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The prognosis for high-grade glioma (HGG) remains dismal and the extent of resection correlates with overall survival and progression free disease. Epidermal growth factor receptor (EGFR) is a biomarker heterogeneously expressed in HGG. We assessed the feasibility of detecting HGG using near-infrared fluorescent antibody targeting EGFR. Mice bearing orthotopic HGG xenografts with modest EGFR expression were imaged in vivo after systemic panitumumab-IRDye800 injection to assess its tumor-specific uptake macroscopically over 14 days, and microscopically ex vivo. EGFR immunohistochemical staining of 59 tumor specimens from 35 HGG patients was scored by pathologists and expression levels were compared to that of mouse xenografts. Intratumoral distribution of panitumumab-IRDye800 correlated with near-infrared fluorescence and EGFR expression. Fluorescence distinguished tumor cells with 90% specificity and 82.5% sensitivity. Target-to-background ratios peaked at 14 h post panitumumab-IRDye800 infusion, reaching 19.5 in vivo and 7.6 ex vivo, respectively. Equivalent or higher EGFR protein expression compared to the mouse xenografts was present in 77.1% HGG patients. Age, combined with IDH-wildtype cerebral tumor, was predictive of greater EGFR protein expression in human tumors. Tumor specific uptake of panitumumab-IRDye800 provided remarkable contrast and a flexible imaging window for fluorescence-guided identification of HGGs despite modest EGFR expression.

We present software-based methods for automatic phase control and for mosaicing high-speed, Lissajous-scanned images. To achieve imaging speeds fast enough for mosaicing, we first increase the image update rate tenfold from 3 to 30 Hz, then vertically interpolate each sparse image in real-time to eliminate fixed pattern noise. We validate our methods by imaging fluorescent beads and automatically maintaining phase control over the course of one hour. We then image fixed mouse brain tissues at varying update rates and compare the resulting mosaics. Using reconstructed image data as feedback for phase control eliminates the need for phase sensors and feedback controllers, enabling long-term imaging experiments without additional hardware. Mosaicing subsampled images results in video-rate imaging speeds, nearly fully recovered spatial resolution, and millimeter-scale fields of view.

Current clinical brain tumour therapy practices are based on tumour resection and post-operative chemotherapy or X-ray radiation. Resection requires technically challenging open-skull surgeries that can lead to major neurological deficits and, in some cases, death. Treatments with X-ray and chemotherapy, on the other hand, cause major side-effects such as damage to surrounding normal brain tissues and other organs. Here we report the development of an integrated nanomedicine-bioelectronics brain-machine interface that enables continuous and on-demand treatment of brain tumours, without open-skull surgery and toxicological side-effects on other organs. Near-infrared surface plasmon characteristics of our gold nanostars enabled the precise treatment of deep brain tumours in freely behaving mice. Moreover, the nanostars' surface coating enabled their selective diffusion in tumour tissues after intratumoral administration, leading to the exclusive heating of tumours for treatment. This versatile remotely controlled and wireless method allows the adjustment of nanoparticles' photothermal strength, as well as power and wavelength of the therapeutic light, to target tumours in different anatomical locations within the brain.

Background: The prognosis for high-grade glioma (HGG) remains dismal and extent of resection correlates with overall survival and progression free disease. Epidermal growth factor receptor (EGFR) is a biomarker heterogeneously expressed in HGG. We assessed the feasibility of detecting HGG using near-infrared fluorescent antibody targeting EGFR. Methods: Mice bearing orthotopic HGG xenografts with modest EGFR expression were imaged in vivo after systemic panitumumab-IRDye800 injection to assess its tumor-specific uptake macroscopically over 14 days, and microscopically ex vivo. EGFR immunohistochemical staining of 59 tumor specimens from 35 HGG patients during was scored by pathologists and expression levels were compared to that of mouse xenografts. Results: Intratumoral distribution of pan800 correlated with near-infrared fluorescence and EGFR expression. Fluorescence distinguished tumor cells with 90% specificity and 82.5% sensitivity. Target-to-background ratios peaked at 14 hours post panitumumab-IRDye800 infusion, reaching 19.5 in vivo and 7.6 ex vivo, respectively. Equivalent or higher EGFR protein expression compared to the mouse xenografts was present in 77.1% HGG patients. Age, combined with IDH-wildtype cerebral tumor, was predictive of greater EGFR protein expression in human tumors. Conclusion: Tumor specific uptake of pan800 provided remarkable contrast and a flexible imaging window for fluorescence-guided identification of HGGs despite modest EGFR expression.

