We use a compact and flexible multiphoton microendoscope (MPME) to acquire in vivo images of unstained liver, kidney, and colon from an anesthetized rat. The device delivers femtosecond pulsed 800 nm light from the core of a raster-scanned dual-clad fiber (DCF), which is focused by a miniaturized gradient-index lens assembly into tissue. Intrinsic fluorescence and second-harmonic generation signal from the tissue is epi-collected through the core and inner clad of the same DCF. The MPME has a rigid distal tip of 3 mm in outer diameter and 4 cm in length. The image field-of-view measures 115 μm by 115 μm and was acquired at 4.1 frames/s with 75 mW illumination power at the sample. Organs were imaged after anesthetizing Sprague-Dawley rats with isofluorane gas, accessing tissues via a ventral-midline abdominal incision, and isolating the organs with tongue depressors. In vivo multiphoton images acquired from liver, kidney, and colon using this device show features similar to that of conventional histology slides, without motion artifact, in ∼ 75% of imaged frames. To the best of our knowledge, this is the first demonstration of multiphoton imaging of unstained tissue from a live subject using a compact and flexible MPME device.
Multiphoton microscopic endoscopy (MPM-E) is a promising medical in vivo diagnostic imaging technique because it
captures intrinsic fluorescence and second harmonic generation signals to reveal anatomical and histological information
about disease states in tissue. However, maximizing light collection from multiphoton endoscopes remains a challenge:
weak nonlinear emissions from endogenous structures, miniature optics, large imaging depths, and light scattering in
tissue all hamper light collection. The quantity of light that may be collected using a dual-clad fiber system from
scattering phantoms that mimic the properties of the in vivo environment is measured. In this experiment, 800nm
excitation light from a Ti:Sapphire laser is dispersion compensated and focused through a SM800 optical fiber and lens
system into the tissue phantom. Emission light from the phantom passes through the lens system, reflects off the dichroic
and is then collected by a second optical fiber actuated by a micromanipulator. The lateral position of the collection fiber
varies, measuring the distribution of emitted light 2000μm on either side of the focal point reimaged to the object plane.
This spatial collection measurement is performed at depths up to 200μm from the phantom surface. The tissue phantoms
are composed of a 15.8 μM fluorescein solution mixed with microspheres, approximating the scattering properties of
human bladder and dermis tissue. Results show that commercially available dual-clad optical fibers collect more than
47% of the total emission returning to the object plane from both phantoms. Based on these results, initial MPM-E
devices will image the surface of epithelial tissues.
Scanning fiber endoscope (SFE) technology has shown promise as a minimally invasive optical imaging tool.
To date, it is capable of capturing full-color 500-line images, at 15 Hz frame rate in vivo, as a 1.6 mm diameter
endoscope. The SFE uses a singlemode optical fiber actuated at mechanical resonance to scan a light spot over
tissue while backscattered or fluorescent light at each pixel is detected in time series using several multimode
optical fibers. We are extending the capability of the SFE from a RGB reflectance imaging device to a
diagnostic tool by imaging laser induced fluorescence (LIF) in tissue, allowing for correlation of endogenous
fluorescence to tissue state. Design of the SFE for diagnostic imaging is guided by a comparison of single point
spectra acquired from an inflammatory bowel disease (IBD) model to tissue histology evaluated by a
pathologist. LIF spectra were acquired by illuminating tissue with a 405 nm light source and detecting intrinsic
fluorescence with a multimode optical fiber. The IBD model used in this study was mdr1a-/- mice, where IBD
was modulated by infection with Helicobacter bilis. IBD lesions in the mouse model ranged from mild to
marked hyperplasia and dysplasia, from the distal colon to the cecum. A principle components analysis (PCA)
was conducted on single point spectra of control and IBD tissue. PCA allowed for differentiation between
healthy and dysplastic tissue, indicating that emission wavelengths from 620 - 650 nm were best able to
differentiate diseased tissue and inflammation from normal healthy tissue.
