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1.
J Biomed Opt ; 17(2): 021102, 2012 Feb.
Article in English | MEDLINE | ID: mdl-22463020

ABSTRACT

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 µm(2) 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.


Subject(s)
Endoscopes, Gastrointestinal , Image Enhancement/instrumentation , Lenses , Microscopy, Confocal/instrumentation , Signal Processing, Computer-Assisted/instrumentation , Equipment Design , Equipment Failure Analysis , Humans , Infrared Rays , Miniaturization
2.
J Acoust Soc Am ; 129(4): 1837-50, 2011 Apr.
Article in English | MEDLINE | ID: mdl-21476640

ABSTRACT

This work reports on an optical hydrophone that is insensitive to hydrostatic pressure, yet capable of measuring acoustic pressures as low as the background noise in the ocean in a frequency range of 1 Hz to 100 kHz. The miniature hydrophone consists of a Fabry-Perot interferometer made of a photonic-crystal reflector interrogated with a single-mode fiber and is compatible with existing fiber-optic technologies. Three sensors with different acoustic power ranges placed within a sub-wavelength sized hydrophone head allow a high dynamic range in the excess of 160 dB with a low harmonic distortion of better than -30 dB. A method for suppressing cross-coupling between sensors in the same hydrophone head is also proposed. A prototype was fabricated, assembled, and tested. The sensitivity was measured from 100 Hz to 100 kHz, demonstrating a sound-pressure-equivalent noise spectral density down to 12 µPa/Hz(1/2), a flatband wider than 10 kHz, and very low distortion.


Subject(s)
Acoustics/instrumentation , Miniaturization , Models, Theoretical , Oceanography/instrumentation , Optical Fibers , Elasticity , Lasers , Oceans and Seas , Optics and Photonics , Pressure , Seawater , Silicon , Temperature , Transducers
3.
J Invest Dermatol ; 131(5): 1061-6, 2011 May.
Article in English | MEDLINE | ID: mdl-21191407

ABSTRACT

Advancing molecular therapies for the treatment of skin diseases will require the development of new tools that can reveal spatiotemporal changes in the microanatomy of the skin and associate these changes with the presence of the therapeutic agent. For this purpose, we evaluated a handheld dual-axis confocal (DAC) microscope that is capable of in vivo fluorescence imaging of skin, using both mouse models and human skin. Individual keratinocytes in the epidermis were observed in three-dimensional image stacks after topical administration of near-infrared (NIR) dyes as contrast agents. This suggested that the DAC microscope may have utility in assessing the clinical effects of a small interfering RNA (siRNA)-based therapeutic (TD101) that targets the causative mutation in pachyonychia congenita (PC) patients. The data indicated that (1) formulated indocyanine green (ICG) readily penetrated hyperkeratotic PC skin and normal callused regions compared with nonaffected areas, and (2) TD101-treated PC skin revealed changes in tissue morphology, consistent with reversion to nonaffected skin compared with vehicle-treated skin. In addition, siRNA was conjugated to NIR dye and shown to penetrate through the stratum corneum barrier when topically applied to mouse skin. These results suggest that in vivo confocal microscopy may provide an informative clinical end point to evaluate the efficacy of experimental molecular therapeutics.


Subject(s)
Contrast Media , Skin Diseases/diagnosis , Animals , Humans , Indocyanine Green , Keratinocytes/pathology , Mice , Microscopy, Confocal/instrumentation , Microscopy, Confocal/methods , Microscopy, Fluorescence/instrumentation , Microscopy, Fluorescence/methods , Pachyonychia Congenita/drug therapy , Pachyonychia Congenita/pathology , RNA, Small Interfering/therapeutic use , Skin/pathology , Skin Diseases/pathology
4.
J Biomed Opt ; 15(3): 036027, 2010.
Article in English | MEDLINE | ID: mdl-20615029

ABSTRACT

Transgenic reporter mice and advances in imaging instrumentation are enabling real-time visualization of cellular mechanisms in living subjects and accelerating the development of novel therapies. Innovative confocal microscope designs are improving their utility for microscopic imaging of fluorescent reporters in living animals. We develop dual-axis confocal (DAC) microscopes for such in vivo studies and create mouse models where fluorescent proteins are expressed in the skin for the purpose of advancing skin therapeutics and transdermal delivery tools. Three-dimensional image volumes, through the different skin compartments of the epidermis and dermis, can be acquired in several seconds with the DAC microscope in living mice, and are comparable to histologic analyses of reporter protein expression patterns in skin sections. Intravital imaging with the DAC microscope further enables visualization of green fluorescent protein (GFP) reporter gene expression in the skin over time, and quantification of transdermal delivery of small interfering RNA (siRNA) and therapeutic efficacy. Visualization of transdermal delivery of nucleic acids will play an important role in the development of innovative strategies for treating skin pathologies.


