David R. Rivera

Compact and flexible raster scanning multiphoton endoscope capable of imaging unstained tissue

We present a compact and flexible endoscope (3-mm outer diameter, 4-cm rigid length) that utilizes a miniaturized resonant/nonresonant fiber raster scanner and a multielement gradient-index lens assembly for two-photon excited intrinsic fluorescence and second-harmonic generation imaging of biological tissues. The miniaturized raster scanner is fabricated by mounting a commercial double-clad optical fiber (DCF) onto two piezo bimorphs that are aligned such that their bending axes are perpendicular to each other. Fast lateral scanning of the laser illumination at 4.1 frames/s (512 lines per frame) is achieved by simultaneously driving the DCF cantilever at its resonant frequency in one dimension and nonresonantly in the orthogonal axis. The implementation of a DCF into the scanner enables simultaneous delivery of the femtosecond pulsed 800-nm excitation source and epi-collection of the signal. Our device is able to achieve a field-of-view (FOVxy) of 110 μm by 110 μm with a highly uniform pixel dwell time. The lateral and axial resolutions for two-photon imaging are 0.8 and 10 μm, respectively. The endoscope’s imaging capabilities were demonstrated by imaging ex vivo mouse tissue through the collection of intrinsic fluorescence and second-harmonic signal without the need for staining. The results presented here indicate that our device can be applied in the future to perform minimally invasive in vivo optical biopsies for medical diagnostics.

In vivo imaging of unstained tissues using long gradient index lens multiphoton endoscopic systems

We characterize long (up to 285 mm) gradient index (GRIN) lens endoscope systems for multiphoton imaging. We fabricate a portable, rigid endoscope system suitable for imaging unstained tissues, potentially deep within the body, using a GRIN lens system of 1 mm diameter and 8 cm length. The portable device is capable of imaging a ~200 µm diameter field of view at 4 frames/s. The lateral and axial resolution in water is 0.85 µm and 7.4 µm respectively. In vivo images of unstained tissues in live, anesthetized rats using the portable device are presented. These results show great promise for GRIN endoscopy to be used clinically.

In vivo imaging of unstained tissues using a compact and flexible multiphoton microendoscope

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.

Use of a lensed fiber for a large-field-of-view, high-resolution, fiber-scanning microendoscope

We report the application of a lensed fiber to a miniaturized fiber raster scanner in order to reduce the fibers output beam size, thereby allowing for a compact and flexible endoscope capable of a large field of view (FOV) and high spatial resolution. For a proof of principle, the fabricated lensed fiber scanner is paired with a miniaturized gradient-index assembly to achieve a one-photon lateral resolution of 1.1 μm with a FOV that has a diameter of 440 μm.

Miniature varifocal objective lens for endomicroscopy.

A miniature catadioptric lens for endoscopic imaging based on the principle of wavelength division multiplexing is presented. We demonstrate change of the magnification and the field of view (FOV) of the lens without any mechanical adjustment of the optical elements. The lens provides magnifications of ~-1.5× at 406-750 nm and ~-0.2× at 800 nm. The lens is used to demonstrate large-FOV (1.3 mm) reflectance imaging and high-resolution (0.57 μm) multiphoton fluorescence imaging of unstained mouse tissues.

Dual modality endomicroscope with optical zoom capability

We present a miniature endomicroscope that combines large field-of-view (FOV) (1.15 mm) reflectance imaging with high-resolution (~0.5 μm) multiphoton intrinsic fluorescence imaging. We acquired in vivo and ex vivo images of unstained normal and tumor-laden tissues by using the large-FOV mode to navigate to the site of interest and then switching to the high-resolution modality to resolve cellular details.

