Optimizing modulation frequency for structured illumination in a fiber-optic microendoscope to image nuclear morphometry in columnar epithelium.

Fiber-optic microendoscopes have shown promise to image the changes in nuclear morphometry that accompany the development of precancerous lesions in tissue with squamous epithelium such as in the oral mucosa and cervix. However, fiber-optic microendoscopy image contrast is limited by out-of-focus light generated by scattering within tissue. The scattering coefficient of tissues with columnar epithelium can be greater than that of squamous epithelium resulting in decreased image quality. To address this challenge, we present a small and portable microendoscope system capable of performing optical sectioning using structured illumination (SI) in real-time. Several optical phantoms were developed and used to quantify the sectioning capabilities of the system. Columnar epithelium from cervical tissue specimens was then imaged ex vivo, and we demonstrate that the addition of SI achieves higher image contrast, enabling visualization of nuclear morphology.

Accuracy and interrater reliability for the diagnosis of Barrett's neoplasia among users of a novel, portable high-resolution microendoscope.

The high-resolution microendoscope (HRME) is a novel imaging modality that may be useful in the surveillance of Barrett's esophagus in low-resource or community-based settings. In order to assess accuracy and interrater reliability of microendoscopists in identifying Barrett's-associated neoplasia using HRME images, we recruited 20 gastroenterologists with no microendoscopic experience and three expert microendoscopists in a large academic hospital in New York City to interpret HRME images. They prospectively reviewed 40 HRME images from 28 consecutive patients undergoing surveillance for metaplasia and low-grade dysplasia and/or evaluation for high-grade dysplasia or cancer. Images were reviewed in a blinded fashion, after a 4-minute training with 11 representative images. All imaged sites were biopsied and interpreted by an expert pathologist. Sensitivity of all endoscopists for identification of high-grade dysplasia or cancer was 0.90 (95% confidence interval [CI]: 0.88-0.92) and specificity was 0.82 (95% CI: 0.79-0.85). Positive and negative predictive values were 0.72 (95% CI: 0.68-0.77) and 0.94 (95% CI: 0.92-0.96), respectively. No significant differences in accuracy were observed between experts and novices (0.90 vs. 0.84). The kappa statistic for all raters was 0.56 (95% CI: 0.54-0.58), and the difference between groups was not significant (0.64 vs. 0.55). These data suggest that gastroenterologists can diagnose Barrett's-related neoplasia on HRME images with high sensitivity and specificity, without the aid of prior microendoscopy experience.

High resolution microendoscopy for classification of colorectal polyps.

BACKGROUND AND STUDY AIMS: It can be difficult to distinguish adenomas from benign polyps during routine colonoscopy. High resolution microendoscopy (HRME) is a novel method for imaging colorectal mucosa with subcellular detail. HRME criteria for the classification of colorectal neoplasia have not been previously described. Study goals were to develop criteria to characterize HRME images of colorectal mucosa (normal, hyperplastic polyps, adenomas, cancer) and to determine the accuracy and interobserver variability for the discrimination of neoplastic from non-neoplastic polyps when these criteria were applied by novice and expert microendoscopists. METHODS: Two expert pathologists created consensus HRME image criteria using images from 68 patients with polyps who had undergone colonoscopy plus HRME. Using these criteria, HRME expert and novice microendoscopists were shown a set of training images and then tested to determine accuracy and interobserver variability. RESULTS: Expert microendoscopists identified neoplasia with sensitivity, specificity, and accuracy of 67 % (95 % confidence interval [CI] 58 % - 75 %), 97 % (94 % - 100 %), and 87 %, respectively. Nonexperts achieved sensitivity, specificity, and accuracy of 73 % (66 % - 80 %), 91 % (80 % - 100 %), and 85 %, respectively. Overall, neoplasia were identified with sensitivity 70 % (65 % - 76 %), specificity 94 % (87 % - 100 %), and accuracy 85 %. Kappa values were: experts 0.86; nonexperts 0.72; and overall 0.78. CONCLUSIONS: Using the new criteria, observers achieved high specificity and substantial interobserver agreement for distinguishing benign polyps from neoplasia. Increased expertise in HRME imaging improves accuracy. This low-cost microendoscopic platform may be an alternative to confocal microendoscopy in lower-resource or community-based settings.

Pre-clinical evaluation of fluorescent deoxyglucose as a topical contrast agent for the detection of Barrett's-associated neoplasia during confocal imaging.