The tumor microenvironment (TME) can be defined as non-transformed stromal elements within the tumor, effecting their growth, invasion, and metastatic ability. Cancer cells leave the primary tumor during metastasis, then spread and colonize in distant organs. In breast cancer, metastasis mechanisms are not yet fully understood within the TME. However, epithelial to mesenchymal transition (EMT) is a known essential step in metastasis, in which patterned epithelial cells transform into migratory cells with mesenchymal phenotypes. Developing state-of-the-art multiparametric in vivo imaging devices increases the opportunities to observe dynamic cellular properties and their interactions with the surrounding stroma and TME during the critical steps of invasion and metastasis. Currently available commercial imaging technologies only enable in vivo imaging under anesthesia in a fixed posture. In contrast, real-time confocal imaging of the EMT and TME is not possible in animals. We have adapted our miniature dual-axis confocal (DAC) fluorescent microscope to track breast cancer cells having fluorescent reporters indicating key molecular splicing events during EMT to overcome these limitations. This project aims to study EMT more thoroughly using rat breast cancer MTLn3 cell lines by mimicking in vivo scenarios through 3D in vitro mono/co-culture spheroids, 3D multi-spheroid matrix overlay models, and 3D sandwich. We have engineered MTLn3 cells with fluorescent reporters correlating with different EMT stages, depending on their invasiveness levels. The ultra-low attachment method is used to engineer 3D MTLn3 spheroids, embed within TME matrices, and monitor advanced in vitro live-cell imaging techniques for up to 10 days. Another goal is to use miniature DAC microscopes, implanted in animal models, to study the fundamental changes occurring during carcinoma progression. This transdisciplinary project's final goal is to optimize the DAC microscope as an implantable and insertable device to investigate EMT-associated splicing events in the TME of orthotopic rat breast carcinoma models. This will be achieved by growing engineered MTLn3 cells and spheroids in their mammary fat pads and following microscopic changes for a certain period. This work will contribute to further developing the miniature implantable DAC microscopes for 3D high resolution, multiparametric imaging to reveal dynamic changes within the TME during carcinoma progression. Citation Format: Ehsanul Hoque Apu, Seock-Jin Chung, Michael J. Mandella, Frank Schonig, Frank B. Gertler, Zhen Qiu, Christopher H. Contag. Implantable and insertable miniature dual-axis confocal (DAC) microscope to detect epithelial-mesenchymal transition (EMT) in the breast cancer microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB243.

We developed a handheld line-scanned dual-axis confocal microscope for real-time optical biopsy. The device utilizes a novel MEMS-based scanning mechanism for field-flattening, and provides high-speed (16 Hz) fluorescence imaging with sub-nuclear resolution in tissue.

A miniature line-scanned (LS) dual-axis confocal (DAC) microscope, with a 12-mm diameter distal tip, has been developed for high-speed (>15 Hz) microscopic imaging of tissue surfaces up to a depth of ~ 150 µm.

Atherosclerosis is a progressive inflammatory condition caused by an unstable lesion, called thin-cap fibro atheromata (TCFA) that underlies coronary artery disease (CAD)—one of the leading causes of death worldwide. Therefore, early clinical diagnosis and effective risk stratification is important for CAD management as well as preventing progression to catastrophic events. However, early detection could be difficult due to their small size, motion, obscuring 18F-FDG uptake by adjacent myocardium, and complex morphological/biological features. To overcome these limitations, we developed a catheter-based Circumferential-Intravascular-Radioluminescence-Photoacoustic-Imaging (CIRPI) system that can detect vulnerable plaques in coronary arteries and characterizes them with respect to pathology and biology. Our CIRPI system combined two imaging modalities: Circumferential Radioluminescence Imaging (CRI) and PhotoAcoustic Tomography (PAT) within a novel optical probe. The probe’s CaF2:Eu based scintillating imaging window provides a 360° view of human (n = 7) and murine carotid (n = 10) arterial plaques by converting β-particles into visible photons during 18F-FDG decay. A 60× and 63× higher radioluminescent signals were detected from the human and murine plaque inflammations, respectively, compared to the control. The system’s photoacoustic imaging provided a comprehensive analysis of the plaque compositions and its morphologic information. These results were further verified with IVIS-200, immunohistochemical analysis, and autoradiography.