Miniaturized fiber raster scanner for endoscopy

A miniaturized scanning mechanism is a crucial component in the creation of endoscopes for microscopic imaging. Several groups have developed resonant scanners (e.g., spiral or Lissajous scan pattern), but these suffer from limitations in non-uniform spatial coverage and sampling time, in comparison to a raster scanner. Additionally, a resonant scanner lacks the ability to perform line-scan imaging, a crucial capability in measuring a variety of fast, dynamic physiological phenomena (e.g., blood flow, molecular diffusion, etc.). However, current miniaturized raster scanners are limited in terms of their physical dimensions and scan speed. We demonstrate a novel hybrid resonant/non-resonant miniaturized raster scanner, fabricated by mounting a double clad fiber onto two perpendicularly oriented piezo bimorphs. The fiber scanner has a total length of 2.6cm, a width/thickness [less than or equal to] 1mm, achieves [greater than or equal to] fiber tip deflection for both the resonant and non-resonant axes, and allows for imaging at approximately 4 frames per second (512 lines per frame). An essentially uniform spatial coverage and sampling time can be achieved by utilizing the middle portion (e.g., middle 500 μm) of the resonant scanning range. The small size allows for the fiber scanner to be easily packaged along with miniaturized lenses to form an endoscope for microscopic imaging. We bonded a stiffening fiber alongside the vibrating fiber to break its cylindrical symmetry. Thus, only one vibration mode is excited, generating a purely linear spatial motion. In order to demonstrate the fiber scanners imaging capabilities we have taken transmission and fluorescence images, in which the double clad fibers inner clad is used for fluorescence collection.

Intravital Multiphoton Endoscopy

When a patient has symptoms that could be indicative of cancer a clinical oncologist will perform a series of tests to reach an accurate diagnosis. The gold standard method for reaching a final diagnosis frequently involves locating and extracting tissue biopsies, which are then processed and studied at the cellular level by a pathologist. From this procedure a pathology report will be compiled that contains the diagnosis of the cancer type and grade. Deep penetration, low-resolution, imaging tests (e.g., magnetic resonance imaging MRI) scans, computed tomography (CT) scans, Ultrasound, etc.) can track the extent and spread of disease and serve as an initial screen for disease in tissues that are not easily accessed using conventional biopsy or optical imaging techniques (e.g. periphery lung, pancreas, small intestine). However, to date, these low resolution imaging tests have not been able to be used as stand alone diagnostic tests because they lack the ability to visualize disease at the cellular level and thus have been unable to match the sensitivity and specificity of diagnosis based upon biopsied tissue processed into histopathology slides. Finally, by compiling the data obtained from all these tests, the oncologist can determine the stage of the cancer (i.e., the severity or extent of the cancer) and use this information to provide the patient with a prognosis and course of treatment.

High resolution, large field-of-view endomicroscope with optical zoom capability

Here, we present a miniature endomicroscope that combines large field-of-view (FOV) (1.15 mm) reflectance modality and high-spatial resolution (~ 0.5 um) multiphoton imaging. The essential element of the endoscope is a 3 mm outside diameter (OD), catadioptric zoom lens based on the idea of separating the optical paths of excitation light with different wavelengths. The two imaging modes are switched by changing the wavelength of the excitation light and, therefore, the optical zoom operation is achieved without any mechanical adjustment at the endoscope distal end. We aligned in free space the zoom lens with a previously demonstrated miniaturized resonant/non-resonant fiber raster scanner. We tested the performance and confirmed the high resolution and large FOV of this miniature device by imaging a US Air Force test target in transmission. We acquired ex vivo images of unstained rodent tissues by using the large FOV mode to navigate to the site of interest and then using the high resolution modality to image with cellular details. The demonstrated endomicroscope with optical zoom capability is a significant step toward developing clinical optical tools for real time tissue diagnostics.

A Miniature Endomicroscope with Optical Zoom Capability

We report a dual-modality miniature endomicroscope that combines large field-of-view (1.15 mm) scattering and high-resolution (~0.5 μm) multiphoton fluorescence imaging. We acquired in vivo and ex vivo images of unstained rodent tissues using both modalities.