoscope (1.68m), with an extra length for maneuverability. At the proximal end of the endoscope, the SMF and wires terminate in connectors that connect to the fiber optic and wired ports of the base station. The installation contains a laser, a filter unit, a high-voltage amplifier and a photomultiplier detector (PMT). The amplifier supplies power and drive signals to the scanner. The optical filter unit couples the laser excitation to the SMF and passes the fluorescence to the PMT.
Endoscopes are reprocessed after each clinical procedure using the STERRAD sterilization process and can withstand up to 18 cycles without failure. For the OPA solution, no signs of damage were observed after more than 10 disinfection cycles. OPA’s results outperformed STERRAD’s, suggesting that the life of endoscopes could be extended by high-level disinfection rather than re-sterilization.
Image resolution was determined from the point spread function using fluorescent beads with a diameter of 0.1 μm. For lateral and axial resolution, a full width at half maximum (FWHM) of 1.1 and 13.6 µm, respectively, was measured (Fig. 2a, b).
Image options. The lateral (a) and axial (b) resolution of the focusing optics are characterized by the point spread function (PSF) measured using fluorescent microspheres with a diameter of 0.1 μm. The measured full width at half maximum (FWHM) was 1.1 and 13.6 µm, respectively. Inset: Expanded views of a single microsphere in the transverse (XY) and axial (XZ) directions are shown. (c) Fluorescent image obtained from a standard (USAF 1951) target strip (red oval) showing that groups 7-6 can be clearly resolved. (d) Image of 10 µm diameter dispersed fluorescent microspheres showing an image field of view of 250 µm×250 µm. The PSFs in (a, b) were built using MATLAB R2019a (https://www.mathworks.com/). (c, d) Fluorescent images were collected using LabVIEW 2021 (https://www.ni.com/).
Fluorescent images from standard resolution lenses clearly distinguish the set of columns in groups 7-6, which maintains high lateral resolution (Fig. 2c). The field of view (FOV) of 250 µm × 250 µm was determined from images of 10 µm diameter fluorescent beads dispersed on coverslips (Fig. 2d).
An automated method for PMT gain control and phase correction is implemented in a clinical imaging system to reduce motion artifacts from endoscopes, colon peristalsis, and patient breathing. Image reconstruction and processing algorithms have been described previously14,15. The PMT gain is controlled by a proportional-integral (PI) controller to prevent intensity saturation16. The system reads the maximum pixel intensity for each frame, calculates the proportional and integral responses, and determines PMT gain values ​​to ensure that the pixel intensity is within the allowable range.
During in vivo imaging, phase mismatch between scanner movement and control signal can cause image blur. Such effects may occur due to changes in the temperature of the device inside the human body. White light images showed that the endoscope was in contact with normal colonic mucosa in vivo (Figure 3a). Blurring of misaligned pixels can be seen in raw images of normal colonic mucosa (Figure 3b). After treatment with proper phase and contrast adjustment, subcellular features of the mucosa could be distinguished (Fig. 3c). For additional information, raw confocal images and processed real-time images are shown in Fig. S1, and the image reconstruction parameters used for real-time and post-processing are presented in Table S1 and Table S2.
Image processing. (a) Wide-angle endoscopic image showing an endoscope (E) placed in contact with normal (N) colonic mucosa to collect in vivo fluorescent images after fluorescein administration. (b) Wander in the X and Y axes during scanning can cause misaligned pixels to blur. For demonstration purposes, a large phase shift is applied to the original image. (c) After post-processing phase correction, mucosal details can be assessed, including crypt structures (arrows), with a central lumen (l) surrounded by the lamina propria (lp). Single cells can be distinguished, including colonocytes (c), goblet cells (g), and inflammatory cells (arrows). See additional video 1. (b, c) Images processed using LabVIEW 2021.
Confocal fluorescence images have been obtained in vivo in several colonic diseases to demonstrate the wide clinical applicability of the instrument. Wide-angle imaging is first performed using white light to detect grossly abnormal mucosa. The endoscope is then advanced through the working channel of the colonoscope and brought into contact with the mucosa.