The availability of confocal endomicroscopy motivates the development of optical contrast agents that can delineate the morphologic and metabolic features of gastrointestinal neoplasia. This study evaluates 2-NBDG, a fluorescent deoxyglucose, the uptake of which is associated with increased metabolic activity, in the identification of Barrett's-associated neoplasia. Surveillance biopsies from patients with varying pathologic grades of Barrett's esophagus were incubated ex vivo at 37°C with 2-NBDG and imaged with a fluorescence confocal microscope. Images were categorized as neoplastic (high grade dysplasia, esophageal adenocarcinoma) or metaplastic (intestinal metaplasia, low grade dysplasia) based on the degree of glandular 2-NBDG uptake. Classification accuracy was assessed using histopathology as the gold standard. Forty-four biopsies were obtained from twenty-six patients; 206 sites were imaged. The glandular mean fluorescence intensity of neoplastic sites was significantly higher than that of metaplastic sites (p<0.001). Chronic inflammation was associated with increased 2-NBDG uptake in the lamina propria but not in glandular epithelium. Sites could be classified as neoplastic or not with 96% sensitivity and 90% specificity based on glandular mean fluorescence intensity. Classification accuracy was not affected by the presence of inflammation. By delineating the metabolic and morphologic features of neoplasia, 2-NBDG shows promise as a topical contrast agent for confocal imaging. Further in vivo testing is needed to determine its performance in identifying neoplasia during confocal endomicroscopic imaging.

Emerging roles for multimodal optical imaging in early cancer detection: a global challenge.

Medical imaging technologies have become increasingly important in the clinical management of cancer, and now play key roles in cancer screening, diagnosis, staging, and monitoring response to treatment. Standard imaging modalities such as MRI, PET, and CT require significant financial resources and infrastructure, which limits access to these modalities to those patients in high-resource settings. In contrast, optical imaging strategies, with the potential for reduced cost and enhanced portability, are emerging as additional tools to facilitate the early detection and diagnosis of cancer. This article presents a vision for an expanding role for optical imaging in global cancer management, including screening, early detection at the point-of-care, biopsy guidance, and real-time histology. Multi-modal optical imaging - the combination of widefield and high resolution imaging - has the potential to aid in the detection and management of precancer and early cancer for traditionally underserved populations. Several recent widefield and high-resolution optical imaging technologies are described, along with requirements for implementing such devices into lower-resource - settings.

Confocal microscopy and molecular-specific optical contrast agents for the detection of oral neoplasia.

Using current clinical diagnostic techniques, it is difficult to visualize tumor morphology and architecture at the cellular level, which is necessary for diagnostic localization of pathologic lesions. Optical imaging techniques have the potential to address this clinical need by providing real-time, sub-cellular resolution images. This paper describes the use of dual mode confocal microscopy and optical molecular-specific contrast agents to image tissue architecture, cellular morphology, and sub-cellular molecular features of normal and neoplastic oral tissues. Fresh tissue slices were prepared from 33 biopsies of clinically normal and abnormal oral mucosa obtained from 14 patients. Reflectance confocal images were acquired after the application of 6% acetic acid, and fluorescence confocal images were acquired after the application of a fluorescence contrast agent targeting the epidermal growth factor receptor (EGFR). The dual imaging modes provided images similar to light microscopy of hematoxylin and eosin and immunohistochemistry staining, but from thick fresh tissue slices. Reflectance images provided information on the architecture of the tissue and the cellular morphology. The nuclear-to-cytoplasmic (N/C) ratio from the reflectance images was at least 7.5 times greater for the carcinoma than the corresponding normal samples, except for one case of highly keratinized carcinoma. Separation of carcinoma from normal and mild dysplasia was achieved using this ratio (p<0.01). Fluorescence images of EGFR expression yielded a mean fluorescence labeling intensity (FLI) that was at least 2.7 times higher for severe dysplasia and carcinoma samples than for the corresponding normal sample, and could be used to distinguish carcinoma from normal and mild dysplasia (p<0.01). Analyzed together, the N/C ratio and the mean FLI may improve the ability to distinguish carcinoma from normal squamous epithelium.

A pilot study for a screening trial of cervical fluorescence spectroscopy.

