We demonstrate improved optical sectioning in light sheet fluorescence microscopy using tunable structured illumination (SI) frequencies to optimize image quality in scattering specimens. The SI patterns are generated coherently using a one-dimensional spatial light modulator for maximum pattern contrast, and the pattern spatial frequency is adjustable up to half the incoherent cutoff frequency of our detection objective. At this frequency, we demonstrate background reductions of 2 orders of magnitude.
MEMS based microendoscopes have become important imaging tools for early cancer diagnosis and precise tumor resection. Due to various technical challenges, few microendoscopes have been translated to clinics or applied to human patients. Through synergistic collaborations, we have developed novel MEMS scanner enabled microendoscopic multispectral (640nm to 780nm) three- dimensional dual-axis confocal fluorescent imaging system for translational applications, including early cancer detection and staging on colorectal cancer, molecular imaging guided surgical navigation on head and neck cancer. Based on dual-axis confocal microscopic architecture, we have miniaturized the imaging system with compact form-factor by integrating micro-optics and a patterned gold coated MEMS scanners, which have been custom-made and mass-produced in the nanofabrication foundry. The metal coating of the scanning mirror provide over 80% high reflectivity over near infra-red range. Both axes of the MEMS scanner could perform large tilting angle (> 6 degree mechanical scan angle) at DC and resonant mode. By advanced computational imaging approach, we have achieved real-time cross-sectional imaging in either raster or lissajous pattern scanning with fast frame rate (> 10 Hz) with large field-of-view (> 600 microns). Advanced real-time mosaicing algorithm has been developed to achieve broader view in millimeter scale. By utilizing molecular contrast probes conjugated with fluorescence dye, we have successfully demonstrated multi-spectral ex-vivo and in-vivo imaging on small animal tumor models and human tissue specimens, aimed for both early cancer detection and molecular imaging guided surgical navigation.
Wide-field fluorescent imaging for fluorescence molecular guidance has become a promising technique for use in imaging guided surgical navigation, but quick and intuitive microscopic inspection of fluorescent hot spots is still needed to confirm local disease states of tissues. To address this unmet need, we have developed a clinically translatable dual-modality handheld surgical microscope that incorporates both, wide-field (mesoscopic) fluorescence imaging and high-resolution (microscopic) horizontal optical-sectioning. This is accomplished by integrating a commercially available wide-field fiberscope, modified for two-color (660nm and 785nm) fluorescent detection, into a compact package (5.5 mm dia.) which also contains a dual-axis confocal (DAC) microscope. DAC microscopy is a high-sensitivity, high-resolution fluorescent imaging technology that benefits from the specificity of molecular probes, and enables interrogation of deeper regions of tissue by performing optical-sectioning of tissue. The DAC microscope has been designed with custom catadioptric micro-lenses to provide broadband multispectral capability for fluorescence imaging of multiple fluorophores over a broad spectral range (VIS to NIR), and also uses a novel MEMS-based scanning system for horizontal sectioning, and thus enables access to deeper regions of tissue at resolutions comparable to histological analysis. Large field-of-view (mm scale) is further provided by image mosaicing. The instrument thus provides simultaneous mesoscopic and microscopic fluorescence imaging over a broad spectral range for intuitively performing fast in-vivo search and microscopic confirmation of optical molecular markers in tissue, which is a capability that will become increasingly important for precise tumor resection in oncology as more optical molecular markers become approved for human use.
Considerable efforts have been recently undertaken to develop miniature optical-sectioning microscopes for in vivo microendoscopy and point-of-care pathology. These devices enable in vivo interrogation of disease as a real-time and noninvasive alternative to gold-standard histopathology, and therefore could have a transformative impact for the early detection of cancer as well as for guiding tumor-resection procedures. Regardless of the specific modality, various trade-offs in size, speed, field of view, resolution, contrast, and sensitivity are necessary to optimize a device for a particular application. Here, a miniature MEMS-based line-scanned dual-axis confocal (LS-DAC) microscope, with a 12-mm diameter distal tip, has been developed for point-of-care pathology. The dual-axis architecture has demonstrated superior rejection of out-of-focus and multiply scattered photons compared to a conventional single-axis confocal configuration. The use of line scanning enables fast frame rates (≥15 frames/sec), which mitigates motion artifacts of a handheld device during clinical use. We have developed a method to actively align the illumination and collection beams in this miniature LS-DAC microscope through the use of a pair of rotatable alignment mirrors. Incorporation of a custom objective lens, with a small form factor for in vivo application, enables the device to achieve an axial and lateral resolution of 2.0 and 1.1 microns, respectively. Validation measurements with reflective targets, as well as in vivo and ex vivo images of tissues, demonstrate that this high-speed LS-DAC microscope can achieve high-contrast imaging of fluorescently labeled tissues with sufficient sensitivity for applications such as oral cancer detection and guiding brain-tumor resections.