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.

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.

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.

Early and comprehensive endoscopic detection of colonic dysplasia – the most clinically significant precursor lesion to colorectal adenocarcinoma – provides an opportunity for timely, minimally-invasive intervention to prevent malignant transformation. Here, the development and evaluation of biodegradable near-infrared fluorescent silica nanoparticles (FSN) is described that have the potential to improve adenoma detection during fluorescence-assisted white-light colonoscopic surveillance in rodent and human-scale models of colorectal carcinogenesis. FSNs are biodegradable (t(1/2) of 2.7 weeks), well-tolerated, and enable detection and delineation of adenomas as small as 0.5 mm(2) with high tumor-to-background ratios. Furthermore, in the human-scale, APC(1311/+) porcine model, the clinical feasibility and benefit of using FSN-guided detection of colorectal adenomas using video-rate fluorescence-assisted white-light endoscopy is demonstrated. Since nanoparticles of similar size (e.g., 100–150-nm) or composition (i.e., silica, silica/gold hybrid) have already been successfully translated to the clinic, and, clinical fluorescent/white light endoscopy systems are becoming more readily available, there is a viable path towards clinical translation of the proposed strategy for early colorectal cancer detection and prevention in high-risk patients.

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.

We demonstrate vertical cross-sectional (XZ-plane) images of near-infrared (NIR) fluorescence with a handheld dual axes confocal endomicroscope that reveals specific binding of a Cy5.5-labeled peptide to pre-malignant colonic mucosa. This view is perpendicular to the tissue surface, and is similar to that used by pathologists. The scan head is 10 mm in outer diameter (OD), and integrates a one dimensional (1-D) microelectromechanical systems (MEMS) X-axis scanner and a bulky lead zirconate titanate (PZT) based Z-axis actuator. The microscope images in a raster-scanning pattern with a ±6 degrees (mechanical) scan angle at ~3 kHz in the X-axis (fast) and up to 10 Hz (0–400 μm) in the Z-axis (slow). Vertical cross-sectional fluorescence images are collected with a transverse and axial resolution of 4 and 5 μm, respectively, over a field-of-view of 800 μm (width) × 400 μm (depth). NIR vertical cross-sectional fluorescence images of fresh mouse colonic mucosa demonstrate histology-like imaging performance with this miniature instrument.

In this paper, we present a novel 2-D microelectromechanical systems (MEMS) scanner that enables dual-axes confocal microscopy. Dual-axes confocal microscopy provides high resolution and long working distance, while also being well suited for miniaturization and integration into endoscopes for in vivo imaging. The gimbaled MEMS scanner is fabricated on a double silicon-on-insulator (SOI) wafer (a silicon wafer bonded on a SOI wafer) and is actuated by self-aligned vertical electrostatic combdrives. Maximum optical deflections of plusmn4.8deg and plusmn5.5deg are achieved in static mode for the outer and inner axes, respectively. Torsional resonant frequencies are at 500 Hz and 2.9 kHz for the outer and inner axes, respectively. The imaging capability of the MEMS scanner is successfully demonstrated in a breadboard setup. Reflectance images with a field of view of are achieved at 8 frames/s. The transverse resolutions are 3.94 mum and 6.68 mum for the horizontal and vertical dimensions, respectively.

There is a need for miniature optical-sectioning microscopes to enable in vivo interrogation of tissues as a real-time and noninvasive alternative to gold-standard histopathology. Such devices could have a transformative impact for the early detection of cancer as well as for guiding tumor-resection procedures. Miniature confocal microscopes have been developed by various researchers and corporations to enable optical sectioning of highly scattering tissues, all of which have necessitated various trade-offs in size, speed, depth selectivity, field of view, resolution, image contrast, and sensitivity. In this study, a miniature line-scanned (LS) dual-axis confocal (DAC) microscope, with a 12-mm diameter distal tip, has been developed for clinical point-of-care pathology. The dual-axis architecture has demonstrated an advantage over the conventional single-axis confocal configuration for reducing background noise from out-of-focus and multiply scattered light. The use of line scanning enables fast frame rates (16 frames/sec is demonstrated here, but faster rates are possible), which mitigates motion artifacts of a hand-held device during clinical use. We have developed a method to actively align the illumination and collection beams in a 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 clinical use, enables our device to achieve an optical-sectioning thickness 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 the clinical potential of this high-speed optical-sectioning microscopy device.