Scanning fiber optical endoscopy shows promise as a small, inexpensive imaging tool. Using this method of image acquisition, a scanning fiber is actuated at mechanical resonance, projecting a light spot across an imaged surface. Light backscattered from scanned spots is measured to form an image. The acquired image field of view, resolvable pixels, and frame rate are dependent on the dynamics of the optical fiber used as a resonant scanner. A finite-element analysis (FEA) model was constructed to predict scanning fiber dynamics, and compared with experimental results. A scanning fiber microfabrication process was developed that allows for the controlled manufacture of fiber scanners. Experimental results confirm that the theoretical model was accurate in predicting the system transfer function with less than 6% error in amplitude and less than 10% error in resonant frequency at the first two resonant modes of a cylindrical and a microfabricated fiber. The scanning fiber microfabrication process proved to be capable of repeatably etching notches in optical fibers as small as 2.00±0.05 mm in length and 15±2 µm in diameter. FEA was used to predict the effect of geometry change on microfabricated fiber scan dynamics, yielding candidate designs chosen for enhanced performance of future scanning endoscopes.
A cantilevered singlemode optical fiber is base-excited to create 2D amplitude-modulated resonant motion as a basis for a scanning fiber endoscope (SFE). Over the past few years, prototype SFEs have been developed with smaller sizes of the distal rigid tip which houses the fiber scanner. Our current prototype is 2 mm in diameter with 15 mm rigid length at the tip of a highly flexible shaft. A spiral scan pattern at 40 degrees field of view generates 250 rings (500 lines) at greater than 10 frames per second with negligible distortion at 10 micron resolution. Future SFEs will use microfabrication techniques to sculpt the optical fiber cantilever to form tapered and microlensed tips for the purpose of increasing field of view without increasing electrical power. Microfabrication of complex optical fiber geometries is guided by linear and nonlinear dynamic models of the resonant motion of these fiberoptic scanners. Linear finite element analysis (FEA) is used to match low amplitude motions of tapered and notched fiber geometries, indicating that more flexible regions or hinges can be designed into future fiber scanners for increased amplitude of motion without sacrificing frequency. Nonlinear models of the fiber dynamics are developed and the results help predict the more complex behavior of microfabricated fiber scanners at wider fields of view. Thus, sophisticated fiber dynamics models are used to guide the development of more efficient scanning fiber image acquisition sensors and systems, such as ultrathin flexible SFEs and low-cost sensors.
Our goal is to produce a micro-optical scanner at the tip of an ultrathin flexible endoscope with an overall diameter of 1 mm. Using a small diameter piezoelectric tube actuator, a cantilevered optical fiber can be driven in mechanical resonance to scan a beam of light in a space-filling, spiral scan pattern. By knowing and/or controlling the fiber position and acquiring backscattered intensity with a photodetector, an image is acquired. A microfabrication process of computer-controlled acid etching is used to reduce the mass along the fiber scanner shaft to allow for high scan amplitude and frequency. A microlens (<1 mm diameter) is fabricated on the end of the optical fiber to reduce divergence of the scanned optical beam. This added mass of the microlens at the free end of the fiber causes the location of the second vibratory node to shift to near the focal length of the microlens. The result is a microlens undergoing angular rotation along two axes with minimal lateral microlens displacement. Preliminary experimental results indicate that this method of optical beam scanning can deliver laser energy over wide fields of view (>50 degrees full angle), up to video scan rates (>10 KHz), while maintaining a scanner diameter of 1 mm. A comparison can be made to bi-axial mirror scanners being fabricated as a MEMS device (micro-electro-mechanical system). Based on the opto-mechanical performance of these microlensed fiber scanners, flexible catheter scopes are possible for new microendoscopies that combine imaging with laser diagnoses.
Flexible endoscopes currently used in medicine have a fundamental tradeoff. Either resolution or field of view (FOV) is sacrificed when the scope diameter is less than 3 mm, since the minimum pixel size is usually greater than 4 microns in a pixel-array such as a camera or fiber bundle. Thus, the number of pixels within the image plane determines the minimum size of a conventional scope. However, an image plane is not required for image acquisition using a scanning single-fiber scope. Both high resolution and wide FOV are possible in a scanning single-fiber scope of 1 to 2 mm in diameter. The technical challenge is to produce a two- dimensional scanned beam of light at the distal tip of the scope. By manipulating a resonant fiberoptic cantilever as the optical scanner, various 2-D scan patterns can be produced. The general design concepts and analyses of the fiberoptic scanner for scaling to small size and high resolution/FOV are reviewed. In our initial experimental tests, the size of the photon detector in a fiberoptic scanning scope is demonstrated to not affect image resolution, unlike existing endoscopes with pixel-based detector systems.
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