The current paradigm for chemotherapy in the treatment of colorectal cancer (CRC) is based on the maximum-tolerated dose approach (MTD), which uses high doses of cytotoxic agents with the aim of killing tumor cells. However, the toxicity of MTD chemotherapy requires drug delivery in a cyclic manner with rest periods that allow the tumor to recover. In contrast, metronomic chemotherapy (MET) uses low but continuous doses that target the endothelial cells of tumors, thereby directly affecting the development of new blood vessels (angiogenesis). Because the potential impact of MET when used as neoadjuvant chemotherapy (NAC) in CRC has not been explored and contrasted with the effects in perfusion of the standard MTD regimen, this study uses an azoxymethane-induced primary model of CRC to assess changes in perfusion and angiogenic markers. Diffuse reflectance spectroscopy (DRS) was used to longitudinally quantify oxygen saturation (StO2) and compare these changes with the change in expression of VEGF-A between MET and MTD treatments. In the MTD group, an increase in StO2 from 65 to 81% was observed by week 4 of treatment and a reduction of VEGF-A expression from 81% to 16% was observed between weeks 4 and 6.
Ulcerative Colitis (UC) is an idiopathic autoimmune inflammatory disorder of the colon mucosa characterized by leukocyte infiltration into the submucosa and ulcer formation in the epithelium, followed by epithelial cell proliferation and restitution of the mucosal barrier. Optical methods sensitive to tissue oxygen demand and epithelial cell metabolism are ideally suited for clinical guidance of disease progression and remission in UC. Here we present endoscopic reporters of submucosal oxygen demand (i.e. inflammatory-related changes) and epithelial cell metabolism (i.e. mucosal re-epithelialization) within the ulcer bed and surrounding microenvironment in a murine model of UC as putative biomarkers of mucosal healing.
KEYWORDS: Tumors, Data modeling, Diffuse reflectance spectroscopy, Reflectivity, Error control coding, In vivo imaging, Tissues, Magnesium, Endoscopy, Data acquisition
Significance: Many studies in colorectal cancer (CRC) use murine ectopic tumor models to determine response to treatment. However, these models do not replicate the tumor microenvironment of CRC. Physiological information of treatment response derived via diffuse reflectance spectroscopy (DRS) from murine primary CRC tumors provide a better understanding for the development of new drugs and dosing strategies in CRC.
Aim: Tumor response to chemotherapy in a primary CRC model was quantified via DRS to extract total hemoglobin content (tHb), oxygen saturation (StO2), oxyhemoglobin, and deoxyhemoglobin in tissue.
Approach: A multimodal DRS and imaging probe (0.78 mm outside diameter) was designed and validated to acquire diffuse spectra longitudinally—via endoscopic guidance—in developing colon tumors under 5-fluoruracil (5-FU) maximum-tolerated (MTD) and metronomic regimens. A filtering algorithm was developed to compensate for positional uncertainty in DRS measurements
Results: A maximum increase in StO2 was observed in both MTD and metronomic chemotherapy-treated murine primary CRC tumors at week 4 of neoadjuvant chemotherapy, with 21 ± 6 % and 17 ± 6 % fold changes, respectively. No significant changes were observed in tHb.
Conclusion: Our study demonstrates the feasibility of DRS to quantify response to treatment in primary CRC models.
Immunotherapy, an emerging field in cancer therapeutics, in colon cancer aims to reduce pre-surgical tumor burden by regulating host immune checkpoints, and when used in combination with neoadjuvant chemotherapy, may improve tumor therapeutic response. One such immune checkpoint is CCL2 (monocyte chemoattractant protein-1)-mediated recruitment of monocytes, which differentiate into tumor-associated macrophages (TAMs) in the tumor microenvironment that promote angiogenesis and tumorigenesis. Thus, CCL2 blockade may play an anti-tumor role via effects on tumor perfusion. However, there have been no studies investigating CCL2 blockade immunotherapy combined with chemotherapy in an animal model of colon cancer. Furthermore, there is a need to longitudinally assess tumor therapeutic response throughout treatment. In this study, CT26 murine colon carcinoma was injected into the flanks of Balb/c mice (n=80) to form tumor allografts. Mice in the key experimental group received combined chemotherapy (5-flurouracil) and immunotherapy (anti-CCL2), with appropriate controls. Tumor therapeutic response was monitored using diffuse reflectance spectroscopy (DRS) by measuring the tumor perfusion metrics, hemoglobin concentration and oxygenation. End-point immunohistochemical analysis was used to quantify TAM fraction (CD68 and DAPI), TAM polarization (iNOS and CD206), and hypoxia (pimonidazole) to spatially and temporally correlate to DRS results. The central hypothesis was that decreasing TAMs via CCL2 blockade alters tumor perfusion, thereby increasing tumor response to 5-fluorouracil. This study may potentially demonstrate an effective immunotherapy approach (CCL2 blockade) and a viable method to longitudinally and non-invasively assess tumor therapeutic response to such immunotherapy (DRS) in mouse allograft models of colon cancer.