Miniature dual-axis confocal (DAC) microscopes are being developed for the early detection of oral cancers and for guiding brain tumor resection. The design of MEMS-scanned DAC microscopes, and their analysis and optimization are described.

Pathologists evaluate histology sectioned perpendicular to the tissue surface, or vertical cross-section. This orientation (XZ-plane) enables evaluation of mucosal differentiation in the basilar-to-luminal direction. Current endomicroscopes use a conventional (single axis) optical design.1 Imaging is limited to horizontal cross-sections (XY-plane) where the micro-anatomy is frequently similar across the field-of-view (FOV). In the dual axes configuration, light is delivered and collected off-axis, and images can be detected over a much larger range of intensities.2 Molecular images collected using fluorescence can improve specificity for disease detection and reveal functional properties about tissue.3 Proper interpretation of these images requires correlation with the micro-anatomy. We aim to demonstrate the simultaneous collection of two fluorescence images in vivo in vertical cross-sections using a dual axes confocal endomicroscope. An overlay of molecular and anatomical images from normal and dysplastic mouse colonic mucosa will be displayed in real time.

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.

A MEMS based handheld multi-spectral confocal microscope has been developed as a noninvasive imaging instrument for cervical cancer screening. Tissue imaging depth of up to 400 μm was demonstrated. The maximum speed of image collection is up to 10 Hz with field of view (FOV) around 200×158 pixel2 size. Our handheld prototype is able to visualize cancer cells with cellular resolution (transverse resolution = 5 μm and axial resolution = 6 μm). Biological imaging demonstration was carried out on cell culture and tissues of human cervix.

Endoscopic imaging is an invaluable diagnostic tool allowing minimally invasive access to tissues deep within the body. It has played a key role in screening colon cancer and is credited with preventing deaths through the detection and removal of precancerous polyps. However, conventional white-light endoscopy offers physicians structural information without the biochemical information that would be advantageous for early detection and is essential for molecular typing. To address this unmet need, we have developed a unique accessory, noncontact, fiber optic-based Raman spectroscopy device that has the potential to provide real-time, multiplexed functional information during routine endoscopy. This device is ideally suited for detection of functionalized surface-enhanced Raman scattering (SERS) nanoparticles as molecular imaging contrast agents. This device was designed for insertion through a clinical endoscope and has the potential to detect and quantify the presence of a multiplexed panel of tumor-targeting SERS nanoparticles. Characterization of the Raman instrument was performed with SERS particles on excised human tissue samples, and it has shown unsurpassed sensitivity and multiplexing capabilities, detecting 326-fM concentrations of SERS nanoparticles and unmixing 10 variations of colocalized SERS nanoparticles. Another unique feature of our noncontact Raman endoscope is that it has been designed for efficient use over a wide range of working distances from 1 to 10 mm. This is necessary to accommodate for imperfect centering during endoscopy and the nonuniform surface topology of human tissue. Using this endoscope as a key part of a multiplexed detection approach could allow endoscopists to distinguish between normal and precancerous tissues rapidly and to identify flat lesions that are otherwise missed.

Miniature microscopes are being developed to examine tissue in situ for early anatomic and molecular indicators of disease, in real time, and at cellular resolution. These new devices will lead to a shift from the current diagnostic paradigm of biopsy followed by histopathology and recommended therapy, to one of non-invasive point-of-care diagnosis with the possibility of treatment in the same session. This potential revolution in disease management may have a major impact on the training of future physicians to include the use and interpretation of real-time in vivo microscopic data, and will also affect the emerging fields of telepathology and telemedicine. Implementation of new technologies into clinical practice is a complex process that requires multidisciplinary communication and collaboration among clinicians, engineers and scientists. As such, our aim is to provide a forward-looking view of the critical issues facing the development of new technologies and directing clinical education. Here, we focus on the use of in vivo microscopy for detection of malignant and pre-malignant lesions as well as for guiding therapy. We will highlight some of the areas in which in vivo microscopy could address unmet clinical needs, and then review the technological challenges that are being addressed, or need to be addressed, for in vivo microscopy to become an effective clinical tool.