Subject(s)
Drug Delivery Systems , Microscopy, Confocal/methods , RNA, Small Interfering/administration & dosage , Animals , Foot , Gene Expression/drug effects , Genes, Reporter , Green Fluorescent Proteins/administration & dosage , Green Fluorescent Proteins/chemistry , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Mice , Mice, Transgenic , RNA, Small Interfering/chemistry , RNA, Small Interfering/genetics , Skin
5.
J Biomed Opt ; 15(2): 026029, 2010.
Article in English | MEDLINE | ID: mdl-20459274

ABSTRACT

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 500x500 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.


Subject(s)
Algorithms , Craniotomy/instrumentation , Image Enhancement/instrumentation , Lenses , Microscopy, Confocal/instrumentation , Surgery, Computer-Assisted/instrumentation , Animals , Mice , Miniaturization , Reproducibility of Results , Sensitivity and Specificity
6.
Opt Lett ; 34(7): 875-7, 2009 Apr 01.
Article in English | MEDLINE | ID: mdl-19340156

ABSTRACT

We predict theoretically and confirm experimentally that the Kerr-induced phase drift of a fiber optic gyroscope (FOG) operated with a laser instead of a broadband source is virtually eliminated when the sensing coil is made of an air-core photonic-bandgap fiber. This is the first demonstration of a laser-driven FOG with a Kerr-induced drift low enough to meet the inertial navigation requirement for a 10-h transcontinental flight.

7.
Biophys J ; 96(6): 2405-14, 2009 Mar 18.
Article in English | MEDLINE | ID: mdl-19289065

ABSTRACT

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.


Subject(s)
Biomarkers/metabolism , Cell Membrane/metabolism , Imaging, Three-Dimensional/methods , Microscopy, Confocal/methods , Antibodies , Breast Neoplasms/metabolism , Cell Line , Collagen , Drug Combinations , Female , Flow Cytometry , Fluorescence , Humans , Laminin , Proteoglycans , Receptor, ErbB-2/metabolism
8.
J Biomed Opt ; 13(3): 034020, 2008.
Article in English | MEDLINE | ID: mdl-18601565

ABSTRACT

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.


Subject(s)
Gastric Mucosa/cytology , Image Enhancement/instrumentation , Intestinal Mucosa/cytology , Microscopy, Confocal/instrumentation , Microscopy, Fluorescence/instrumentation , Nephelometry and Turbidimetry/instrumentation , Optics and Photonics/instrumentation , Humans , In Vitro Techniques , Phantoms, Imaging
9.
Opt Express ; 16(10): 7224-32, 2008 May 12.
Article in English | MEDLINE | ID: mdl-18545427

ABSTRACT

We present a handheld dual-axes confocal microscope that is based on a two-dimensional microelectromechanical systems (MEMS) scanner. It performs reflectance and fluorescence imaging at 488 nm wavelength, with three-dimensional imaging capability. The fully packaged microscope has a diameter of 10 mm and acquires images at 4 Hz frame rate with a maximum field of view of 400 microm x 260 microm. The transverse and axial resolutions of the handheld probe are 1.7 microm and 5.8 microm, respectively. Capability to perform real time small animal imaging is demonstrated in vivo in transgenic mice.


Subject(s)
Microscopy, Confocal/methods , Microscopy, Fluorescence/methods , Animals , Computers , Diagnostic Imaging/instrumentation , Diagnostic Imaging/methods , Equipment Design , Green Fluorescent Proteins/metabolism , Image Processing, Computer-Assisted , Imaging, Three-Dimensional , Mice , Mice, Transgenic , Optics and Photonics , Photons , Software
10.
Opt Lett ; 32(12): 1674-6, 2007 Jun 15.
Article in English | MEDLINE | ID: mdl-17572743

ABSTRACT

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.