Colorectal cancer (CRC) ranks fourth in terms of newly diagnosed cases in the United States (135,430 in 2017); patients with locally advanced disease (Stage II and III) receive 5-fluorouracil (5-FU) and external beam radiotherapy-based neoadjuvant therapy (NAT) prior to surgical resection. However, there are no clinically accepted methods to assess in vivo therapeutic response to NAT.
Optical methods based on diffuse reflectance spectroscopy (DRS) have shown significant promise in predicting response to NAT in breast cancer, but the anatomy of the distal colon requires the use of endoscopically-deployable methods. We have developed a small-diameter (0.78 mm) multimodal optical imaging and diffuse reflectance spectroscopy (DRS) probe which can be deployed via the biopsy port of a commercial veterinary colonoscope (Karl Storz COLOView) to be used in a chemically-induced (azoxymethane (AOM)) orthotopic model.
Total diffuse reflectance measured by the probe was correlated with the reduced scattering (μ’s(λ)) and absorption coefficients (μa(λ)) for λ = 450 – 800nm via a look-up table (LUT). Liquid phantoms were used to create the LUT and validate the measured μ’s and μa values. The LUT has a maximum total reflectance of 0.14 and ranges for μa and μ’s are 0-10 cm-1and 3-18 cm-1, respectively. Error for μ’s and μa has been 10.7±8.8% and 7.9±5.3%, respectively. For the imaging component, circular active area diameter is 325 μm and center-to-center fiber spacing of 3.3 μm.
Building on previous work this DRS approach enables quantification of total hemoglobin (Hb) content, oxygen saturation (SaO2), estimates mean vessel diameter and scattering component, and allows for co-registered highresolution image data of superficial mucosa in vivo of tumor perfusion and microstructure, which can translate to the clinic to help physicians determine the response of tumors to therapy.
Diffuse reflectance spectroscopy (DRS) is a probe-based spectral biopsy technique used in cancer studies to quantify tissue reduced scattering (μs′) and absorption (μa) coefficients and vary in source–detector separation (SDS) to fine-tune sampling depth. In subcutaneous murine tumor allografts or xenografts, a key design requirement is ensuring that the source light interrogates past the skin layer into the tumor without significantly sacrificing signal-to-noise ratio (target of ≥15 dB). To resolve this requirement, a DRS probe was designed with four SDSs (0.75, 2.00, 3.00, and 4.00 mm) to interrogate increasing tissue volumes between 450 and 900 nm. The goal was to quantify percent errors in extracting μa and μs′, and to quantify sampling depth into subcutaneous Balb/c-CT26 colon tumor allografts. Using an optical phantom-based experimental method, lookup-tables were constructed relating μa,μs′, diffuse reflectance, and sampling depth. Percent errors were <10 % and 5% for extracting μa and μs′, respectively, for all SDSs. Sampling depth reached up to 1.6 mm at the first Q-band of hemoglobin at 542 nm, the key spectral region for quantifying tissue oxyhemoglobin concentration. This work shows that the DRS probe can accurately extract optical properties and the resultant physiological parameters such as total hemoglobin concentration and tissue oxygen saturation, from sufficient depth within subcutaneous Balb/c-CT26 colon tumor allografts. Methods described here can be generalized for other murine tumor models. Future work will explore the feasibility of the DRS in quantifying volumetric tumor perfusion in response to anticancer therapies.