Wide-field endoscopy, confocal endomicroscopy, and histology (H&E) images are shown for colonic neoplasia, including tubular adenoma and hyperplastic polyp. Wide-field endoscopy, confocal endomicroscopy, and histology (H&E) images are shown for colonic neoplasia, including tubular adenoma and hyperplastic polyp. Широкопольная эндоскопия, конфокальная эндомикроскопия и гистологические (H&E) изображения показаны для неоплазии толстой кишки, включая тубулярную аденому и гиперпластический полип. Colonic endoscopy, confocal endomicroscopy, and histological (H&E) imaging are indicated for colonic neoplasia, including tubular adenoma and hyperplastic polyp.显示结肠肿瘤（包括管状腺瘤和增生性息肉）的广角内窥镜检查、共聚焦显微内窥镜检查和组织学(H&E) 图像。共设计脚肠化(图像管状躰化和增生性息肉)的广角内刵霱录共共共光在微微全在圕别具和结果学(H&E) image. Широкопольная эндоскопия, конфокальная микроэндоскопия и гистологические (H&E) изображения, показывающие опухоли толстой кишки, включая тубулярные аденомы и гиперпластические полипы. Broad-field endoscopy, confocal microendoscopy, and histological (H&E) images showing tumors of the colon, including tubular adenomas and hyperplastic polyps. Tubular adenomas showed loss of normal crypt architecture, reduction in the size of goblet cells, distortion of the crypt lumen, and thickening of the lamina propria (Fig. 4a-c). Hyperplastic polyps showed stellate architecture of crypts, few goblet cells, slit-like lumen of crypts, and irregular lamellar crypts (Fig. 4d-f).
Image of mucosal thick skin in vivo. Representative white light endoscopy, confocal endomicroscope, and histology (H&E) images are shown for (ac) adenoma, (df) hyperplastic polyp, (gi) ulcerative colitis, and (jl) Crohn’s colitis. Representative white light endoscopy, confocal endomicroscope, and histology (H&E) images are shown for (ac) adenoma, (df) hyperplastic polyp, (gi) ulcerative colitis, and (jl) Crohn’s colitis. Типичные изображения эндоскопии в белом свете, конфокального эндомикроскопа и гистологии (H&E) показаны для (ac) аденомы, (df) гиперпластического полипа, (gi) язвенного колита и (jl) колита Крона. Typical white-light endoscopy, confocal endomicroscope, and histology (H&E) images are shown for (ac) adenoma, (df) hyperplastic polyp, (gi) ulcerative colitis, and (jl) Crohn’s colitis.显示了(ac) 腺瘤、(df) 增生性息肉、(gi) 溃疡性结肠炎和(jl) 克罗恩结肠炎的代表性白光内窥镜检查、共聚焦内窥镜检查和组织学(H&E) 图像。 It shows(ac) 躰真、(df) 增生性息肉、(gi) 苏盖性红肠炎和(jl) 克罗恩红肠炎的体育性白光内肠肠炎性、共公司内肠肠炎性和电视学( H&E) image. Представлены репрезентативные эндоскопия в белом свете, конфокальная эндоскопия и гистология (ac) аденомы, (df) гиперпластического полипоза, (gi) язвенного колита и (jl) колита Крона (H&E). Representative white-light endoscopy, confocal endoscopy, and histology of (ac) adenoma, (df) hyperplastic polyposis, (gi) ulcerative colitis, and (jl) Crohn’s colitis (H&E) are shown. (B) shows a confocal image obtained in vivo from a tubular adenoma (TA) using an endoscope (E). This precancerous lesion shows loss of normal crypt architecture (arrow), distortion of the crypt lumen (l), and crowding of the crypt lamina propria (lp). Colonocytes (c), goblet cells (g), and inflammatory cells (arrows) can also be identified. Smt. Supplementary Video 2. (e) shows a confocal image obtained from a hyperplastic polyp (HP) in vivo. This benign lesion demonstrates a stellate crypt architecture (arrow), a slit-like crypt lumen (l), and an irregularly shaped lamina propria (lp). Colonocytes (c), several goblet cells (g) and inflammatory cells (arrows) can also be identified. Smt. Supplementary Video 3. (h) shows confocal images acquired in ulcerative colitis (UC) in vivo. This inflammatory condition shows distorted crypt architecture (arrow) and prominent goblet cells (g). Feathers of fluorescein (f) are extruded from epithelial cells, reflecting increased vascular permeability. Numerous inflammatory cells (arrows) are seen in the lamina propria (lp). Smt. Supplementary Video 4. (k) shows a confocal image obtained in vivo from a region of Crohn’s colitis (CC). This inflammatory condition shows distorted crypt architecture (arrow) and prominent goblet cells (g). Feathers of fluorescein (f) are extruded from epithelial cells, reflecting increased vascular permeability. Numerous inflammatory cells (arrows) are seen in the lamina propria (lp). Smt. Supplementary Video 5. (b, d, h, l) Images processed using LabVIEW 2021.
