Miniature Objective Lens Research Articles

Fiber-optic large-depth 3D chromatic confocal endomicroscopy.

Current endoscopy techniques have difficulties to provide both high resolution and large imaging depth, which significantly hinders the early diagnosis of gastric cancer. Here, we developed a label-free, large-depth, three-dimensional (3D) chromatic reflectance confocal endomicroscopy. In order to solve the problem of insufficient imaging depth of traditional chromatic confocal microscopy, a customized miniature objective lens both with large chromatic focal shift and correction for spherical aberration was used to focus light of different wavelengths at different depths of the sample simultaneously, and a fiber bundle containing 50000 single-mode cores was used to collect the confocal reflectance signal. To acquire detailed information along the axial direction at a faster speed, a high-speed multi-pixel spectrometer was used to realize simultaneous detection of multi-depth signals. Specifically, we have built up a label-free fiber-optic 3D chromatic reflectance confocal endomicroscopy, with 2.3 µm lateral resolution, imaging depth of 570 µm in 3D phantom and 220 µm in tissue, and 1.5 Hz 3D volumetric frame rate. We have demonstrated that the fiber-optic 3D chromatic confocal endomicroscopy can be used to image human gastric tissues ex vivo, and provide important morphological information for diagnosis without labeling. These results show the great potential of the fiber-optic 3D chromatic confocal endomicroscopy for gastric cancer diagnosis.

Low-cost, chromatic confocal endomicroscope for cellular imaging in vivo.

We have developed a low-cost, chromatic confocal endomicroscope (CCE) that can image a cross-section of the tissue at cellular resolution. In CCE, a custom miniature objective lens was used to focus different wavelengths into different tissue depths. Therefore, each tissue depth was encoded with the wavelength. A custom miniature spectrometer was used to spectrally-disperse light reflected from the tissue and generate cross-sectional confocal images. The CCE prototype had a diameter of 9.5 mm and a length of 68 mm. Measured resolution was high, 2 µm and 4 µm for lateral and axial directions, respectively. Effective field size was 468 µm. Preliminary results showed that CCE can visualize cellular details from cross-sections of the tissue in vivo down to the tissue depth of 100 µm.

Miniaturized multimodal multiphoton microscope for simultaneous two-photon and three-photon imaging with a dual-wavelength Er-doped fiber laser.

A multimodal multiphoton microscopy (MPM) is developed to acquire both two-photon microscopy (2PM) and three-photon microscopy (3PM) signals. A dual-wavelength Er-doped fiber laser is used as the light source, which provides the fundamental pulse at 1580 nm to excite third harmonic generation (THG) and the frequency-doubled pulse at 790 nm to excite intrinsic two-photon excitation fluorescence (TPEF) and second harmonic generation (SHG). Due to their different contrast mechanisms, the TPEF, SHG, and THG images can acquire complementary information about tissues, including cells, collagen fibers, lipids, and interfaces, all label-free. The compact MPM imaging probe is developed using miniature objective lens and a micro-electro-mechanical scanner. Furthermore, the femtosecond laser pulses are delivered by a single mode fiber and the signals are collected by a multimode fiber, which makes the miniaturized MPM directly fiber-coupled, compact, and portable. Design considerations on using the dual excitation wavelengths are discussed. Multimodal and label-free imaging by TPEF, SHG, and THG are demonstrated on biological samples. The miniaturized multimodal MPM is shown to have great potential for label-free imaging of thick and live tissues.