Spatial frequency domain imaging (SFDI) is a widefield, noncontact, and label-free imaging modality that is currently being explored as a new tool for longitudinal tracking of cancer therapies in the preclinical setting. We describe a two-layer look-up-table (LUT) inversion algorithm for SFDI that better accounts for the skin (top layer) and tumor (bottom layer) tissue geometry in subcutaneous tumor models. Monte Carlo (MC) simulations were conducted natively in the spatial frequency domain, avoiding discretization errors associated with Fourier or Hankel transforms of conventional MC simulation results. The two-layer LUT was validated using two-layer tissue mimicking optical phantoms, in which the optical property extractions of the bottom (tumor) layer were determined to be within 20% and 11% of the true values for μa and μs′, respectively. A sensitivity analysis was conducted to evaluate how imperfect top layer estimates affect bottom-layer optical property extractions. Finally, the two-layer LUT was used to reanalyze a prior longitudinal data set, which revealed larger therapy-induced changes in optical scattering and a more hypoxic tumor environment compared to the homogeneous LUT. The two-layer LUT described here improves the accuracy of subcutaneous tumor imaging, and the general methodology can be applied for arbitrary multilayer SFDI applications.
Spatial Frequency Domain Imaging (SFDI) is a Diffuse Optical Imaging (DOI) technique that is well suited for preclinical functional imaging. Recently, we have shown that SFDI can successfully be used for longitudinal monitoring of a prostate subcutaneous tumor xenograft, where we have applied a look-up-table (LUT) based approach to extract tissue absorption (μa) and scattering properties (μs’). This LUT assumes a semi-infinite homogeneous medium and simulates reflectance (Rd) in spatial domain, and scales Rd for all μa and μs’ of interest from a single Monte Carlo simulation. However, converting Rd to spatial frequency domain (SFD) and scaling for μs’ may introduces unacceptable errors. Most importantly, the homogeneous model fails to mimic the actual physiology of a subcutaneous tumor, which can be described as a two-layer medium with a thin skin layer above the tumor layer. To overcome these limitations, we have developed a Monte Carlo based two-layer LUT with a wide range of tumor (bottom) layer optical properties, and fixed skin (top) properties. The two-layer LUT will be validated by two-layer silicone phantoms and tested for sensitivity to inaccurate layer assumptions. Additionally, the homogeneous and two-layer LUTs will be used on a large mouse tumor database (n=54 mice monitored over 3 months) to identify how the two-layer LUT can improve accuracy of SFDI by more accurately reflecting in vivo physiology, and reducing discretization and scaling errors. Improved SFDI findings in small animals, in the long run, will help establish clinical DOI tools for early detection of chemotherapy efficacy during treatment.
Locally advanced adenocarcinomas located in the distal rectum are commonly treated via 5-fluorouracil (5-FU)-based neoadjuvant chemoradiation therapy (CRT). The occurrence of pre-operative pathological complete response, or the absence of any histological evidence of residual cancer, is seen in 15-27% of rectal cancer cases. Response to chemotherapeutic agents varies between patients, introducing the need for a system to predict optimal drug combinations. We propose a method of utilizing optical metabolic imaging of in vitro, primary tumor-derived, three-dimensional organoid culture to create specific drug sensitivity profiles, and to rapidly assess a patient’s potential response to drugs. Murine xenografts were developed in Swiss athymic nude mice, using human colorectal adenocarcinoma cell lines, implanted in the flank (RKO, ATCC). Tumors were excised upon reaching a volume of 500mm3 and processed for organoid culture. Organoids were subjected to longitudinal metabolic imaging of metabolic cofactors FAD and NADH for seven days. The resulting images were used to yield an optical redox value on a cell-by-cell basis, determined by the fluorescence intensity ratio of FAD/(FAD+NADH). This data infers proliferative index of the organoids. Beginning on day three, a control vehicle dimethyl sulfoxide, or the cytotoxic agent 5-FU, was added to the organoid growth media in wells, with metabolic imaging performed the same as previously stated. The optical redox values decreased due to the addition of 5-FU, which targets rapidly dividing cells and induces apoptosis. The changes in the optical redox histograms were correlated to markers of cell proliferation (Ki-67) and apoptosis (cleaved caspase-3).