A similar set of images of colonic inflammation is shown, including ulcerative colitis (UC) (Figure 4g-i) and Crohn’s colitis (Figure 4j-l). The inflammatory response is thought to be characterized by distorted crypt structures with protruding goblet cells. Fluorescein is squeezed out of epithelial cells, reflecting increased vascular permeability. A large number of inflammatory cells can be seen in the lamina propria.
We have demonstrated the clinical application of a flexible fiber-coupled confocal laser endoscope that uses a distally positioned MEMS scanner for in vivo image acquisition. At resonant frequency, frame rates up to 20 Hz can be achieved using a high-density Lissajous scan mode to reduce motion artifacts. The optical path is folded to provide beam expansion and a numerical aperture sufficient to achieve a lateral resolution of 1.1 µm. Fluorescent images of histological quality were obtained during routine colonoscopy of normal colonic mucosa, tubular adenomas, hyperplastic polyps, ulcerative colitis, and Crohn’s colitis. Single cells can be identified, including colonocytes, goblet cells, and inflammatory cells. Mucosal features such as crypt structures, crypt cavities, and lamina propria can be distinguished. The precision hardware is micro-machined to ensure precise alignment of the individual optical and mechanical components within the 2.4mm diameter x 10mm length instrument. The optical design reduces the length of the rigid distal tip sufficiently to permit direct passage through a standard size (3.2 mm diameter) working channel in medical endoscopes. Therefore, regardless of the manufacturer, the device can be widely used by doctors at the place of residence. Excitation was performed at λex = 488 nm to excite fluorescein, an FDA approved dye, to obtain high contrast. The instrument was reprocessed without problems for 18 cycles using clinically accepted sterilization methods.
Two other instrument designs have been clinically validated. Cellvizio (Mauna Kea Technologies) is a probe-based confocal laser endoscope (pCLE) that uses a bundle of multimode coherent fiber optic cables to collect and transmit fluorescence images1. A galvo mirror located on the base station performs a lateral scan at the proximal end. Optical sections are collected in the horizontal (XY) plane with a depth of 0 to 70 µm. Microprobe kits are available from 0.91 (19 G needle) to 5 mm in diameter. A lateral resolution of 1 to 3.5 µm was achieved. Images were collected at a frame rate of 9 to 12 Hz with a one-dimensional field of view from 240 to 600 µm. The platform has been used clinically in a variety of areas including the bile duct, bladder, colon, esophagus, lungs, and pancreas. Optiscan Pty Ltd has developed an endoscope-based confocal laser endoscope (eCLE) with a scanning engine built into the insertion tube (distal end) of a professional endoscope (EC-3870K, Pentax Precision Instruments) 17 . The optical section was carried out using a single-mode fiber, and side scanning was carried out using a cantilever mechanism through a resonant tuning fork. A Shape Memory Alloy (Nitinol) actuator is used to create axial displacement. The total diameter of the confocal module is 5 mm. For focusing, a GRIN lens with a numerical aperture of NA = 0.6 is used. Horizontal images were acquired with lateral and axial resolutions of 0.7 and 7 µm, respectively, at a frame rate of 0.8–1.6 Hz and a field of view of 500 µm × 500 µm.
We demonstrate subcellular resolution in vivo fluorescence imaging acquisition from the human body through a medical endoscope using a distal end MEMS scanner. Fluorescence provides high image contrast, and ligands that bind to cell surface targets can be labeled with fluorophores to provide molecular identity for improved disease diagnosis18. Other optical techniques for in vivo microendoscopy are also being developed. OCT uses the short coherence length from a broadband light source to collect images in the vertical plane with depths >1 mm19. OCT uses the short coherence length from a broadband light source to collect images in the vertical plane with depths >1 mm19. ОКТ использует короткую длину когерентности широкополосного источника света для сбора изображений в вертикальной плоскости с глубиной >1 мм19. OCT uses the short coherence length of a broadband light source to acquire images in the vertical plane with >1 mm depth19. OCT 使用宽带光源的短相干长度来收集垂直平面中深度> 1 mm19 的图像。 1 mm19 的图像。 ОКТ использует короткую длину когерентности широкополосного источника света для сбора изображений на глубине >1 мм19 в вертикальной плоскости. OCT uses the short coherence length of a broadband light source to acquire images >1 mm19 in the vertical plane. However, this low-contrast approach relies on backscattered light collection and image resolution is limited by speckle artifacts. Photoacoustic endoscopy generates in vivo images based on rapid thermoelastic expansion in tissue after absorption of a laser pulse that generates sound waves20. This approach has demonstrated imaging depths >1 cm in human colon in vivo to monitor therapy. This approach has demonstrated imaging depths >1 cm in human colon in vivo to monitor therapy. Этот подход продемонстрировал глубину визуализации > 1 см в толстой кишке человека in vivo для мониторинга терапии. This approach has demonstrated an imaging depth of >1 cm in the human colon in vivo for therapy monitoring.这种方法已经证明在体内人结肠中成像深度> 1 厘米以监测治疗。这种方法已经证明在体内人结肠中成像深度> 1 Этот подход был продемонстрирован на глубине изображения > 1 см в толстой кишке человека in vivo для мониторинга терапии. This approach has been demonstrated at imaging depths >1 cm in the human colon in vivo to monitor therapy. The contrast is mainly produced by hemoglobin in the vasculature. Multiphoton endoscopy generates high-contrast fluorescence images when two or more NIR photons hit tissue biomolecules simultaneously21. This approach can achieve imaging depths >1 mm with low phototoxicity. This approach can achieve imaging depths >1 mm with low phototoxicity. Этот подход может обеспечить глубину изображения > 1 мм с низкой фототоксичностью. This approach can provide image depth > 1 mm with low phototoxicity.这种方法可以实现>1 毫米的成像深度，光毒性低。这种方法可以实现>1 毫米的成像深度，光毒性低。 Этот подход может обеспечить глубину изображения > 1 мм с низкой фототоксичностью. This approach can provide image depth > 1 mm with low phototoxicity. High intensity femtosecond laser pulses are required and this method has not been clinically proven during endoscopy.