High-resolution multimodal flexible coherent Raman endoscope

Coherent Raman scattering microscopy is a fast, label-free, and chemically specific imaging technique that shows high potential for future in vivo optical histology. However, the imaging depth in tissues is limited to the sub-millimeter range because of absorption and scattering. Realization of coherent Raman imaging using a fiber endoscope system is a crucial step towards imaging deep inside living tissues and providing information that is inaccessible with current microscopy tools. Until now, the development of coherent Raman endoscopy has been hampered by several issues, mainly related to the fiber delivery of the excitation pulses and signal collection. Here, we present a flexible, compact, coherent Raman, and multimodal nonlinear endoscope (4.2 mm outer diameter, 71 mm rigid length) based on a resonantly scanned hollow-core Kagomé-lattice double-clad fiber. The fiber design enables distortion-less, background-free delivery of femtosecond excitation pulses and back-collection of nonlinear signals through the same fiber. Sub-micrometer spatial resolution over a large field of view is obtained by combination of a miniature objective lens with a silica microsphere lens inserted into the fiber core. We demonstrate high-resolution, high-contrast coherent anti-Stokes Raman scattering, and second harmonic generation endoscopic imaging of biological tissues over a field of view of 320 µm at a rate of 0.8 frames per second. These results pave the way for intraoperative label-free imaging applied to real-time histopathology diagnosis and surgery guidance.

Compact fiber-based multi-photon endoscope working at 1700 nm.

We present the design, implementation and performance analysis of a compact multi-photon endoscope based on a piezo electric scanning tube. A miniature objective lens with a long working distance and a high numerical aperture (≈ 0.5) is designed to provide a diffraction limited spot size. Furthermore, a 1700 nm wavelength femtosecond fiber laser is used as an excitation source to overcome the scattering of biological tissues and reduce water absorption. Therefore, the novel optical system along with the unique wavelength allows us to increase the imaging depth. We demonstrate that the endoscope is capable of performing third and second harmonic generation (THG/SHG) and three-photon excitation fluorescence (3PEF) imaging over a large field of view (> 400 μm) with high lateral resolution (2.2 μm). The compact and lightweight probe design makes it suitable for minimally-invasive in-vivo imaging as a potential alternative to surgical biopsies.

Miniature objective lens with variable focus for confocal endomicroscopy

Spectrally encoded confocal microscopy (SECM) is a reflectance confocal microscopy technology that can rapidly image large areas of luminal organs at microscopic resolution. One of the main challenges for large-area SECM imaging in vivo is maintaining the same imaging depth within the tissue when patient motion and tissue surface irregularity are present. In this paper, we report the development of a miniature vari-focal objective lens that can be used in an SECM endoscopic probe to conduct adaptive focusing and to maintain the same imaging depth during in vivo imaging. The vari-focal objective lens is composed of an aspheric singlet with an NA of 0.5, a miniature water chamber, and a thin elastic membrane. The water volume within the chamber was changed to control curvature of the elastic membrane, which subsequently altered the position of the SECM focus. The vari-focal objective lens has a diameter of 5 mm and thickness of 4 mm. A vari-focal range of 240 μm was achieved while maintaining lateral resolution better than 2.6 μm and axial resolution better than 26 μm. Volumetric SECM images of swine esophageal tissues were obtained over the vari-focal range of 260 μm. SECM images clearly visualized cellular features of the swine esophagus at all focal depths, including basal cell nuclei, papillae, and lamina propria.

Reflectance confocal endomicroscope with optical axial scanning for in vivo imaging of the oral mucosa.

This paper presents the design and evaluation of a reflectance confocal laser endomicroscope using a miniature objective lens within a rigid probe in conjunction with an electrically tunable lens for axial scanning. The miniature lens was characterized alone as well as in the endoscope across a 200 µm axial scan range using the tunable lens. The ability of the confocal endoscope to probe the human oral cavity is demonstrated by imaging of the oral mucosa in vivo. The results indicate that reflectance confocal endomicroscopy has the potential to be used in a clinical setting and guide diagnostic evaluation of biological tissue.

Achromatic miniature lens system for coherent Raman scattering microscopy

We discuss the design and performance of a miniature objective lens optimized for coherent Raman scattering microscopy. The packaged lens assembly has a numerical aperture of 0.51 in water and an outer diameter of 8 mm. The lens system exhibits minimum chromatic aberrations, and produces coherent Raman scattering images with sub-micrometer lateral resolution (0.648 μm) using near-infrared excitation pulses. We demonstrate that despite the small dimensions of the miniature objective, the performance of this lens system is comparable to standard microscope objective lenses, offering opportunities for miniaturizing coherent Raman scattering imaging probes without sacrificing the image quality.