Topical application and quantification of targeted, surface-enhanced Raman scattering (SERS) nanoparticles offer a new technique that has the potential for early detection of epithelial cancers of hollow organs. Although less toxic than intravenous delivery, the additional washing required to remove unbound nanoparticles cannot necessarily eliminate nonspecific pooling. Therefore, we developed a real-time, ratiometric imaging technique to determine the relative concentrations of at least two spectrally unique nanoparticle types, where one serves as a nontargeted control. This approach improves the specific detection of bound, targeted nanoparticles by adjusting for working distance and for any nonspecific accumulation following washing. We engineered hardware and software to acquire SERS signals and ratios in real time and display them via a graphical user interface. We report quantitative, ratiometric imaging with nanoparticles at pM and sub-pM concentrations and at varying working distances, up to 50 mm. Additionally, we discuss optimization of a Raman endoscope by evaluating the effects of lens material and fiber coating on background noise, and theoretically modeling and simulating collection efficiency at various working distances. This work will enable the development of a clinically translatable, noncontact Raman endoscope capable of rapidly scanning large, topographically complex tissue surfaces for small and otherwise hard to detect lesions.
Our work demonstrates a MEMS based handheld dual-axis confocal microscope for cervical cancer screening. Imaging demonstration is performed with plant and animal tissue biopsies. The data is collected and displayed in real time with 2-5 Hz frame rates.
Near-infrared confocal microendoscopy is a promising technique for deep in vivo imaging of tissues and can generate high-resolution cross-sectional images at the micron-scale. We demonstrate the use of a dual-axis confocal (DAC) near-infrared fluorescence microendoscope with a 5.5-mm outer diameter for obtaining clinical images of human colorectal mucosa. High-speed two-dimensional en face scanning was achieved through a microelectromechanical systems (MEMS) scanner while a micromotor was used for adjusting the axial focus. In vivo images of human patients are collected at 5 frames/sec with a field of view of 362×212 μm2 and a maximum imaging depth of 140 μm. During routine endoscopy, indocyanine green (ICG) was topically applied a nonspecific optical contrasting agent to regions of the human colon. The DAC microendoscope was then used to obtain microanatomic images of the mucosa by detecting near-infrared fluorescence from ICG. These results suggest that DAC microendoscopy may have utility for visualizing the anatomical and, perhaps, functional changes associated with colorectal pathology for the early detection of colorectal cancer.
A fluorescence confocal microscope incorporating a 1.8-mm-diam gradient-index relay lens is developed for in vivo histological guidance during resection of brain tumors. The microscope utilizes a dual-axis confocal architecture to efficiently reject out-of-focus light for high-contrast optical sectioning. A biaxial microelectromechanical system (MEMS) scanning mirror is actuated at resonance along each axis to achieve a large field of view with low-voltage waveforms. The unstable Lissajous scan, which results from actuating the orthogonal axes of the MEMS mirror at highly disparate resonance frequencies, is optimized to fully sample 500×500 pixels at two frames per second. Optically sectioned fluorescence images of brain tissues are obtained in living mice to demonstrate the utility of this microscope for image-guided resections.