In this prototype, the scanner performs only lateral deflection, so the optical part is in the horizontal (XY) plane. The device is capable of operating at a higher frame rate (20 Hz) than the galvanic mirrors (12 Hz) in the Cellvizio system. Increase the frame rate to reduce motion artifacts and decrease the frame rate to boost the signal. High-speed and automated algorithms are needed to mitigate large motion artifacts caused by endoscopic motion, respiratory motion, and intestinal motility. Parametric resonant scanners have been shown to achieve axial displacements in excess of hundreds of microns22. Images can be collected in vertical plane (XZ), perpendicular to the mucosal surface, to provide the same view as that of histology (H&E). Images can be collected in vertical plane (XZ), perpendicular to the mucosal surface, to provide the same view as that of histology (H&E). Изображения могут быть получены в вертикальной плоскости (XZ), перпендикулярной поверхности слизистой оболочки, чтобы обеспечить такое же изображение, как при гистологии (H&E). Images can be taken in a vertical plane (XZ) perpendicular to the mucosal surface to provide the same image as in histology (H&E).可以在垂直于粘膜表面的垂直平面(XZ) 中收集图像，以提供与组织学(H&E) 相同的视图。可以在垂直于粘膜表面的垂直平面(XZ) 中收集图像，以提供与组织学(H&E) Изображения могут быть получены в вертикальной плоскости (XZ), перпендикулярной поверхности слизистой оболочки, чтобы обеспечить такое же изображение, как при гистологическом исследовании (H&E). Images can be taken in a vertical plane (XZ) perpendicular to the mucosal surface to provide the same image as a histological examination (H&E). The scanner can be placed in a post-objective position where the illumination beam falls along the main optical axis to reduce sensitivity to aberrations8. Nearly diffraction-limited focal volumes can deviate over arbitrarily large fields of view. Random access scanning can be performed to deflect reflectors to user-defined positions9. The field of view can be reduced to highlight arbitrary areas of the image, improving the signal-to-noise ratio, contrast, and frame rate. Scanners can be mass-produced using simple processes. Hundreds of devices can be made on each silicon wafer to increase production for low cost mass production and wide distribution.