Fluorescence spectroscopy is a promising technology for detection of epithelial precancers and cancers. In preparation for a multicenter phase II screening trial, a pilot trial was conducted to test data collection and patient examination procedures, use data forms, time procedures, and identify problems with preliminary data analysis. Women 18 years of age and older underwent a questionnaire, a complete history, and a physical examination, including a pan-colposcopy of the lower genital tract. A fiber-optic probe measured fluorescence excitation-emission matrices at 1-3 cervical sites for 58 women. The data collection procedures, data forms, and procedure times worked well, although collection times for all the clinical data take an average of 28 min. The clinical team followed procedures well, and the data could be retrieved from the database at all sites. The multivariate analysis algorithm correctly identified squamous normal tissue 99% of the time and columnar normal tissue only 7%. The assessment of ploidy from monolayer samples was not accurate in this small sample. The study was successful as a pilot trial. We learned who participated, who withdrew, how often abnormalities were present, and that algorithms that have worked extremely well in previous studies do not work as well when a few study parameters are changed. The current algorithm for diagnosis identified squamous normal tissue very accurately and did less well for columnar normal tissue. Inflammation may be an explanation for this phenomenon. Fluorescence spectroscopy is a promising technology for the detection of epithelial precancers and cancers. The screening trial of fluorescence and reflectance spectroscopy was successful.

Detection and diagnosis of oral neoplasia with an optical coherence microscope.

The use of high resolution, in vivo optical imaging may offer a clinically useful adjunct to standard histopathologic techniques. A pilot study was performed to investigate the diagnostic capabilities of optical coherence microscopy (OCM) to discriminate between normal and abnormal oral tissue. Our objective is to determine whether OCM, a technique combining the subcellular resolution of confocal microscopy with the coherence gating and heterodyne detection of optical coherence tomography, has the same ability as confocal microscopy to detect morphological changes present in precancers of the epithelium while providing superior penetration depths. We report our results using OCM to characterize the features of normal and neoplastic oral mucosa excised from 13 subjects. Specifically, we use optical coherence and confocal microscopic images obtained from human oral biopsy specimens at various depths from the mucosal surface to examine the optical properties that distinguish normal and neoplastic oral mucosa. An analysis of penetration depths achieved by the OCM and its associated confocal arm found that the OCM consistently imaged more deeply. Extraction of scattering coefficients from reflected nuclear intensity is successful in nonhyperkeratotic layers and shows differentiation between scattering properties of normal and dysplastic epithelium and invasive cancer.

Molecular imaging of carcinogenesis with immuno-targeted nanoparticles.

Molecular characterization of cancer could have important clinical benefits such as earlier cancer detection based on molecular characterization, the ability to predict the risk of cancer progression, real time margin detection, the ability to rationally select molecular therapy and to monitor response to the therapy. We present a new class of molecular specific contrast agents for optical imaging of carcinogenesis in vivo - gold nanoparticles conjugated with monoclonal antibodies specific for cancer biomarkers.

Optical systems for in vivo molecular imaging of cancer.

Progress toward a molecular characterization of cancer would have important clinical benefits; thus, there is an important need to image the molecular features of cancer in vivo. In this paper, we describe a comprehensive strategy to develop inexpensive, rugged and portable optical imaging systems for molecular imaging of cancer, which couples the development of optically active contrast agents with advances in functional genomics of cancer. We describe initial results obtained using optically active contrast agents to image the expression of three well known molecular signatures of neoplasia: including over expression of the epidermal growth factor receptor (EGFR), matrix metallo-proteases (MMPs), and oncoproteins associated with human papillomavirus (HPV) infection. At the same time, we are developing inexpensive, portable optical systems to image the morphologic and molecular signatures of neoplasia noninvasively in real time. These real-time, portable, inexpensive systems can provide tools to characterize the molecular features of cancer in vivo.

Near real time in vivo fibre optic confocal microscopy: sub-cellular structure resolved.

We have built a fibre optic confocal reflectance microscope capable of imaging biological tissue in near real time. The measured lateral resolution is 3 micro m and axial resolution is 6 micro m. Images of epithelial cells, excised tissue biopsies, and the human lip in vivo have been obtained at 15 frames s-1. Both cell morphology and tissue architecture can be appreciated from images obtained with this microscope. This device has the potential to enable reflected light confocal imaging of internal organs for in situ detection of pathology.

Understanding the contributions of NADH and collagen to cervical tissue fluorescence spectra: modeling, measurements, and implications.