Subject(s)
Microscopy, Confocal/instrumentation , Optics and Photonics , Anisotropy , Chemical Phenomena , Chemistry, Physical , Equipment Design , Microscopy, Confocal/methods , Models, Statistical , Models, Theoretical , Monte Carlo Method , Photons , Scattering, Radiation
11.
Opt Lett ; 32(3): 256-8, 2007 Feb 01.
Article in English | MEDLINE | ID: mdl-17215937

ABSTRACT

The first, to our knowledge, miniature dual-axes confocal microscope has been developed, with an outer diameter of 10 mm, for subsurface imaging of biological tissues with 5-7 microm resolution. Depth-resolved en face images are obtained at 30 frames per second, with a field of view of 800 x 100 microm, by employing a two-dimensional scanning microelectromechanical systems mirror. Reflectance and fluorescence images are obtained with a laser source at 785 nm, demonstrating the ability to perform real-time optical biopsy.


Subject(s)
Fiber Optic Technology/instrumentation , Image Enhancement/instrumentation , Micromanipulation/instrumentation , Microscopy, Confocal/instrumentation , Spectrophotometry, Infrared/instrumentation , Computer Systems , Electronics , Equipment Design , Equipment Failure Analysis , Mechanics , Micromanipulation/methods , Microscopy, Confocal/methods , Miniaturization , Reproducibility of Results , Sensitivity and Specificity , Spectrophotometry, Infrared/methods , Transducers
12.
Opt Express ; 15(9): 5342-59, 2007 Apr 30.
Article in English | MEDLINE | ID: mdl-19532788

ABSTRACT

Using an eigenmode decomposition technique, we numerically determine the backreflection coefficient of the modes of air-core photonic bandgap fibers for flat terminations. This coefficient is found to be very small for the fundamental air-guided mode, of the order of 10(-5) to 10(-6), in contrast with the surface and bulk modes, which exhibit significantly higher reflections, by about three to four orders of magnitude. For the Crystal Fibre HC-1550-2 fiber, we find a reflection coefficient of 1.9x10(-6) for an air termination, and approximately 3.3% for a silica termination. We also find that the Fresnel approximation is ill suited for the determination of the modal reflection coefficient, and instead propose a more accurate new formula based on an averaged modal index.

13.
J Biomed Opt ; 11(5): 054019, 2006.
Article in English | MEDLINE | ID: mdl-17092168

ABSTRACT

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-microm resolution, at tissue depths up to 610 microm. Post-objective scanning enables a large field of view (610 x 640 microm), 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.


Subject(s)
Anatomy, Cross-Sectional/instrumentation , Colonic Neoplasms/pathology , Image Enhancement/instrumentation , Imaging, Three-Dimensional/instrumentation , Microscopy, Confocal/instrumentation , Anatomy, Cross-Sectional/methods , Equipment Design , Equipment Failure Analysis , Humans , Microscopy, Confocal/methods , Reproducibility of Results , Sensitivity and Specificity
14.
J Opt Soc Am A Opt Image Sci Vis ; 23(7): 1669-77, 2006 Jul.
Article in English | MEDLINE | ID: mdl-16783430

ABSTRACT

We present a simple processing technique that uses the concept of minimum-phase functions to improve frequency-domain optical coherence tomography systems. Our approach removes the autocorrelation noise and therefore increases both the accessible depth range and the recovery accuracy. To our knowledge, this is the first time that the concept of minimum-phase functions has been applied to improve optical coherence tomography.


Subject(s)
Algorithms , Artifacts , Image Enhancement/methods , Image Interpretation, Computer-Assisted/methods , Imaging, Three-Dimensional/methods , Tomography, Optical Coherence/methods , Information Storage and Retrieval/methods , Reproducibility of Results , Sensitivity and Specificity
15.
Nano Lett ; 6(3): 355-60, 2006 Mar.
Article in English | MEDLINE | ID: mdl-16522022

ABSTRACT

Optically resonant metallic bowtie nanoantennas are utilized as fabrication tools for the first time, resulting in the production of polymer resist nanostructures <30 nm in diameter at record low incident multiphoton energy densities. The nanofabrication is accomplished via nonlinear photopolymerization, which is initiated by the enhanced, confined optical fields surrounding the nanoantenna. The position, size, and shape of the resist nanostructures directly correlate with rigorous finite-difference time-domain computations of the field distribution, providing a nanometer-scale measurement of the actual field confinement offered by single optical nanoantennas. In addition, the size of the photoresist regions yields strong upper bounds on photoacid diffusion and resist resolution in SU-8, demonstrating a technique that can be generalized to the study of many current and yet-to-be-developed photoresist systems.