There exists a broad range of techniques that can be used to classify and count white blood cells in a point-of-care (POC) three-part leukocyte differential test. Improvements in lenses, light sources, and cameras for image-based POC systems have renewed interest in acridine orange (AO) as a contrast agent, whereby subpopulations of leukocytes can be differentiated by colorimetric analysis of AO fluorescence emission. We evaluated the effect on test accuracy using different AO staining and postprocessing methods in the context of an image-based POC colorimetric cell classification scheme. Thirty blood specimens were measured for percent cell counts using our POC system and a conventional hematology analyzer for comparison. Controlling the AO concentration used during whole-blood staining, the incubation time with AO, and the colorimetric ratios among the three population of leukocytes yielded a percent deviation of 0.706%, −1.534%, and −0.645% for the lymphocytes, monocytes, and granulocytes, respectively. Overall, we demonstrated that a redshift in AO fluorescence was observed at elevated AO concentrations, which lead to reproducible inaccuracy of cell counts. This study demonstrates there is a need for a strict control of the AO staining and postprocessing methods to improve test accuracy in these POC systems.
Qualitative screening for colorectal polyps via fiber bundle microendoscopy imaging has shown promising results, with studies reporting high rates of sensitivity and specificity, as well as low interobserver variability with trained clinicians. A quantitative image quality control and image feature extraction algorithm (QFEA) was designed to lessen the burden of training and provide objective data for improved clinical efficacy of this method. After a quantitative image quality control step, QFEA extracts field-of-view area, crypt area, crypt circularity, and crypt number per image. To develop and validate this QFEA, a training set of microendoscopy images was collected from freshly resected porcine colon epithelium. The algorithm was then further validated on ex vivo image data collected from eight human subjects, selected from clinically normal appearing regions distant from grossly visible tumor in surgically resected colorectal tissue. QFEA has proven flexible in application to both mosaics and individual images, and its automated crypt detection sensitivity ranges from 71 to 94% despite intensity and contrast variation within the field of view. It also demonstrates the ability to detect and quantify differences in grossly normal regions among different subjects, suggesting the potential efficacy of this approach in detecting occult regions of dysplasia.
Spatially–resolved diffuse reflectance (SRDR) measurements provide photon path information, and enable layered tissue analysis. This paper presents experimental SRDR measurements on two-layer PDMS skin tissue-mimicking phantoms of varying top layer thicknesses, and bulk phantoms of varying optical properties using concentric multi-pixel photodiode array (CMPA) probes, and corresponding forward Monte Carlo simulations. The CMPA is the most densely packed semiconductor SRDR probe reported to date. Signal contrasts between the single layer phantom and bi-layer phantoms with varying top layer thicknesses are as high as 80%. The mean error between the Monte Carlo simulations and the experiment is less than 6.2 %.
Two-photon excitation of label-free tissue is of increasing interest, as advances have been made in endoscopic clinical application of multiphoton microscopy, such as second harmonic generation (SHG) scanning endoscopy used to monitor cervical collagen in mice1. We used C57BL mice as a model to investigate the progression of gastrointestinal structures, specifically glandular area and circularity. We used multiphoton microscopy to image ex-vivo label-free murine colon, focusing on the collagen structure changes over time, in mice ranging from 10 to 20 weeks of age. Series of images were acquired within the colonic and intestinal tissue at depth intervals of 20 microns from muscularis to the epithelium, up to a maximum depth of 180 microns.
The imaging system comprised a two-photon laser tuned to 800nm wavelength excitation, and the SHG emission was filtered with a 400/40 bandpass filter before reaching the photomultiplier tube. Images were acquired at 15 frames per second, for 200 to 300 cumulative frames, with a field of view of 261um by 261um, and 40mW at sample. Image series were compared to histopathology H&E slides taken from adjacent locations. Quantitative metrics for determining differences between murine glandular structures were applied, specifically glandular area and circularity.