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.

We describe a 2-D MEMS scanner for a handheld multispectral confocal microscope for early detection of cervical cancer. The MEMS scanner has an inner gimbal design with torsional springs separated from the reflectors to reduce light loss while maintaining chip size to 3.25 × 3.25 mm2. The devices are large-scale batch fabricated using a double layer SOI process. The scanner has electrostatic optical deflection angles of 3.25° for the inner axis at 75V and ±1.6° for the outer axis at 60V. The device has resonance frequencies of 2.84kHz and 452Hz for the inner and outer axis torsional modes respectively.

We demonstrated a handheld multispectral fluorescence confocal microscope for cervical cancer diagnostic using dual-axis confocal microscope architecture and a microelectromechnical systems scanner. The real time images are acquired with frame rate upto 15 Hz.

Our work demonstrated a MEMS based handheld dual-axis confocal microscope for cervical cancer screening. Imaging demonstration is performed with plants and animal tissue biopsies. The data is collected and displayed in real time with 2–5 Hz.

An achromatic version of a micro-mirror-scanned surgical dual-axis confocal microscope has been developed for ratiometric quantification of biomarker expression as revealed by the topical application of fluorescent contrast agents.

This paper describes a 3-D MEMS scanning system for 3.2mm-diameter dual-axis confocal (DAC) microendoscopes. Co-operation of a 2-D lateral scanner and a 1-D depth scanner enables fast 3-D microscopy over a volume that measures 340μm by 236μm by 286μm.

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.

Confocal microscopy is an indispensable tool for three-dimensional (3-D) imaging in both intravital and clinical settings. Because of its optical sectioning property achieved from its optical pinhole, it allows serial image stacking of each focus image plane to re-create volumetric imaging. Conventional single-axis confocal (SAC) microscopes have a tradeoff between resolution, field of view (FOV), and objective lens size, since a high numerical aperture (NA) lens is needed for sufficient resolution, and a long focal length is needed for a large FOV and working distance (WD). A dual-axes confocal (DAC) microscope architecture has been proposed utilizing two overlapping low NA beams, which effectively decouples these tradeoffs [1]. The DAC microscope offers several advantages over the SAC design. First, the higher NA required by the SAC design implies a smaller WD and smaller FOV. In contrast, the DAC design has a long WD, providing room for a postobjective MEMS scanner. Second, in the SAC design, the transverse resolution is substantially better than the axial resolution, while the DAC design provides balanced resolutions in all spatial dimensions. Third, the DAC design has superior optical sectioning because light scattered along the illumination path outside the focal volume couples to the output spatial filter with very low efficiency, enhancing both detection sensitivity and dynamic range. Previously, MEMS-scanner-based DAC microscopes have been demonstrated in a tabletop setup [2], and then in the first miniaturized proto-type system on a V-block with 1300- and 488-nm-wavelength, respectively [3, 4].

A 1.8-mm diameter gradient-index relay lens has been incorporated into a miniature dual-axis confocal microscope for image-guided surgery. A biaxial MEMS mirror is actuated at resonance along each axis to create a non-repeating Lissajous scan pattern for achieving large fields of view.

We demonstrate microelectromechanical systems (MEMS) based near infrared fluorescence dual-axes confocal (DAC) microscopes in both a 10-mm microscope and a 5-mm endoscope for three-dimensional (3-D) cellular imaging of both ex vivo and in vivo samples.

We have developed a two-color ratiometric microscopy technique that not only reduces image contrast due to nonspecific probe accumulation, but also provides a quantitative 3D map of cellular biomarker expression.

We demonstrate MEMS based near infrared fluorescence dual-axes confocal (DAC) imaging in a 10-mm-diameter microscope for three-dimensional (3-D) imaging of in vivo human skin. The probe is used to reveal mosaicing two-dimensional (2-D) en face sub-surface histology at depths of up to 250 µm. The probe has a maximum field of view (FOV) of 800×450 µm2 and a maximum acquisition rate of 10 frames/second. The transverse and axial resolutions are 5 µm and 7 µm, respectively.