Subject(s)
Nanostructures/chemistry , Nanotechnology/instrumentation , Nanotechnology/methods , Color , Microscopy, Atomic Force , Nanostructures/ultrastructure , Optics and Photonics
16.
J Chem Phys ; 124(6): 61101, 2006 Feb 14.
Article in English | MEDLINE | ID: mdl-16483189

ABSTRACT

Single metallic bowtie nanoantennas provide a controllable environment for surface-enhanced Raman scattering (SERS) of adsorbed molecules. Bowties have experimentally measured electromagnetic enhancements, enabling estimation of chemical enhancement for both the bulk and the few-molecule regime. Strong fluctuations of selected Raman lines imply that a small number of p-mercaptoaniline molecules on a single bowtie show chemical enhancement >10(7), much larger than previously believed, likely due to charge transfer between the Au surface and the molecule. This chemical sensitivity of SERS has significant implications for ultra-sensitive detection of single molecules.


Subject(s)
Aniline Compounds/chemistry , Mercury/chemistry , Nanotechnology/methods , Organometallic Compounds/analysis , Spectrum Analysis, Raman/methods , Absorption , Organometallic Compounds/chemistry , Scattering, Radiation , Sensitivity and Specificity
17.
Opt Express ; 14(7): 2979-93, 2006 Apr 03.
Article in English | MEDLINE | ID: mdl-19516437

ABSTRACT

We describe a finite-difference numerical method that allows us to simulate the modes of air-core photonic-bandgap fibers (PBF) of any geometry in minutes on a standard PC. The modes' effective indices and fields are found by solving a vectorial transverse magnetic-field equation in a matrix form, which can be done quickly because this matrix is sparse and because we reduce its bandwidth by rearranging its elements. The Stanford Photonic-Bandgap Fiber code, which is based on this method, takes about 4 minutes to model 20 modes of a typical PBF on a PC. Other advantages include easy coding, faithful modeling of the abrupt discontinuities in the index profile, high accuracy, and applicability to waveguides of arbitrarily complex profile.

18.
J Biomed Opt ; 9(4): 735-42, 2004.
Article in English | MEDLINE | ID: mdl-15250760

ABSTRACT

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 < or =4.4 microm 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.


Subject(s)
Drosophila/cytology , Drosophila/embryology , Image Enhancement/instrumentation , Microscopy, Confocal/instrumentation , Microscopy, Fluorescence/instrumentation , Muscle, Skeletal/cytology , Neurons/cytology , Animals , Cerebellum/cytology , Equipment Design , Equipment Failure Analysis , Feasibility Studies , Mice , Microscopy, Confocal/methods , Microscopy, Fluorescence/methods
19.
Opt Lett ; 28(20): 1915-7, 2003 Oct 15.
Article in English | MEDLINE | ID: mdl-14587774

ABSTRACT

We present a dual-axes confocal microscope that employs postobjective scanning and low-coherence heterodyne detection to collect vertical cross-sectional images from biological tissue with high axial resolution, reduced noise from scattered light, deep tissue penetration, and a large dynamic range. This architecture can be scaled down to millimeter dimensions with microelectromechanical systems technology for performance of in vivo optical biopsy.


Subject(s)
Biopsy/methods , Microscopy, Confocal , Equipment Design , Esophagus/pathology , Humans
20.
Opt Lett ; 28(6): 414-6, 2003 Mar 15.
Article in English | MEDLINE | ID: mdl-12659264

ABSTRACT

We describe a novel confocal microscope that uses separate low-numerical-aperture objectives with the illumination and collection axes crossed at angle theta from the midline. This architecture collects images in scattering media with high transverse and axial resolution, long working distance, large field of view, and reduced noise from scattered light. We measured transverse and axial (FWHM) resolution of 1.3 and 2.1 microm, respectively, in free space, and confirm subcellular resolution in excised esophageal mucosa. The optics may be scaled to millimeter dimensions and fiber coupled for collection of high-resolution images in vivo.


Subject(s)
Microscopy, Confocal , Models, Theoretical , Humans
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