We present a light sheet confocal microscope (LSCM) capable of high-resolution imaging of cell suspensions in a microfluidic environment. In lieu of conventional pressure-driven flow or mechanical translation of the samples, we have employed a novel method of fluid transport, redox-magnetohydrodynamics (redox-MHD). This method achieves fluid motion by inducing a small current into the suspension in the presence of a magnetic field via electrodes patterned onto a silicon chip. This on-chip transportation requires no moving parts, and is coupled to the remainder of the imaging system. The microscopy system comprises a 450 nm diode 20 mW laser coupled to a single mode fiber and a cylindrical lens that converges the light sheet into the back aperture of a 10x, 0.3 NA objective lens in an epi-illumination configuration. The emission pathway contains a 150 mm tube lens that focuses the light onto the linear sensor at the conjugate image plane. The linear sensor (ELiiXA+ 8k/4k) has three lateral binning modes which enables variable detection aperture widths between 5, 10, or 20 μm, which can be used to vary axial resolution. We have demonstrated redox-MHD-enabled light sheet microscopy in suspension of fluorescent polystyrene beads. This approach has potential as a high-throughput image cytometer with myriad cellular diagnostic applications.
Point-of-care approaches for 3-part leukocyte differentials (granulocyte, monocyte, and lymphocyte), traditionally performed using a hematology analyzer within a panel of tests called a complete blood count (CBC), are essential not only to reduce cost but to provide faster results in low resource areas. Recent developments in lab-on-a-chip devices have shown promise in reducing the size and reagents used, relating to a decrease in overall cost. Furthermore, smartphone diagnostic approaches have shown much promise in the area of point-of-care diagnostics, but the relatively high per-unit cost may limit their utility in some settings. We present here a method to reduce computing cost of a simple epi-fluorescence imaging system using a Raspberry Pi (single-board computer, <$40) to perform a 3-part leukocyte differential comparable to results from a hematology analyzer. This system uses a USB color camera in conjunction with a leukocyte-selective vital dye (acridine orange) in order to determine a leukocyte count and differential from a low volume (<20 microliters) of whole blood obtained via fingerstick. Additionally, the system utilizes a "cloud-based" approach to send image data from the Raspberry Pi to a main server and return results back to the user, exporting the bulk of the computational requirements. Six images were acquired per minute with up to 200 cells per field of view. Preliminary results showed that the differential count varied significantly in monocytes with a 1 minute time difference indicating the importance of time-gating to produce an accurate/consist differential.
In the non-keratinized epithelia, dysplasia typically arises near the basement membrane and proliferates into the upper epithelial layers over time. We present a non-invasive, multimodal technique combining high-resolution fluorescence imaging and broadband sub-diffuse reflectance spectroscopy (sDRS) to monitor health at various tissue layers. This manuscript focuses on characterization of the sDRS modality, which contains two source-detector separations (SDSs) of 374 μm and 730 μm, so that it can be used to extract in vivo optical parameters from human oral mucosa at two tissue thicknesses. First, we present empirical lookup tables (LUTs) describing the relationship between reduced scattering (μs') and absorption coefficients (μa) and absolute reflectance. LUTS were shown to extract μs' and μa with accuracies of approximately 4% and 8%, respectively. We then present LUTs describing the relationship between μs', μa and sampling depth. Sampling depths range between 210-480 and 260-620 μm for the 374 and 730 μm SDSs, respectively. We then demonstrate the ability to extract in vivo μs', μa, hemoglobin concentration, bulk tissue oxygen saturation, scattering exponent, and sampling depth from the inner lip of thirteen healthy volunteers to elucidate the differences in the extracted optical parameters from each SDS (374 and 730 μm) within non-keratinized squamous epithelia.