The burgeoning fields of in vivo three-dimensional (3D) microscopy and endomicroscopy, as well as ex vivo tissue cytometry have introduced new challenges for tissue preparation and staining with exogenous molecular contrast agents. These challenges include effective delivery of the agents, and once delivered, distinguishing between bound verses unbound molecular probes. If applied topically, there are additional issues with rinsing off unbound probe, which can be nonuniform and inefficient in thick tissues, thus leading to ambiguous contrast and a large nonspecific background that may obscure molecule-specific staining. Therefore, we have developed a ratiometric 3D microscopy scheme that not only reduces the effects of nonspecific sources of contrast, but also enables quantification of the relative binding affinity of imaging probes to their biomarker targets. Here we demonstrate this ratiometric approach by simultaneously imaging a HER2/neu (erbB2)-targeted monoclonal antibody labeled with one fluorophore and an isotype-matched negative control antibody labeled with another fluorophore. By taking a pixel-by-pixel calibrated ratio between the signals from each fluorescent image channel, accurate quantification of specific versus nonspecific binding affinity is achieved with cultured cells, yielding data that are in agreement with analyses via flow cytometry. We also demonstrate quantitative 3D microscopic imaging of biomarker expression in tissue models and in thick human biopsy samples of normal, HER2-negative, and HER2-positive breast tumors. This strategy enables rapid, quantitative, and unambiguous volumetric microscopy of biomarker expression in thick tissues, including whole biopsies, and will enable real-time optical assessment of disease markers in the living body.

In this paper, a miniaturized intravital dual-axes confocal (DAC) microscope for investigating gene transfer in skin in vivo was reported. In vivo 3-D imaging not only validates what can be observed in ex vivo tissue sections, but gives spatial and temporal information in a living specimen. This opens up new opportunities in advancing disease and treatment research in the skin.

We demonstrate a dual-axes confocal endoscope in a 5.5 mm diameter package for clinical use. Miniaturization is achieved by using a barbell-shaped, gimbaled, two-dimensional MEMS scanner that is actuated by self-aligned, vertical-comb actuators. The maximum DC optical scan angles are plusmn2.6deg on the inner axis and plusmn0.8deg on the outer axis, and the corresponding resonance frequencies are 3.1 kHz and 1.1 KHz. The maximum imaging rate is 10 frames/second, and the microscope achieves full-width-half-maximum transverse and axial resolutions of 5 mum and 7 mum, respectively when operated in the near infrared wavelength (785 nm).

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.

Towards developing dual-axes confocal microscopy for gastrointestinal disease detection, we quantitatively investigate the optical-sectioning ability of this technology through reflectance experiments in a tissue phantom, and also obtain deep fluorescence image sections of tissue specimens.

We perform Monte Carlo simulations to show that the dual-axes (DA) confocal architecture has superior rejection of multiply scattered photons in tissue to that of single axis. As a result, the DA configuration provides improved signal-to-noise ratio and dynamic range, and thus is sensitive to ballistic photons from deeper within tissue, features that are particularly useful for performing vertical cross-sectional reflectance images in tissue.

We demonstrate a novel microelectromechanical system (MEMS) based Dual-Axes confocal (DAC) microscope in a 10-mm diameter handheld package. Miniaturization is achieved by using a barbell-shaped, gimbaled, two-dimensional (2-D) MEMS scanner that is actuated by self-aligned vertical comb actuators. The maximum DC optical scan angles are plusmn4.25deg on the inner axis and plusmn1.5deg on the outer axis, and the corresponding resonance frequencies are 3.4 kHz and 1.2 kHz. The maximum imaging rate is 5 frames/second. The miniature DAC microscope operating at 785-nm-wavelength achieves full- width-half-maximum (FWHM) transverse and axial resolutions of 4.2 mum and 7 mum, respectively.

We demonstrate reflectance and fluorescence imaging with a handheld dual-axes confocal microscope based on a two-dimensional microelectromechanical system scanner. Three-dimensional imaging capability is shown in tissue, and in vivo imaging of a mouse is performed.

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 resolu tion 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 additio n, 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-ax is (SA) configurations. Simulation results show that the axial response for the dual axes configur ation 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 cross-sectional images, which has significant implications for in vivo imaging. Keywords: scattering, Mie theory, confocal, microscopy, single-axis, dual-axes, Monte Carlo