Microscopes are being developed for use in living animals, and even humans, to image microanatomical changes and
molecular markers that are associated with disease. Phantoms that can be used to evaluate the performance
characteristics of these systems have not been well described or standardized. We have been developing the tools to
evaluate a dual-axis confocal (DAC) microscope design to optimize the features required for in vivo diagnosic imaging,
and these may have features that are useful for evaluation of other such devices. We have performed diffraction-theory
modeling, Monte-Carlo scattering simulations, reflectance experiments in tissue phantoms, and tissue-imaging
validations. First, we determined how scattering from tissue deteriorates the diffraction-limited transverse and vertical
responses in reflectance DAC imaging. Specifically, the vertical and transverse responses of the DAC to a plane
reflector and a knife edge, respectively, were measured at various depths in an Intralipid scattering phantom.
Comparisons were made with both diffraction-theory and Monte-Carlo scattering simulations. Secondly, as a practical
demonstration of deep-tissue fluorescence microscopy, three-dimensional fluorescence images were obtained in thick
human biopsy samples. These results demonstrate that the efficient rejection of scattered light in a DAC microscope
enables deep optical sectioning in tissue. Finally, we will discuss our needs and plans for similar tissue-phantom
experiments to validate the performance of multimodal optical- and ultrasound-imaging platforms under development.
As devices are developed for the imaging of epithelial surfaces and substructures, standardized phantoms that represent
the multilayered anatomical features of these tissues will need to be developed.
Miniature endoscopic microscopes, with subcellular imaging capabilities, will enable in vivo detection of molecularly-targeted fluorescent probes for early disease detection. To optimize a dual-axis confocal microscope (DACM) design for this purpose, we use a tabletop instrument to determine the ability of this technology to perform optical sectioning deep within tissue. First, we determine how tissue scattering deteriorates the diffraction-limited transverse and vertical responses in reflectance imaging. Specifically, the vertical response of a DACM to a plane reflector is measured at various depths in a scattering phantom and compared with diffraction theory and Monte Carlo scattering simulations. Similarly, transverse line scans across a knife-edge target are performed at various depths in a scattering phantom. Second, as a practical demonstration of deep-tissue fluorescence microscopy that corroborates the findings from our scattering experiments, 3-D fluorescence images are obtained in thick human gastrointestinal mucosal specimens. Our results demonstrate efficient rejection of scattered light in a DACM, which enables deep optical sectioning in tissue with subcellular resolution that can distinguish between normal and premalignant pathologies.
Here we describe a simple optical design for a MEMS-based dual-axes fiber optic confocal scanning microscope that has
been miniaturized for handheld imaging of tissues, and which is capable of being further scaled to smaller dimensions
for endoscope compatibility while preserving its field-of-view (FOV), working distance, and resolution. Based on the
principle of parallel beams that are focused by a single parabolic mirror to a common point, the design allows the use of
replicated optical components mounted and aligned within a rugged cylindrical housing that is designed for use as a
handheld tissue microscope. A MEMS scanner is used for high speed scanning in the X-Y plane below the tissue
surface. An additional design feature is a mechanism for controlling a variable working distance, thus producing a scan
in the Z direction and allowing capture of 3-D volumetric images of tissue. The design parameters that affect the
resolution, FOV, and working distance are analyzed using ASAPTM optical modeling software and verified by
experimental results. Other features of this design include use of a solid immersion lens (SIL), which enhances both
resolution and FOV, and also provides index matching between the optics and the tissue.
Tissue scattering has a significant effect on the image resolution and light collection efficiency in confocal microscopy.
The dual-axes (DA) confocal architecture has many advantages including high axial resolution with low numerical
aperture lenses and long working distance for use in vivo as a microendoscope. In addition, less scattered light along the
illumination path may be collected and introduced as noise. In this paper, we use Monte Carlo tissue scattering
simulations to compare the dual-axes and conventional single-axis (SA) configurations. Simulation results show that the
axial response for the dual axes configuration varies with pinhole size and optical thickness of scattering media in a way
that differs from the single axis architecture. The DA configuration is able to filter out efficiently multiply-scattered
photons and out-of-focus light, thus allowing imaging with greater tissue penetration depths to provide vertical crosssectional
images, which has significant implications for in vivo imaging.