The folded light path reduces the size of the rigid distal tip, making it easy to use the endoscope as an accessory during routine colonoscopy. In the fluorescent images shown, subcellular features of the mucosa can be seen to distinguish tubular adenomas (precancerous) from hyperplastic polyps (benign). These results suggest that endoscopy can reduce the number of unnecessary biopsies23. General complications associated with surgery can be reduced, monitoring intervals can be optimized, and histological analysis of minor lesions can be minimized. We also show in vivo images of patients with inflammatory bowel disease, including ulcerative colitis (UC) and Crohn’s colitis. Conventional white light colonoscopy provides a macroscopic view of the mucosal surface with limited ability to accurately assess mucosal healing. Endoscopy can be used in vivo to evaluate the efficacy of biological therapies such as anti-TNF24 antibodies. Accurate in vivo assessment can also reduce or prevent disease recurrence and complications such as surgery and improve quality of life. No serious adverse reactions have been reported in clinical studies associated with the use of fluorescein-containing endoscopes in vivo25. The laser power on the mucosal surface was limited to <2 mW to minimize risk for thermal injury and meet the FDA requirements for non-significant risk26 per 21 CFR 812. The laser power on the mucosal surface was limited to <2 mW to minimize the risk for thermal injury and meet the FDA requirements for non-significant risk26 per 21 CFR 812. Мощность лазера на поверхности слизистой оболочки была ограничена до <2 мВт, чтобы свести к минимуму риск термического повреждения и соответствовать требованиям FDA относительно незначительного риска26 согласно 21 CFR 812. The laser power at the mucosal surface was limited to <2 mW to minimize the risk of thermal damage and meet FDA requirements for negligible risk26 under 21 CFR 812.粘膜表面的激光功率限制在<2 mW，以最大限度地降低热损伤风险，并满足FDA 21 CFR 812 对非重大风险26 的要求。粘膜表面的激光功率限制在<2 mW Мощность лазера на поверхности слизистой оболочки была ограничена до <2 мВт, чтобы свести к минимуму риск термического повреждения и соответствовать требованиям FDA 21 CFR 812 относительно незначительного риска26. The laser power at the mucosal surface was limited to <2 mW to minimize the risk of thermal damage and meet FDA 21 CFR 812 requirements for negligible risk26.
The design of the instrument can be modified to improve image quality. Special optics are available to reduce spherical aberration, improve image resolution and increase working distance. The SIL can be tuned to better match the refractive index of the tissue (~1.4) to improve light coupling. The drive frequency can be adjusted to increase the lateral angle of the scanner and widen the image field of view. You can use automated methods to remove frames of an image with significant movement to mitigate this effect. A field-programmable gate array (FPGA) with high-speed data acquisition will be used to provide high-performance real-time full-frame correction. For greater clinical utility, automated methods must correct for phase shift and motion artifacts for real-time image interpretation. A monolithic 3-axis parametric resonant scanner can be implemented to introduce axial scanning 22 . These devices have been developed to achieve unprecedented vertical displacement >400 µm by tuning the drive frequency in a regime that features mixed softening/stiffening dynamics27. These devices have been developed to achieve unprecedented vertical displacement >400 µm by tuning the drive frequency in a regime that features mixed softening/stiffening dynamics27. Эти устройства были разработаны для достижения беспрецедентного вертикального смещения > 400 мкм путем настройки частоты возбуждения в режиме, который характеризуется смешанной динамикой смягчения/жесткости27. These devices have been designed to achieve an unprecedented vertical displacement of >400 µm by setting the drive frequency in a mode that is characterized by mixed soft/hard dynamics27.这些设备的开发是为了通过在具有混合软化/硬化动力学的状态下调整驱动频率来实现前所未有的>400 µm 的垂直位移27。这些 设备 的 开发 是 为了 在 具有 混合 软化 硬化 硬化 学 学 状态 下 调整 驱动频率 来 实现 的> 400 µm 的 垂直 位移 27。 Эти устройства были разработаны для достижения беспрецедентных вертикальных смещений >400 мкм путем настройки частоты срабатывания в режиме со смешанной кинетикой размягчения/затвердевания27. These devices have been designed to achieve unprecedented vertical displacements >400 µm by adjusting the trigger frequency in mixed softening/hardening kinetics mode27. In the future, vertical transverse imaging may help in staging early cancer (T1a). A capacitive sensing circuit can be implemented to track scanner movement and correct for phase shift 28 . Automatic phase calibration using a sensor circuit can replace manual instrument calibration prior to use. Instrument reliability can be improved by using more reliable instrument sealing techniques to increase the number of processing cycles. MEMS technology promises to accelerate the use of endoscopes for visualizing the epithelium of hollow organs, diagnosing disease, and monitoring treatment in a minimally invasive way. With further development, this new imaging modality could become a low-cost solution to be used as an adjunct to medical endoscopes for immediate histological examination and could eventually replace traditional pathological analysis.
Ray tracing simulations were performed using ZEMAX optical design software (version 2013) to determine the parameters of the focusing optics. Design criteria include near-diffractive axial resolution, working distance = 0 µm, and field of view (FOV) greater than 250 × 250 µm2. For excitation at a wavelength λex = 488 nm, a single-mode fiber (SMF) was used. Achromatic doublets are used to reduce the variance of the fluorescence collection (Figure 5a). The beam passes through the SMF with a mode field diameter of 3.5 μm and without truncation passes through the center of the reflector with an aperture diameter of 50 μm. Use a hard immersion (hemispherical) lens with a high refractive index (n = 2.03) to minimize incident beam spherical aberration and ensure full contact with the mucosal surface. Focusing optics provides a total NA = 0.41, where NA = nsinα, n is the refractive index of the tissue, α is the maximum beam convergence angle. The diffraction-limited lateral and axial resolutions are 0.44 and 6.65 µm, respectively, using NA = 0.41, λ = 488 nm, and n = 1.3313. Only commercially available lenses with outer diameter (OD) ≤ 2 mm were considered. The optical path is folded, and the beam leaving the SMF passes through the central aperture of the scanner and is reflected back by a fixed mirror (0.29 mm in diameter). This configuration shortens the length of the rigid distal end to facilitate forward passage of the endoscope through the standard (3.2 mm diameter) working channel of medical endoscopes. This feature makes it easy to use as an accessory during routine endoscopy.