OBJECTIVE: At 380 nm excitation, cervical tissue fluorescence spectra demonstrate characteristic changes with both patient age and the presence of dysplasia. A Monte Carlo model was developed in order to quantitatively examine how intrinsic NADH and collagen fluorescence, in combination with tissue scattering and absorption properties, yield measured tissue spectra. METHODS: Excitation-emission matrices were measured for live cervical cells and collagen gel phantoms. Fluorescence microscopy of fresh tissue sections was performed to obtain the location and density of fluorophores as a function of patient age and the presence of dysplasia. A Monte Carlo model was developed which incorporated measurements of fluorophore line shapes and spatial distributions. RESULTS: Modeled spectra were consistent with clinical measurements and indicate that an increase in NADH fluorescence and decrease in collagen fluorescence create clinically observed differences between normal and dysplastic tissue spectra. Model predictions were most sensitive to patient age and epithelial thickness. CONCLUSIONS: Monte Carlo techniques provide an important means to investigate the combined contributions of multiple fluorophores to measured emission spectra. The approach will prove increasingly valuable as a more sophisticated understanding of in vivo optical properties is developed.

Fast and noninvasive fluorescence imaging of biological tissues in vivo using a flying-spot scanner.

We have developed a flying-spot scanner (FSS), for fluorescence imaging of tissues in vivo. The FSS is based on the principles of single-pixel illumination and detection via a raster scanning technique. The principal components of the scanner are a laser light source, a pair of horizontal and vertical scanning mirrors to deflect the laser light in these respective directions on the tissue surface, and a photo multiplier tube (PMT) detector. This paper characterizes the performance of the FSS for fluorescence imaging of tissues in vivo. First, a signal-to-noise ratio (SNR) analysis is presented. This is followed by characterization of the experimental SNR, linearity and spatial resolution of the FSS. Finally, the feasibility of tissue fluorescence imaging is demonstrated using an animal model. In summary, the performance of the FSS is comparable to that of fluorescence-imaging systems based on multipixel illumination and detection. The primary advantage of the FSS is the order-of-magnitude reduction in the cost of the light source and detector. However, the primary disadvantage of the FSS its significantly slower frame rate (1 Hz). In applications where high frame rates are not critical, the FSS will represent a low-cost alternative to multichannel fluorescence imaging-systems.

Fluorescence spectroscopy for in vivo characterization of ovarian tissue.

BACKGROUND AND OBJECTIVE: The objective of this study was to explore whether fluorescence spectroscopy signatures differed between normal variations within the ovary, benign neoplasms, and ovarian cancer. STUDY DESIGN/MATERIALS AND METHODS: Ovarian tissue fluorescence emission spectra were collected sequentially at 18 excitation wavelengths ranging from 330 to 500 nm from 11 patients undergoing oophorectomy and assembled into fluorescence excitation emission matrices (EEMs); biopsies corresponded to the area interrogated. Spectral areas that could differentiate normal ovary, benign neoplasms, and cancers were evaluated, using histopathology as the reference standard. RESULTS: The most promising measurements are (1) the integrated fluorescence intensity from 400 to 430 nm excitation at 460 nm emission, and (2) the ratios of fluorescence intensities at 330 nm excitation, 385 and 500 nm emission, and at 375 and 415 nm excitation, 460 nm emission. Simple systems to visualize these optical signatures at laparoscopy could be designed. CONCLUSION: Fluorescence spectroscopy may have the ability to distinguish ovarian cancers from normal ovarian structures and benign neoplasms, as well as differentiate between normal variations and metaplastic structures and should be further explored as a device for the early detection of ovarian cancers.

Biomarker modulation in a nonhuman rhesus primate model for ovarian cancer chemoprevention.

OBJECTIVE: The objective of this study was to explore whether a nonhuman primate model could be developed to test drugs for the prevention of ovarian cancer. METHODS: Nineteen adult female Rhesus macaques were given fenretinide (4HPR), oral contraceptives (OCP), the combination (4HPR + OCP), or no medication for 3 months. Exploratory laparotomy was done pre- and postdrug to assess intermediary biomarkers of neoplastic phenotype, proliferation, response pathways, and growth-regulatory and metabolic markers. Fluorescence emission spectra were plotted for each group pre- and postdrug and means were overlaid on these plots and normalized. Fluorescence intensities were compared using the 2-tailed Student t test, (P = 0.1-0.01). RESULTS: All monkeys tolerated drugs and surgeries without difficulty. Histochemical markers showed no significant trend. However, fluorescence spectroscopy showed increased intensity at 450 nm excitation, 550 nm emission correlating with increased FAD presence. The 4HPR group (P = 0.01) showed higher intensity than the OCP group (P = 0.05-0.07) when compared with the controls. Decreased emission was seen at 350 nm excitation, 450 nm emission correlating with decreased NAD(P)H presence. The OCP group showed the largest change (P < 0.01), and the control group showed the smallest change. CONCLUSIONS: The nonhuman primate is an excellent model to test drug effect on the ovarian surface epithelium and merits additional study. Fluorescence spectroscopy was the most sensitive marker for drug activity and the apparent increase in NAD and FAD in the 4HPR group is consistent with the effect of 4HPR observed in cell culture. The differences between the OCP and the 4HPR groups suggest a different mechanism of activity of these drugs.

