Alexandre R. Tumlinson

Miniature endoscope for simultaneous optical coherence tomography and laser-induced fluorescence measurement

We have designed a multimodality system that combines optical coherence tomography (OCT) and laser-induced fluorescence (LIF) in a 2.0-mm-diameter endoscopic package. OCT provides approximately 18-microm resolution cross-sectional structural information over a 6-mm field. LIF spectra are collected sequentially at submillimeter resolution across the same field and provide histochemical information about the tissue. We present the use of a rod prism to reduce the asymmetry in the OCT beam caused by a cylindrical window. The endoscope has been applied to investigate mouse colon cancer in vivo.

Endoscope-tip interferometer for ultrahigh resolution frequency domain optical coherence tomography in mouse colon

Frequency domain optical coherence tomography (FD-OCT) allows interferometer topologies with simplified system construction and handling. Problems of dispersion and polarization matching between the sample and reference arms, as well as beamsplitter spectral non-uniformity, are mitigated when the interferometer is wholly contained in the endoscope tip. A common path set-up, using a reference reflection originating from the inside surface of the glass envelope at the distal end of the endoscope, and an alternative approach with more efficient collection of the reference light using a novel beamsplitter design have been developed. High speed (20,000 A-lines/s) ultrahigh axial resolution (2.4 mum) tomograms of mouse colon have been acquired using a 2 mm outer diameter endoscope in vivo. The FD-OCT system uses a compact mode-locked Ti:Al(2)O(3) laser emitting a broad spectrum (160 nm full-width-half-maximum) centered at 800 nm in combination with a CCD based, spectrally sensitive detector.

Dual modality instrument for simultaneous optical coherence tomography imaging and fluorescence spectroscopy

We develop a dual-modality device that combines the anatomical imaging capabilities of optical coherence tomography (OCT) with the functional capabilities of laser-induced fluorescence (LIF) spectroscopy. OCT provides cross-sectional images of tissue structure to a depth of up to 2 mm with approximately 10-microm resolution. LIF spectroscopy provides histochemical information in the form of emission spectra from a given tissue location. The OCT subsystem utilizes a superluminescent diode with a center wavelength of 1300 nm, whereas a helium cadmium laser provides the LIF excitation source at wavelengths of 325 and 442 nm. Preliminary data are obtained on eight postmortem aorta samples, each 10 mm in length. OCT images and LIF spectra give complementary information from normal and atherosclerotic portions of aorta wall. OCT images show structures such as intima, media, internal elastic lamina, and fibrotic regions. Emission spectra ratios of 520/490 (325-nm excitation) and 595/635 (442-nm excitation) could be used to identify normal and plaque regions with 97 and 91% correct classification rates, respectively. With miniaturization of the delivery probe and improvements in system speed, this dual-modality device could provide a valuable tool for identification and characterization of atherosclerotic plaques.

Task-based imaging of colon cancer in the Apc Min/+ mouse model

Optical coherence tomography (OCT), laser-induced fluorescence (LIF), and laser-scanning confocal microscopy (LSCM) were used for the task of multimodal study of healthy and adenomatous mouse colon. The results from each modality were compared with histology, which served as the gold standard. The Apc(Min/+) genetic mouse model of colon cancer was compared with wild-type mice. In addition, a special diet was used for the task of studying the origins of a 680 nm autofluorescent signal that was previously observed in colon. The study found close agreement among each of the modalities and with histology. All four modalities were capable of identifying diseased tissue accurately. The OCT and LSCM images provided complementary structural information about the tissue, while the autofluorescence signal measured by LIF and LSCM provided biochemical information. OCT and LIF were performed in vivo and nondestructively, while the LSCM and histology required extraction of the tissue. The magnitude of the 680 nm signal correlates with chlorophyll content in the mouse diet, suggesting that the autofluorescent compound is a dietary metabolite.

In vivo ultrahigh-resolution optical coherence tomography of mouse colon with an achromatized endoscope.

Endoscopic ultrahigh-resolution optical coherence tomography (OCT) enables collection of minimally invasive cross-sectional images in vivo, which may be used to facilitate rapid development of reliable mouse models of colon disease as well as assess chemopreventive and therapeutic agents. The small physical scale of mouse colon makes light penetration less problematic than in other tissues and high resolution acutely necessary. In our 2-mm diameter endoscopic time domain OCT system, isotropic ultrahigh-resolution is supported by a center wavelength of 800 nm and full-width-at-half-maximum bandwidth of 150 nm (mode-locked titanium:sapphire laser) combined with 1:1 conjugate imaging of a small core fiber. A pair of KZFSN5/SFPL53 doublets provides excellent color correction to support wide bandwidth throughout the imaging depth. A slight deviation from normal beam exit angle suppresses collection of the strong back reflection at the exit window surface. Our system achieves axial resolution of 3.2 microm in air and 4.4-microm lateral spot diameter with 101-dB sensitivity. Microscopic features too small to see in mouse tissue with conventional resolution systems, including colonic crypts, are clearly resolved. Resolution near the cellular level is potentially capable of identifying abnormal crypt formation and dysplastic cellular organization.