Folded light guide and endoscope packaging. (a) The excitation beam exits the OBC and passes through the central aperture of the scanner. The beam is expanded and reflected from a fixed circular mirror back into the scanner for lateral deflection. The focusing optics consist of a pair of achromatic doublet lenses and a solid immersion (hemispherical) lens providing contact with the mucosal surface. ZEMAX 2013 (https://www.zemax.com/) for optical design and ray tracing simulation. (b) Shows the location of various instrument components, including single mode fiber (SMF), scanner, mirrors, and lenses. Solidworks 2016 (https://www.solidworks.com/) was used for 3D modeling of the endoscope packaging.
An SMF (#460HP, Thorlabs) with a mode field diameter of 3.5 µm at a wavelength of 488 nm was used as a “hole” for spatial filtering of defocused light (Fig. 5b). The SMFs are enclosed in flexible polymer tubes (#Pebax 72D, Nordson MEDICAL). A length of approximately 4 meters is used to ensure sufficient distance between the patient and the imaging system. A pair of 2 mm MgF2 coated achromatic doublet lenses (#65568, #65567, Edmund Optics) and a 2 mm uncoated hemispherical lens (#90858, Edmund Optics) were used to focus the beam and collect fluorescence. Insert a stainless steel end tube (4 mm long, 2.0 mm OD, 1.6 mm ID) between the resin and the outer tube to isolate scanner vibration. Use medical adhesives to protect the instrument from body fluids and handling procedures. Use heat shrink tubing to protect the connectors.
The compact scanner is made on the principle of parametric resonance. Etch a 50 µm aperture at the center of the reflector to transmit the excitation beam. Using a set of quadrature comb-driven drives, the expanded beam is deflected transversely in the orthogonal direction (XY plane) in Lissajous mode. A data acquisition board (#DAQ PCI-6115, NI) was used to generate analog signals to control the scanner. Power was provided by a high voltage amplifier (#PDm200, PiezoDrive) via thin wires (#B4421241, MWS Wire Industries). Make wiring on the electrode armature. The scanner operates at frequencies close to 15 kHz (fast axis) and 4 kHz (slow axis) to achieve FOV up to 250 µm × 250 µm. Video can be shot at a frame rate of 10, 16, or 20 Hz. These frame rates are used to match the repetition rate of the Lissajous scan pattern, which depends on the value of the X and Y excitation frequencies of the scanner29. Details of the trade-offs between frame rate, pixel resolution, and scan pattern density are presented in our previous work14.
A solid state laser (#OBIS 488 LS, coherent) provides λex = 488 nm to excite fluorescein for image contrast (Fig. 6a). Optical pigtails are connected to the filter unit via FC/APC connectors (loss 1.82 dB) (Fig. 6b). The beam is deflected by a dichroic mirror (#WDM-12P-111-488/500:600, Oz Optics) in the SMF through another FC/APC connector. In accordance with 21 CFR 812, incident power to tissue is limited to a maximum of 2 mW to meet FDA requirements for negligible risk. Fluorescence was passed through a dichroic mirror and a long transmission filter (#BLP01-488R, Semrock). Fluorescence was transmitted to a photomultiplier tube (PMT) detector (#H7422-40, Hamamatsu) via an FC/PC connector using a ~1 m long multimode fiber with a 50 µm core diameter. Fluorescent signals were amplified with a high speed current amplifier (#59-179, Edmund Optics). Special software (LabVIEW 2021, NI) has been developed for real-time data acquisition and image processing. The laser power and PMT gain settings are determined by the microcontroller (#Arduino UNO, Arduino) using a special printed circuit board. The SMF and wires terminate in connectors and connect to the fiber optic (F) and wired (W) ports on the base station (Figure 6c). The imaging system is contained on a portable cart (Figure 6d). An isolation transformer was used to limit the leakage current to <500 μA. An isolation transformer was used to limit the leakage current to <500 μA. Для ограничения тока утечки до <500 мкА использовался изолирующий трансформатор. An isolation transformer was used to limit the leakage current to <500 µA.使用隔离变压器将泄漏电流限制在<500 μA。 <500 μA。 Используйте изолирующий трансформатор, чтобы ограничить ток утечки до <500 мкА. Use an isolation transformer to limit the leakage current to <500µA.