A fiber-optic system for dual-modality photoacoustic microscopy and confocal fluorescence microscopy using miniature components

Imaging of the cells and microvasculature simultaneously is beneficial to the study of tumor angiogenesis and microenvironments. We designed and built a fiber-optic based photoacoustic microscopy (PAM) and confocal fluorescence microscopy (CFM) dual-modality imaging system. To explore the feasibility of this all-optical device for future endoscopic applications, a microelectromechanical systems (MEMS) scanner, a miniature objective lens, and a small size optical microring resonator as an acoustic detector were employed trying to meet the requirements of miniaturization. Both the lateral resolutions of PAM and CFM were quantified to be 8.8μm. Axial resolutions of PAM and CFM were experimentally measured to be 19μm and 53μm, respectively. The experiments on ex vivo animal bladder tissues demonstrate the good performance of this system in imaging not only microvasculature but also cellular structure, suggesting that this novel imaging technique holds potential for improved diagnosis and guided treatment of bladder cancer.

A compact fiber-optic SHG scanning endomicroscope and its application to visualize cervical remodeling during pregnancy

We report the development of an all-fiber-optic scanning endomicroscope capable of high-resolution second harmonic generation (SHG) imaging of biological tissues and demonstrate its utility for monitoring the remodeling of cervical collagen during gestation in mice. The endomicroscope has an overall 2.0 mm diameter and consists of a single customized double-clad fiber, a compact rapid two-dimensional beam scanner, and a miniature compound objective lens for excitation beam delivery, scanning, focusing, and efficient SHG signal collection. Endomicroscopic SHG images of murine cervical tissue sections at different stages of normal pregnancy reveal progressive, quantifiable changes in cervical collagen morphology with resolution similar to that of bench-top SHG microscopy. SHG endomicroscopic imaging of ex vivo murine and human cervical tissues through intact epithelium has also been performed. Our findings demonstrate the feasibility of SHG endomicroscopy technology for staging normal pregnancy, and suggest its potential application as a minimally invasive tool for clinical assessment of abnormal cervical remodeling associated with preterm birth.

Confocal fluorescence microendoscopy of bronchial epithelium

Confocal microendoscopy permits the acquisition of high-resolution real-time confocal images of bronchial mucosa via the instrument channel of an endoscope. We report here on the construction and validation of a confocal fluorescence microendoscope and its use to acquire images of bronchial epithelium in vivo. Our objective is to develop an imaging method that can distinguish preneoplastic lesions from normal epithelium to enable us to study the natural history of these lesions and the efficacy of chemopreventive agents without biopsy removal of the lesion that can introduce a spontaneous regression bias. The instrument employs a laser-scanning engine and bronchoscope-compatible confocal probe consisting of a fiber-optic image guide and a graded-index objective lens. We assessed the potential of topical application of physiological pH cresyl violet (CV) as a fluorescence contrast-enhancing agent for the visualization of tissue morphology. Images acquired ex vivo with the confocal microendoscope were first compared with a bench-top confocal fluorescence microscope and conventional histology. Confocal images from five sites topically stained with CV were then acquired in vivo from high-risk smokers and compared to hematoxylin and eosin stained sections of biopsies taken from the same site. Sufficient contrast in the confocal imagery was obtained to identify cells in the bronchial epithelium. However, further improvements in the miniature objective lens are required to provide sufficient axial resolution for accurate classification of preneoplastic lesions.

Nonlinear optical endoscope based on a compact two axes piezo scanner and a miniature objective lens

We report on a nonlinear optical endoscope that adopts a hollow core photonic crystal fiber for single-mode illumination delivery and a multimode one for signal collection. Femtosecond laser pulses up to 100 mW can be delivered at a centered wavelength of 800 nm. The two-photon fluorescence response of our system is shown to have axial and lateral resolutions of 5.8 microm and 0.6 microm respectively. Fluorescence detection was obtained at different wavelengths between 790 and 840 nm which could allow SHG detection for example. The maximal field-of-view of the acquired images is 420 microm x 420 microm. Detection efficiency is greater by using an avalanche photodiode in comparison to a photo multiplier tube. Results presented here demonstrate the ability of the system to resolve cellular details and the potential of the device for future in vivo imaging diagnosis.

