Caroline Boudoux

Extended-cavity semiconductor wavelength-swept laser for biomedical imaging

We demonstrate a compact high-power rapidly swept wavelength tunable laser source based on a semiconductor optical amplifier and an extended-cavity grating filter. The laser produces excellent output characteristics for biomedical imaging, exhibiting >4-mW average output power, <0.06-nm instantaneous linewidth, and >80-dB noise extinction with its center wavelength swept over 100 nm at 1310 nm at variable repetition rates up to 500 Hz.

Dual-modality needle probe for combined fluorescence imaging and three-dimensional optical coherence tomography

To the best of our knowledge, we present the first needle probe for combined optical coherence tomography (OCT), and fluorescence imaging. The probe uses double-clad fiber (DCF) that guides the OCT signal and fluorescence excitation light in the core and collects and guides the returning fluorescence in the large-diameter multimode inner cladding. It is interfaced to a 1310 nm swept-source OCT system that has been modified to enable simultaneous 488 nm fluorescence excitation and >500 nm emission detection by using a DCF coupler to extract the returning fluorescence signal in the inner cladding with high efficiency. We present imaging results from an excised sheep lung with fluorescein solution infused through the vasculature. We were able to identify alveoli, bronchioles, and blood vessels. The results demonstrate that the combined OCT plus fluorescence needle images provide improved tissue differentiation over OCT alone.

Double-clad fiber coupler for endoscopy.

We present a double-clad fiber coupler (DCFC) for use in endoscopy to reduce speckle contrast, increase signal collection and depth of field. The DCFC is made by fusing and tapering two all silica double-clad fiber (DCF) and allows achromatic transmission of >95% of core illumination (1265nm - 1325nm) as well as collection of >42% of inner cladding diffuse light. Its potential for endoscopy is demonstrated in a spectrally encoded imaging setup which shows speckle reduction by a factor 5, increased signal collection by a factor 9 and enhanced depth of field by 1.8 times. Separation by the DCFC of single- and multi-mode signals allows combining low-speckle reflectance images (25.5 fps) with interferometrically measured depth profiles (post-processed) for of small three-dimensional (3D) features through an all-fiber low loss instrument.

Large area confocal microscopy.

Imaging large tissue areas with microscopic resolution in vivo may offer an alternative to random excisional biopsy. We present an approach for performing confocal imaging of large tissue surface areas using spectrally encoded confocal microscopy (SECM). We demonstrate a single-optical-fiber SECM apparatus, designed for imaging luminal organs, that is capable of imaging with a transverse resolution of 2.1 microm over a subsurface area of 16 cm2 in less than 1 min. Due to the unique probe configuration and scanning geometry, the speed and resolution of this new imaging technology are sufficient for comprehensively imaging large tissues areas at a microscopic scale in times that are appropriate for clinical use.

Comprehensive imaging of gastroesophageal biopsy samples by spectrally encoded confocal microscopy.

BACKGROUND Spectrally encoded confocal microscopy (SECM) is a high-speed reflectance confocal microscopy technique that has the potential to be used for acquiring comprehensive images of the entire distal esophagus endoscopically with subcellular resolution. OBJECTIVE The goal of this study was to demonstrate large-area SECM in upper GI tissues and to determine whether the images contain microstructural information that is useful for pathologic diagnosis. DESIGN A feasibility study. SETTING Gastrointestinal Unit, Massachusetts General Hospital. PATIENTS Fifty biopsy samples from 36 patients undergoing routine EGD were imaged by SECM, in their entirety, immediately after their removal. RESULTS The microstructure seen in the SECM images was similar to that seen by histopathology. Gastric cardia mucosa was clearly differentiated from squamous mucosa. Gastric fundic/body type mucosa showed more tightly packed glands than gastric cardia mucosa. Fundic gland polyps showed cystically dilated glands lined with cuboidal epithelium. The presence of intraepithelial eosinophils was detected with the cells demonstrating a characteristic bilobed nucleus. Specialized intestinal metaplasia was identified by columnar epithelium and the presence of goblet cells. Barretts esophagus (BE) with dysplasia was differentiated from specialized intestinal metaplasia by the loss of nuclear polarity and disorganized glandular architecture. LIMITATIONS Ex vivo, descriptive study. CONCLUSIONS Large-area SECM images of gastroesophageal biopsy samples enabled the visualization of both subcellular and architectural features of various upper GI mucosal types and were similar to the corresponding histopathologic slides. These results suggest that the development of an endoscopic SECM probe is merited.