visualization system. (a) The PMT, laser and amplifier are in the base station. (b) In the filter bank, the laser (blue) is driving over the fiber optic cable through the FC/APC connector. The beam is deflected by a dichroic mirror (DM) into a single mode fiber (SMF) via a second FC/APC connector. Fluorescence (green) travels through the DM and long pass filter (LPF) to the PMT via multimode fiber (MMF). (c) The proximal end of the endoscope is connected to the fiber optic (F) and wired (W) ports of the base station. (d) Endoscope, monitor, base station, computer, and isolation transformer on a portable cart. (a, c) Solidworks 2016 was used for 3D modeling of the imaging system and endoscope components.
The lateral and axial resolution of the focusing optics was measured from the point spread function of fluorescent microspheres (#F8803, Thermo Fisher Scientific) 0.1 µm in diameter. Collect images by translating the microspheres horizontally and vertically in 1 µm steps using a linear stage (# M-562-XYZ, DM-13, Newport). Image stack using ImageJ2 to acquire cross-sectional images of microspheres.
Special software (LabVIEW 2021, NI) has been developed for real-time data acquisition and image processing. On fig. 7 shows an overview of the routines used to operate the system. The user interface consists of data acquisition (DAQ), main panel and controller panel. The data collection panel interacts with the main panel to collect and store raw data, provide input for custom data collection settings, and manage scanner driver settings. The main panel allows the user to select the desired configuration for using the endoscope, including the scanner control signal, video frame rate, and acquisition parameters. This panel also allows the user to display and control the brightness and contrast of the image. Using the raw data as input, the algorithm calculates the optimal gain setting for the PMT and automatically adjusts this parameter using a proportional-integral (PI)16 feedback control system. The controller board interacts with the main board and the data acquisition board to control the laser power and PMT gain.
System software architecture. The user interface consists of modules (1) data acquisition (DAQ), (2) main panel and (3) controller panel. These programs run concurrently and communicate with each other through message queues. The key is MEMS: Microelectromechanical System, TDMS: Technical Data Control Flow, PI: Proportional Integral, PMT: Photomultiplier. Image and video files are saved in BMP and AVI formats, respectively.
A phase correction algorithm is used to calculate the dispersion of image pixel intensities at different phase values ​​to determine the maximum value used to sharpen the image. For real-time correction, the phase scan range is ±2.86° with a relatively large step of 0.286° to reduce computation time. In addition, using parts of the image with fewer samples further reduces the image frame computation time from 7.5 seconds (1 Msample) to 1.88 seconds (250 Ksample) at 10 Hz. These input parameters were chosen to provide adequate image quality with minimal latency during in vivo imaging. Live images and videos are recorded in BMP and AVI formats, respectively. The raw data is stored in the Technical Data Management Flow Format (TMDS).
Post-processing of in vivo images for quality improvement with LabVIEW 2021. Accuracy is limited when using phase correction algorithms during in vivo imaging due to the long computation time required. Only limited image areas and sample numbers are used. In addition, the algorithm does not work well for images with motion artifacts or low contrast and leads to phase calculation errors30. Individual frames with high contrast and no motion artifacts were manually selected for phase fine tuning with a phase scan range of ±0.75° in 0.01° steps. The entire image area was used (eg, 1 Msample of an image recorded at 10 Hz). Table S2 details the image parameters used for real-time and post-processing. After phase correction, a median filter is used to further reduce image noise. Brightness and contrast are further improved by histogram stretching and gamma correction31.
The clinical trials were approved by the Michigan Medical Institutions Review Board and were conducted in the Department of Medical Procedures. This study is registered online with ClinicalTrials.gov (NCT03220711, registration date: 07/18/2017). Inclusion criteria included patients (aged 18 to 100 years) with a previously planned elective colonoscopy, an increased risk of colorectal cancer, and a history of inflammatory bowel disease. Informed consent was obtained from each subject who agreed to participate. Exclusion criteria were patients who were pregnant, had a known hypersensitivity to fluorescein, or were undergoing active chemotherapy or radiation therapy. This study included consecutive patients scheduled for routine colonoscopy and was representative of the Michigan Medical Center population. The study was conducted in accordance with the Declaration of Helsinki.