Handheld subcellular-resolution single-fiber confocal microscope using high-reflectivity two-axis vertical combdrive silicon microscanner

We introduce a handheld single-fiber laser-scanning confocal microscope, incorporating a high-reflectivity two-axis silicon vertical combdrive microscanner, aimed at in vivo early detection of epithelial precancers. The approach adopted is motivated by need for a portable, economical, biopsy-free, early precancer screening technology in low-infrastructure environments. Our microelectromechanical system (MEMS) based handheld probe integrates the microscanners with miniature objective lens system and flexible electrical routing in a forward-imaging configuration, with 4.8 mm distal probe tip outer diameter for unrestricted imaging access in biological sites such as the oral cavity and cervix. Reflectance confocal videos of a USAF 1951 resolution target and biological samples were obtained over 200 microm x 110 microm field of view, with 0.80 and 9.55 microm lateral and axial resolution, at 3.5-5.0 frames per second. With improvements to objective numerical aperture, our probe can enable precise evaluation of nuclear size, density, nucleus-to-cytoplasm ratio and cell density, which are important visual identifiers of epithelial precancers.

Increasing the imaging depth of coherent anti-Stokes Raman scattering microscopy with a miniature microscope objective

A miniature objective lens with a tip diameter of 1.3 mm was used for extending the penetration depth of coherent anti-Stokes Raman scattering (CARS) microscopy. Its axial and lateral focal widths were determined to be 11.4 and 0.86 microm, respectively, by two-photon excitation fluorescence imaging of 200 nm beads at a 735 nm excitation wavelength. By inserting the lens tip into a soft gel sample, CARS images of 2 microm polystyrene beads 5 mm deep from the surface were acquired. The miniature objective was applied to CARS imaging of rat spinal cord white matter with a minimal requirement for surgery.

Fiber-optic confocal microscope using a MEMS scanner and miniature objective lens

We designed and constructed a single-fiber-optic confocal microscope (SFCM) with a microelectromechanical system (MEMS) scanner and a miniature objective lens. Axial and lateral resolution values for the system were experimentally measured to be 9.55 mum and 0.83 mum respectively, in good agreement with theoretical predictions. Reflectance images were acquired at a rate of 8 frames per second, over a 140 mum x 70 mum field-of-view. In anticipation of future applications in oral cancer detection, we imaged ex vivo and in vivo human oral tissue with the SFCM, demonstrating the ability of the system to resolve cellular detail.

Design, assembly, and optical bench testing of a high-numerical-aperture miniature injection-molded objective for fiber-optic confocal reflectance microscopy

The design, analysis, assembly methods, and optical-bench test results for a miniature injection-molded plastic objective lens used in a fiber-optic confocal reflectance microscope are presented. The five-lens plastic objective was tested as a stand-alone optical system before its integration into a confocal microscope for in vivo imaging of cells and tissue. Changing the spacing and rotation of the individual optical elements can compensate for fabrication inaccuracies and improve performance. The system performance of the miniature objective lens is measured by use of an industry-accepted slanted-edge modulation transfer function (MTF) metric. An estimated Strehl ratio of 0.61 and a MTF value of 0.66 at the fiber-optic bundle Nyquist frequency have been obtained. The optical bench testing system is configured to permit interactive optical alignment during testing to optimize performance. These results are part of an effort to demonstrate the manufacturability of low-cost, high-performance biomedical optics for high-resolution in vivo imaging. Disposable endoscopic microscope objectives could help in vivo confocal microscopy technology mature to permit wide-scale clinical screening and detection of early cancers and precancerous lesions.

In vivo fiber-optic confocal reflectance microscope with an injection-molded plastic miniature objective lens

For in vivo optical diagnostic technologies to be distributed to the developed and developing worlds, optical imaging systems must be constructed of inexpensive components. We present a fiber-optic confocal reflectance microscope with a cost-effective injection-molded plastic miniature objective lens for in vivo imaging of human tissues in near real time. The measured lateral resolution is less than 2.2 microm, and the measured axial resolution is 10 microm. Confocal images of ex vivo cervical tissue biopsies and in vivo human lip taken at 15 frames/s demonstrate the microscope's capability of imaging cell morphology and tissue architecture.