Multimodality optical imaging of embryonic heart microstructure

Study of developmental heart defects requires the visualization of the microstructure and function of the embryonic myocardium, ideally with minimal alterations to the specimen. We demonstrate multiple endogenous contrast optical techniques for imaging the Xenopus laevis tadpole heart. Each technique provides distinct and complementary imaging capabilities, including: 1. 3-D coherence microscopy with subcellular (1 to 2 microm) resolution in fixed embryos, 2. real-time reflectance confocal microscopy with large penetration depth in vivo, and 3. ultra-high speed (up to 900 frames per second) that enables real-time 4-D high resolution imaging in vivo. These imaging modalities can provide a comprehensive picture of the morphologic and dynamic phenotype of the embryonic heart. The potential of endogenous-contrast optical microscopy is demonstrated for investigation of the teratogenic effects of ethanol. Microstructural abnormalities associated with high levels of ethanol exposure are observed, including compromised heart looping and loss of ventricular trabecular mass.

Multiplexed two-photon microscopy of dynamic biological samples with shaped broadband pulses

Coherent control can be used to selectively enhance or cancel concurrent multiphoton processes, and has been suggested as a means to achieve nonlinear microscopy of multiple signals. Here we report multiplexed two-photon imaging in vivo with fast pixel rates and micrometer resolution. We control broadband laser pulses with a shaping scheme combining diffraction on an optically-addressed spatial light modulator and a scanning mirror allowing to switch between programmable shapes at kiloHertz rates. Using coherent control of the two-photon excited fluorescence, it was possible to perform selective microscopy of GFP and endogenous fluorescence in developing Drosophila embryos. This study establishes that broadband pulse shaping is a viable means for achieving multiplexed nonlinear imaging of biological tissues.

Asymmetric double-clad fiber couplers for endoscopy.

We present an asymmetric double-clad fiber coupler (A-DCFC) exploiting a disparity in fiber etendues to exceed the equipartition limit (≤50% extraction of inner cladding multi-mode light). The A-DCFC is fabricated using two commercially available fibers and a custom fusion-tapering setup to achieve >70% extraction of multi-mode inner cladding light without affecting (>95% transmission) single-mode light propagation in the core. Imaging with the A-DCFC is demonstrated in a spectrally encoded imaging setup using a weakly backscattering biological sample. Other applications include the combination of optical coherence tomography with weak fluorescent or Raman scattering signals.

Molecular imaging needles: dual-modality optical coherence tomography and fluorescence imaging of labeled antibodies deep in tissue

Molecular imaging using optical techniques provides insight into disease at the cellular level. In this paper, we report on a novel dual-modality probe capable of performing molecular imaging by combining simultaneous three-dimensional optical coherence tomography (OCT) and two-dimensional fluorescence imaging in a hypodermic needle. The probe, referred to as a molecular imaging (MI) needle, may be inserted tens of millimeters into tissue. The MI needle utilizes double-clad fiber to carry both imaging modalities, and is interfaced to a 1310-nm OCT system and a fluorescence imaging subsystem using an asymmetrical double-clad fiber coupler customized to achieve high fluorescence collection efficiency. We present, to the best of our knowledge, the first dual-modality OCT and fluorescence needle probe with sufficient sensitivity to image fluorescently labeled antibodies. Such probes enable high-resolution molecular imaging deep within tissue.

Optical Microscopy of the Pediatric Vocal Fold

OBJECTIVES To compare and contrast 4 optical imaging techniques for evaluating the developing microstructure of the pediatric vocal fold and to identify the optimal strategy for in vivo imaging. DESIGN Prospective study. SETTING Academic medical center. PATIENTS A total of 6 laryngeal specimens: 5 pediatric (ages 10 months to 16 years) (4 from cadavers and 1 from a living patient immediately after laryngectomy) and 1 cadaveric young adult specimen (age, 23 years). INTERVENTION Sequential noninvasive optical imaging of pediatric vocal fold specimens using optical frequency domain imaging (OFDI), angle-resolved OFDI (AR-OFDI), spectrally encoded confocal microscopy (SECM), and full-field optical coherence microscopy (FF-OCM), followed by fixation, sectioning, and histologic analysis of the same specimen for comparison. MAIN OUTCOME MEASURE Correlation between the microstructure observed using the 4 noninvasive optical imaging techniques and with the results of histopathologic analysis for the same specimen. RESULTS A successful in vivo imaging technique for developmental assessment of the pediatric vocal fold would include visualization of distinct layers (epithelium, lamina propria, and muscularis mucosa) and allow for identification of the individual cells composing the layers. The OFDI and AR-OFDI techniques provide a global assessment of the microstructure of the pediatric vocal fold to a depth of 1200 mum but lack the ability to distinguish cellular and subcellular structures. The FF-OCM technique allows for visualization with improved cellular detail (1-mum resolution), but the image acquisition speed is too slow for clinical use. The SECM technique has a faster acquisition rate and shows good cellular and subcellular detail to a depth of 250 mum. CONCLUSIONS The OFDI and SECM techniques were identified as promising and complementary candidates for in vivo cellular and subcellular imaging of the epithelium, basement membrane, and lamina propria of the pediatric vocal fold. To further validate the clinical potential of these techniques, a handheld SECM probe has been developed and demonstrated for in vivo evaluation of the pediatric vocal fold.