Colorectal cancer is the second leading cause of cancer deaths in the United States, affecting more than 130,000 Americans every year1. Determining tumor margins prior to surgical resection is essential to providing optimal treatment and reducing recurrence rates. Colorectal cancer recurrence can occur in up to 20% of cases, commonly within three years after curative treatment. Typically, when colorectal cancers are resected, a margin of normal tissue on both sides of the tumor is required. The minimum margin required for colon cancer is 5 cm and for the lower rectum 2 cm. However, usually more normal tissue is taken on both sides of the tumor because the blood supply to the entire segment is removed with the surgery and therefore the entire segment must be removed. Anastomotic recurrences may result from inadequate margins. Pathologists look at the margins to ensure that there is no residual tumor and this is usually documented in the pathology report. We have developed a portable, point-of-care fiber bundle microendoscopy imaging system for detection of abnormalities in colonic epithelial microstructure. The system comprises a laptop, a modified fiber bundle image guide with a 1mm active area diameter and custom LabVIEW interface, and is approved for imaging surgically resected colon tissue at the University of Arkansas for Medical Sciences. The microendoscopy probe provides high-resolution images of superficial epithelial histology in real-time to assist surgical guidance and to localize occult regions of dysplasia which may not be visible. Microendoscopy images of freshly resected human colonic epithelium were acquired using the microendoscopy device and subsequently mosaicked using custom post-processing software. Architectural changes in the glands were mapped to histopathology H&E slides taken from the precise location of the microendoscopy images. Qualitatively, glandular distortion and placement of image guide was used to map normal and dysplastic areas of the colonic tumor and surrounding region from microendoscopy images to H&E slides. Quantitative metrics for correlating images were also explored and were obtained by analyzing glandular diameter and spatial distribution as well as image texture.
Due to their robust optical properties, biological inertness, and readily adjustable surface chemistry, gold nanostructures have been demonstrated as contrast agents in a variety of biomedical imaging applications. One application is dynamic imaging of live cells using bioconjugated gold nanoparticles to monitor molecule trafficking mechanisms within cells; for instance, the regulatory pathway of epidermal growth factor receptor (EGFR) undergoing endocytosis. In this paper, we have demonstrated a method to track endocytosis of EGFR in MDA-MB-468 breast adenocarcinoma cells using bioconjugated gold nanocages (AuNCs) and multiphoton microscopy. Dynamic imaging was performed using a time series capture of 4 images every minute for one hour. Specific binding and internalization of the bioconjugated AuNCs was observed while the two control groups showed non-specific binding at fewer surface sites, leading to fewer bound AuNCs and no internalization.
Many cases of epithelial cancer originate in basal layers of tissue and are initially undetected by conventional microendoscopy techniques. We present a bench-top, fiber-bundle microendoscope capable of providing high resolution images of surface cell morphology. Additionally, the microendoscope has the capability to interrogate deeper into material by using diffuse reflectance and broadband diffuse reflectance spectroscopy. The purpose of this multimodal technique was to overcome the limitation of microendoscopy techniques that are limited to only visualizing morphology at the tissue or cellular level. Using a custom fiber optic probe, high resolution surface images were acquired using topical proflavine to fluorescently stain non-keratinized epithelia. A 635 nm laser coupled to a 200 μm multimode fiber delivers light to the sample and the diffuse reflectance signal was captured by a 1 mm image guide fiber. Finally, a tungsten-halogen lamp coupled to a 200 μm multimode fiber delivers broadband light to the sample to acquire spectra at source-detector separations of 374, 729, and 1051 μm. To test the instrumentation, a high resolution proflavine-induced fluorescent image of resected healthy mouse colon was acquired. Additionally, five monolayer poly(dimethylsiloxane)-based optical phantoms with varying absorption and scattering properties were created to acquire diffuse reflectance profiles and broadband spectra.
Imaging cells in a microfluidic chamber with an area scan camera is difficult due to motion blur and data loss during frame readout causing discontinuity of data acquisition as cells move at relatively high speeds through the chamber. We have developed a method to continuously acquire high-resolution images of cells in motion through a microfluidics chamber using a high-speed line scan camera. The sensor acquires images in a line-by-line fashion in order to continuously image moving objects without motion blur. The optical setup comprises an epi-illuminated microscope with a 40X oil immersion, 1.4 NA objective and a 150 mm tube lens focused on a microfluidic channel. Samples containing suspended cells fluorescently stained with 0.01% (w/v) proflavine in saline are introduced into the microfluidics chamber via a syringe pump; illumination is provided by a blue LED (455 nm). Images were taken of samples at the focal plane using an ELiiXA+ 8k/4k monochrome line-scan camera at a line rate of up to 40 kHz. The system’s line rate and fluid velocity are tightly controlled to reduce image distortion and are validated using fluorescent microspheres. Image acquisition was controlled via MATLAB’s Image Acquisition toolbox. Data sets comprise discrete images of every detectable cell which may be subsequently mined for morphological statistics and definable features by a custom texture analysis algorithm. This high-throughput screening method, comparable to cell counting by flow cytometry, provided efficient examination including counting, classification, and differentiation of saliva, blood, and cultured human cancer cells.