Before surgery, calibrate the endoscope using 10 µm fluorescent beads (#F8836, Thermo Fisher Scientific) mounted in silicone molds. A translucent silicone sealant (#RTV108, Momentive) was poured into a 3D printed 8 cm3 plastic mold. Drop the water fluorescent beads over the silicone and leave until the water medium dries.
The entire colon was examined using a standard medical colonoscope (Olympus, CF-HQ190L) with white light illumination. After the endoscopist has determined the area of ​​the alleged disease, the area is washed with 5-10 ml of 5% acetic acid, and then with sterile water to remove mucus and debris. A 5 ml dose of 5 mg/ml fluorescein (Alcon, Fluorescite) was injected intravenously or sprayed topically onto the mucosa using a standard cannula (M00530860, Boston Scientific) that was passed through the working channel.
Use an irrigator to flush excess dye or debris from the mucosal surface. Remove the nebulizing catheter and pass the endoscope through the working channel to obtain ante-mortem images. Use wide-field endoscopic guidance to position the distal tip in the target area. The total time used to collect confocal images was <10 min. The total time used to collect confocal images was <10 min. Общее время, затраченное на сбор конфокальных изображений, составило <10 мин. The total time taken to collect confocal images was <10 min. The total acquisition time for confocal images was less than 10 minutes. Endoscopic white light video was processed using the Olympus EVIS EXERA III (CLV-190) imaging system and recorded using an Elgato HD video recorder. Use LabVIEW 2021 to record and save endoscopy videos. After imaging is complete, the endoscope is removed and the tissue to be visualized is excised using biopsy forceps or a snare. The tissues were processed for routine histology (H&E), and evaluated by an expert GI pathologist (HDA). The tissues were processed for routine histology (H&E), and evaluated by an expert GI pathologist (HDA). Ткани были обработаны для обычной гистологии (H&E) и оценены экспертом-патологом желудочно-кишечного тракта (HDA). Tissues were processed for routine histology (H&E) and assessed by an expert gastrointestinal pathologist (HDA).对组织进行常规组织学(H&E) 处理，并由专家GI 病理学家(HDA) 进行评估。对组织进行常规组织学(H&E) 处理，并由专家GI 病理学家(HDA) 进行评估。 Ткани были обработаны для обычной гистологии (H&E) и оценены экспертом-патологом желудочно-кишечного тракта (HDA). Tissues were processed for routine histology (H&E) and assessed by an expert gastrointestinal pathologist (HDA). The spectral properties of fluorescein were confirmed using a spectrometer (USB2000+, Ocean Optics) as shown in Figure S2.
Endoscopes are sterilized after each use by humans (Fig. 8). Cleaning procedures were performed under the direction and approval of the Department of Infection Control and Epidemiology of the Michigan Medical Center and the Central Sterile Processing Unit. Prior to the study, the instruments were tested and validated for sterilization by Advanced Sterilization Products (ASP, Johnson & Johnson), a commercial entity that provides infection prevention and sterilization validation services. Prior to the study, the instruments were tested and validated for sterilization by Advanced Sterilization Products (ASP, Johnson & Johnson), a commercial entity that provides infection prevention and sterilization validation services. Перед исследованием инструменты были протестированы и одобрены для стерилизации компанией Advanced Sterilization Products (ASP, Johnson & Johnson), коммерческой организацией, предоставляющей услуги по профилактике инфекций и проверке стерилизации. Prior to study, instruments were tested and approved for sterilization by Advanced Sterilization Products (ASP, Johnson & Johnson), a commercial organization providing infection prevention and sterilization verification services. Перед исследованием инструменты были стерилизованы и проверены Advanced Sterilization Products (ASP, Johnson & Johnson), коммерческой организацией, которая предоставляет услуги по профилактике инфекций и проверке стерилизации. Instruments were sterilized and inspected prior to study by Advanced Sterilization Products (ASP, Johnson & Johnson), a commercial organization that provides infection prevention and sterilization verification services.
Tool recycling. (a) Endoscopes are placed in trays after each sterilization using the STERRAD processing process. (b) The SMF and wires are terminated with fiber optic and electrical connectors, respectively, which are closed prior to reprocessing.
Clean the endoscopes by doing the following: (1) wipe the endoscope with a lint-free cloth soaked in an enzymatic cleaner from proximal to distal; (2) Immerse the instrument in the enzymatic detergent solution for 3 minutes with water. lint-free fabric. Electrical and fiber optic connectors are covered and removed from the solution; (3) The endoscope is wrapped and placed in the instrument tray for sterilization using STERRAD 100NX, hydrogen peroxide gas plasma. relatively low temperature and low humidity environment.
The datasets used and/or analyzed in the current study are available from the respective authors upon reasonable request.
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