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 (FOV(xy)) 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.

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.

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 fiber's 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.

Multifocal multiphoton endoscope.

We report a miniaturized resonant/non-resonant multi-fiber raster scanner that is paired with a gradient-index lens assembly to achieve a compact and flexible multifocal multiphoton endoscope capable of longitudinal parallel image acquisition. Multiphoton images are obtained simultaneously at three axial depths, separated by ≥4.8 µm, by incorporating three axially offset double clad optical fibers into the miniaturized scanner. The fabricated endoscope has an outer diameter of 3 mm, a rigid length of 4 cm, and acquires images at 4 frames/s per focal plane, with lateral and axial resolutions for two-photon imaging of 0.8 and 10 µm, respectively.

Refractive Outcomes for Cataract Surgery With Toric Intraocular Lenses at a Veterans Affairs Medical Center.

Background: Refractive outcomes for cataract surgery with toric intraocular lenses (IOLs) are not well described in a teaching hospital setting. This study investigated the refractive outcomes of cataract surgery with toric IOLs at an academic-affiliated Veterans Affairs Medical Center (VAMC) and compared the accuracy of 2 biometric formulae for toric IOL power calculation. Methods: A retrospective chart review of patients who received cataract surgery with toric IOLs from November 2013 to May 2018 was conducted. The Holladay 2 and Barrett toric IOL formulae were used to predict the postoperative refraction for each cataract surgery. The main outcome measures were best-corrected visual acuity (BCVA) and the difference in cylinder between the preoperative and postoperative manifest refractions. The accuracy of each biometric formula was also assessed using 2-tailed t tests of the mean absolute error, and subgroup analyses were conducted for short, medium, and long eyes. Results: Of 325 charts reviewed, 283 patients met the inclusion criteria; 87% (248/283) of these surgeries were performed by resident surgeons. The median postoperative BCVA was 20/20, and 92% of patients had a postoperative BCVA of 20/25 or better. There was no statistically significant difference in mean absolute error between the 2 formulae for the entire axial length range (P = .21), as well as the short (P = .94), medium (P = .49), and long axial length (P = .08) groups. Conclusions: To our knowledge, this is the largest study that compared the performance of the Barrett toric and Holladay 2 formulae and the first that made the comparison in a teaching hospital setting. This study suggests that the 2 formulae have similar refractive outcomes across all axial lengths.

Accuracy of biometric formulae for intraocular lens power calculation in a teaching hospital.

AIM: To evaluate the accuracy of three commonly used biometric formulae across different axial lengths (ALs) at one United States Veterans Affairs teaching hospital. METHODS: A retrospective chart review was conducted from November 2013 to May 2018. One eye of each patient who underwent cataract surgery with a monofocal intraocular lens (IOL) was included. The range of postoperative follow-up period was from 3wk to 4mo. The Holladay 2, Barrett Universal II, and Hill-Radial Basis Function (Hill-RBF) formulae were used to predict the postoperative refraction for all cataract surgeries. For each formula, we calculated the prediction errors [including mean absolute prediction error (MAE)] and the percentage of eyes within ±0.25 diopter (D) and ±0.5 D of predicted refraction. We performed subgroup analyses for short (AL<22.0 mm), medium (AL 22.0-25.0 mm), and long eyes (AL>25.0 mm). RESULTS: A total of 1131 patients were screened, and 909 met the inclusion criteria. Resident ophthalmologists were the primary surgeons in 710 (78.1%) cases. We found no statistically significant difference in predictive accuracy among the three formulae over the entire AL range or in the short, medium, and long eye subgroups. Across the entire AL range, the Hill-RBF formula resulted in the lowest MAE (0.384 D) and the highest percentage of eyes with postoperative refraction within ±0.25 D (42.7%) and ±0.5 D (75.5%) of predicted. All three formulae had the highest MAEs (>0.5 D) and lowest percentage within ±0.5 D of predicted refraction (<55%) in short eyes. CONCLUSION: In cataract surgery patients at our teaching hospital, three commonly used biometric formulae demonstrate similar refractive accuracy across all ALs. Short eyes pose the greatest challenge to predicting postoperative refractive error.

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.

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.

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.

Multiphoton tomographic imaging: a potential optical biopsy tool for detecting gastrointestinal inflammation and neoplasia.

Endoscopy is widely used to detect and remove premalignant lesions with the goal of preventing gastrointestinal (GI) cancers. Because current endoscopes do not provide cellular resolution, all suspicious lesions are biopsied and subjected to histologic evaluation. Technologies that facilitate directed biopsies should decrease both procedure-related morbidity and cost. Here we explore the use of multiphoton microscopy (MPM), an optical biopsy tool that relies on intrinsic tissue emissions, to evaluate pathology in both experimental and human GI specimens, using hematoxylin and eosin (H&E)-stained sections from these tissues for comparison. After evaluating the entire normal mouse GI tract, MPM was used to investigate disease progression in mouse models of colitis and colorectal carcinogenesis. MPM provided sufficient histologic detail to identify all relevant substructures in ex vivo normal GI tissue, visualize both acute and resolving stages of colitis, and show the progression of colorectal carcinogenesis. Next, ex vivo specimens from human subjects with celiac sprue, inflammatory bowel disease, and colorectal neoplasia were imaged by MPM. Finally, colonic mucosa in live anesthetized rats was imaged in vivo using a flexible endoscope prototype. In both animal models and human specimens, MPM images showed a striking similarity to the results of H&E staining, as shown by the 100% concordance achieved by the study pathologists' diagnoses. In summary, MPM is a promising technique that accurately visualizes histology in fresh, unstained tissues. Our findings support the continued development of MPM as a technology to enhance the early detection of GI pathologies including premalignant lesions.