Optical phantoms are used in the development of various imaging systems. For certain applications, the development of thin phantoms that simulate the physical size and optical properties of tissue is important. Here, we demonstrate a method for producing thin phantom layers with tunable optical properties using poly(dimethylsiloxane) (PDMS) as a substrate material. The thickness of each layer (between 115 and 880 μm) was controlled using a spin coater. The reduced scattering and absorption coefficients were controlled using titanium dioxide and alcohol–soluble nigrosin, respectively. These optical coefficients were quantified at six discrete wavelengths (591, 631, 659, 691, 731, and 851 nm) at varying concentrations of titanium dioxide and nigrosin using spatial frequency domain imaging. From the presented data, we provide lookup tables to determine the appropriate concentrations of scattering and absorbing agents to be used in the design of PDMS-based phantoms with specific optical coefficients. In addition, heterogeneous phantoms mimicking the layered features of certain tissue types may be fabricated from multiple stacked layers, each with custom optical properties. These thin, tunable PDMS optical phantoms can simulate many tissue types and have broad imaging calibration applications in endoscopy, diffuse optical spectroscopic imaging, and optical coherence tomography, etc.
Current procedures for oral cancer screening typically involve visual inspection of the entire tissue surface at risk under
white light illumination. However, pre-cancerous lesions can be difficult to distinguish from many benign conditions
when viewed under these conditions. We have developed wide-field (macroscopic) imaging system which additionally
images in cross-polarized white light, narrowband reflectance, and fluorescence imaging modes to reduce specular glare,
enhance vascular contrast, and detect disease-related alterations in tissue autofluorescence.
We have also developed a portable system to enable high-resolution (microscopic) evaluation of cellular features within
the oral mucosa in situ. This system is a wide-field epi-fluorescence microscope coupled to a 1 mm diameter, flexible
fiber-optic imaging bundle. Proflavine solution was used to specifically label cell nuclei, enabling the characteristic
differences in N/C ratio and nuclear distribution between normal, dysplastic, and cancerous oral mucosa to be quantified.
This paper discusses the technical design and performance characteristics of these complementary imaging systems. We
will also present data from ongoing clinical studies aimed at evaluating diagnostic performance of these systems for
detection of oral neoplasia.
Early detection of neoplasia in patients with Barrett's esophagus is essential to improve outcomes. The aim of this ex vivo study was to evaluate the ability of high-resolution microendoscopic imaging and quantitative image analysis to identify neoplastic lesions in patients with Barrett's esophagus. Nine patients with pathologically confirmed Barrett's esophagus underwent endoscopic examination with biopsies or endoscopic mucosal resection. Resected fresh tissue was imaged with fiber bundle microendoscopy; images were analyzed by visual interpretation or by quantitative image analysis to predict whether the imaged sites were non-neoplastic or neoplastic. The best performing pair of quantitative features were chosen based on their ability to correctly classify the data into the two groups. Predictions were compared to the gold standard of histopathology. Subjective analysis of the images by expert clinicians achieved average sensitivity and specificity of 87% and 61%, respectively. The best performing quantitative classification algorithm relied on two image textural features and achieved a sensitivity and specificity of 87% and 85%, respectively. This ex vivo pilot trial demonstrates that quantitative analysis of images obtained with a simple microendoscope system can distinguish neoplasia in Barrett's esophagus with good sensitivity and specificity when compared to histopathology and to subjective image interpretation.
Worldwide incidence and mortality rates due to cancer continue to rise, with the burden of disease increasingly shifting
to developing countries. Several optical diagnostic methods are under development to enable earlier detection of cancer,
however, these are primarily intended for use in healthcare facilities in industrialized countries. Using knowledge
gained from early clinical studies with large-scale prototype systems, we have designed and tested low-cost, portable
versions of these instruments. We propose that these systems may be used for early diagnosis and screening in
developing countries, and that pilot clinical studies are warranted in these low-resource settings.
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