Serial endoscopy in azoxymethane treated mice using ultra-high resolution optical coherence tomography.

Purpose: Optical coherence tomography (OCT) is a minimally invasive, depth-resolved imaging tool that can be implemented in a small diameter endoscope for imaging mouse models of colorectal cancer (CRC). In this study, we utilized ultrahigh resolution (UHR) OCT to serially image the lower colon of azoxymethane (AOM) treated A/J mouse models of CRC in order to monitor the progression of neoplastic transformations and determine if OCT is capable of identifying early disease. Experimental Design: Thirteen AOM treated A/J and two control A/J mice were surveyed at four timepoints (8, 14, 22, and 26 weeks post AOM treatment) using a 2.0 mm diameter UHR OCT endoscopic system with 3.2 µm axial and 4.4 µm lateral resolution. Histological samples obtained at the final timepoint served as the diagnostic reference. A blinded expert panel of mouse colon pathologists provided diagnoses from the OCT images based on criteria developed from a separate training set of OCT images. Panel results were compared to histological diagnoses assigned by a blinded pathologist. Results: At the final imaging timepoint, 95% of adenomas and 23% of gastrointestinal neoplasias (38% protruding GINs and 9% non-protruding GINs) were correctly diagnosed. The panel identified 68% of disease foci (95% adenoma, 76% protruding GINs, and 13% non-protruding GINs). Over the OCT imaging timepoints, disease progression followed a typical succession, with normal or GIN preceding adenoma. Conclusions: Endoscopic UHR OCT enabled accurate diagnosis of adenomas, identification of protruding GIN, and non-destructive visualization of CRC progression, providing a tool for cancer research in animal models.

Inherent homogenous media dispersion compensation in frequency domain optical coherence tomography by accurate k-sampling

Depth dependent broadening of the axial point spread function due to dispersion in the imaged media, and algorithms for postprocess correction, have been previously described for both time domain and frequency domain optical coherence tomography. We show that homogeneous media dispersion artifacts disappear when frequency domain samples are acquired with uniform spacing in circular wavenumber, as opposed to uniform sampling in optical frequency. We further explicate the source of this point spread broadening and simulate its magnitude in aqueous media. We experimentally demonstrate media dispersion compensation in high dispersion glass by choosing sample frequencies at equal intervals of media index of refraction divided by vacuum wavelength, and we recover unbroadened reflections without an additional postprocessing step.

An achromatized endoscope for ultrahigh-resolution optical coherence tomography

Mouse models are increasingly important for studying human GI pathology. OCT provides minimally invasive, cross-sectional images that indicate the thickness and scattering density of underlying tissue. We have developed endoscopic ultrahigh resolution OCT (UHR-OCT) to imaging mouse colon in vivo. The reduced scale of the mouse colon makes tissue light penetration much less problematic, and high resolution acutely necessary. Higher lateral resolution requires a departure from the traditional cemented GRIN lens design. We support the need for better chromatic aberration than can be achieved by a GRIN lens using commercial raytracing software. We have designed and built a 2mm diameter endoscopic UHR-OCT system achromatized for 770-1020nm for use with a Titanium:sapphire laser with 260 nm bandwidth at full-width-half-maximum centered at 800 nm while achieving a 4.4um lateral spot dimension at focus. A pair of KZFSN5/SFPL53 doublets provides excellent primary and secondary color correction to maintain wide bandwidth through the imaging depth. A slight deviation from normal beam exit angle suppresses collection of the strong back reflection at the exit window surface. The novel design endoscope was built and characterized for through focus bandwidth, axial resolution, signal to noise, and lateral spot dimension. Performance is demonstrated on in vivo mouse colon. Ultrahigh-resolution images of mouse tissue enable the visualization of microscopic features, including crypts that have previously been observed with standard resolution OCT in humans but were too small to see in mouse tissue. Resolution near the cellular level is potentially capable of identifying abnormal crypt formation and dysplastic cellular organization.

Scatter sensitive microscopic techniques to identify contrasting mucosal structures in ultrahigh-resolution optical coherence tomograms of mouse colon

Optical coherence tomography, optical coherence microscopy, reflectance confocal microscopy, and darkfield microscopy all derive contrast from the intensity of endogenous tissue scatter. We have imaged excised mouse colon tissue with these complimentary technologies to make conclusions about structural origins of scatter in the mouse colonic mucosa observed with endoscopic OCT. We find hyperintense scattering both from the cytoplasm of epithelial cells and from the boundary between epithelia and the lamina propria. We find almost no scatter from the portion of epithelial cells containing the nucleus. These observations substantiate explanations for the appearance of colonic crypts and the luminal surface.

Inherent media dispersion compensation by FD-OCT

Depth dependent broadening of the axial point spread function due to dispersion in the imaged media, and algorithms for postprocess correction have been previously described for both time domain and frequency domain optical coherence tomography. Homogeneous media dispersion artifacts disappear when frequency domain samples are uniformly spaced in circular wavenumber, as opposed to uniform sampling in optical frequency. In this paper, we explicate the source of this point spread broadening and simulate its magnitude in aqueous media. We conclude with a suggestion for interferometric k-triggering which accounts for dispersion in the media.