A dual-axes confocal reflectance microscope has been developed that utilizes a narrowband laser at 1310 nm to achieve high axial resolution, image contrast, field of view, and tissue penetration for distinguishing among normal, hyperplastic, and dysplastic colonic mucosa ex vivo. Light is collected off-axis using a low numerical aperture objective to obtain vertical image sections, with 4- to 5-µm resolution, at tissue depths up to 610 µm. Post-objective scanning enables a large field of view (610×640 µm), and balanced-heterodyne detection provides sensitivity to collect vertical sections at one frame per second. System optics are optimized to effectively reject out-of-focus scattered light without use of a low-coherence gate. This design is scalable to millimeter dimensions, and the results demonstrate the potential for a miniature instrument to detect precancerous tissues, and hence to perform in vivo histopathology.
A dual-axes confocal reflectance microscope has been developed that utilizes a narrowband source at 1310 nm to achieve high axial resolution, image contrast, field of view, and tissue penetration for distinguishing among normal, hyperplastic, and dysplastic colonic mucosa ex vivo. Light is collected off-axis using a low numerical aperture objective to obtain vertical image sections, with 4 to 5-μm resolution, at tissue depths up to 610 μm. Post-objective scanning enables a large field of view (610 x 640 μm) and balanced-heterodyne detection provides sensitivity to collect vertical sections at two frames per second. System optics are optimized to effectively reject out-of-focus scattered light without use of a low-coherence gate. This design is scalable to millimeter dimensions, and the results demonstrate the potential for a miniature instrument to detect pre-cancerous tissues, and hence to perform in vivo histopathology.
We demonstrate a standalone digital pen that writes on regular paper by tracking the writing nib’s absolute position in paper coordinates. The pen writes like a regular pen, but simultaneously captures handwritten information digitally with the aid of a Navigation Engine mounted atop the pen. The Navigation Engine has a wide-field-of-view vision system with a single-viewpoint catadioptric lens for observing the environment as the pen writes. A processor belonging to the Navigation Engine applies computationally efficient navigation algorithms to a stream of images of the pen’s environment to capture the pen’s full movement. The resultant data stream including x-y position of the nib and the pen’s Euler angles facilitate application of this technology to a wide range of tasks. In contrast to all other known digital pen technologies, this pen not only functions like a regular pen, but also provides an electronic copy of the digital writing without using any special paper.
The application of conventional confocal microscopes with high numerical aperture (NA) to in vivo imaging is limited
by the objectiveís large physical dimensions and short working distance. We are developing a confocal microscope that
uses simple low NA lenses oriented in a dual axes configuration for miniaturization and in vivo imaging. This architecture
achieves a long working distance, micron level axial resolution, and reduced noise from scattered light outside the
focal volume. Combined with the novel method of post-objective scanning, this design can be scaled down to millimeter
dimensions. We derive the dual axes response from diffraction theory, and construct two tabletop prototypes to
demonstrate the performance of this approach. We collect images from freshly excised biopsy specimens of human
esophagus and transgenic mouse cerebellum expressing GFP. With horizontal cross-sectional images, we achieve 1 to 2
μm resolution and collect reflectance and fluorescence images. With vertical cross-sectional images, we achieve 4 to 5
μm resolution, dynamic range of 70 dB, and tissue penetration over 1 mm. An instrument miniaturized with this configuration
could be used for in vivo cellular and molecular imaging.
KEYWORDS: Luminescence, Microscopes, Confocal microscopy, Signal to noise ratio, Green fluorescent protein, Tissues, Objectives, In vivo imaging, Lenses, Mirrors
We present a novel confocal microscope that has dual-axis architecture and biaxial postobjective scanning for the collection of fluorescence images from biological specimens. This design uses two low-numerical-aperture lenses to achieve high axial resolution and long working distance, and the scanning mirror located distal to the lenses rotates along the orthogonal axes to produce arc-surface images over a large field of view (FOV). With fiber optic coupling, this microscope can potentially be scaled down to millimeter dimensions via microelectromechanical systems (MEMS) technology. We demonstrate a benchtop prototype with a spatial resolution 4.4 µm that collects fluorescence images with a high SNR and a good contrast ratio from specimens expressing GFP. Furthermore, the scanning mechanism produces only small differences in aberrations over the image FOV. These results demonstrate proof of concept of the dual-axis confocal architecture for in vivo molecular and cellular imaging.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.