Fluorescence spectroscopy of epithelial tissue throughout the dysplasia-carcinoma sequence in an animal model: spectroscopic changes precede morphologic changes.

BACKGROUND AND OBJECTIVE: The hamster cheek pouch carcinogenesis model, using chronic treatments of dimethylbenz[alpha]anthracene (DMBA) was used as a model system to investigate changes in epithelial tissue autofluorescence throughout the dysplasia-carcinoma sequence. STUDY DESIGN/MATERIALS AND METHODS: Fluorescence emission spectra were measured weekly from 42 DMBA-treated animals and 20 control animals at 337, 380, and 460 nm excitation. A subset of data in which histopathology was available was used to develop diagnostic algorithms to separate neoplastic and non-neoplastic tissue. The change in fluorescence intensity over time was examined in all samples at excitation-emission wavelength pairs identified as diagnostically useful. RESULTS: Algorithms based on autofluorescence can separate neoplastic and non-neoplastic tissue with 95% sensitivity and 93% specificity. Greatest contributions to diagnostic algorithms are obtained at 380 nm excitation, and 430, 470, and 600 nm emission. Changes in fluorescence intensity are apparent as early as 3 weeks after initial treatment with DMBA, whereas morphologic changes associated with dysplasia occur on average at 7.5-12.5 weeks after initial treatment. CONCLUSIONS: Fluorescence spectroscopy provides a potential tool to identify biochemical changes associated with dysplasia and hyperplasia, which precede morphologic changes observed in histologically stained sections.

Reflectance spectroscopy for in vivo characterization of ovarian tissue.

BACKGROUND AND OBJECTIVE: To explore whether reflectance spectroscopy can differentiate normal ovary, benign neoplasms, and ovarian cancer. STUDY DESIGN/MATERIALS AND METHODS: Reflectance spectra (390-600 nm) were measured at three source-detector separations (SDS) in vivo at 64 sites in 16 patients undergoing oophorectomy. Parameters with largest statistical differences were identified. Based on these parameters algorithms were developed and evaluated. RESULTS: Promising parameters were the reflectance intensity from 540 to 580 nm (SDS, 1.1 mm), the slope of the reflectance spectrum from 490 to 520 nm (SDS, 1.1 mm), the slope from 510 to 530 nm (SDS, 2.1 mm), and the slope from 510 to 530 (SDS, 3 mm). Average sensitivity and specificity were 86 +/- 6% and 79 +/- 5% to separate normal ovary from benign neoplasms and cancers. Average sensitivity and specificity were 86 +/- 4% and 80 +/- 8% to separate ovarian cancers from benign neoplasms and normal ovary. CONCLUSION: Reflectance spectroscopy should be further investigated for ovarian cancer screening.

Autofluorescence microscopy of fresh cervical-tissue sections reveals alterations in tissue biochemistry with dysplasia.

Fluorescence spectroscopy offers an effective, noninvasive approach to the detection of precancers in multiple organ sites. Clinical studies have demonstrated that fluorescence spectroscopy can provide highly sensitive, specific and cost-effective diagnosis of cervical precancers. However, the underlying biochemical mechanisms responsible for differences in the fluorescence spectra of normal and dysplastic tissue are not fully understood. We designed a study to assess the differences in autofluorescence of normal and dysplastic cervical tissue. Transverse, fresh tissue sections were prepared from colposcopically normal and abnormal biopsies in a 34-patient study. Autofluorescence images were acquired at 380 and 460 nm excitation. Results showed statistically significant increases in epithelial fluorescence intensity (arbitrary units) at 380 nm excitation in dysplastic tissue (106 +/- 39) relative to normal tissue (85 +/- 30). The fluorophore responsible for this increase is possibly reduced nicotinamide adenine dinucleotide. Stromal fluorescence intensities in the dysplastic samples decreased at both 380 nm (102 +/- 34 [dysplasia] vs 151 +/- 44 [normal]) and 460 nm excitation (93 +/- 35 [dysplasia] vs 137 +/- 49 [normal]), wavelengths at which collagen is excited. Decreased redox ratio (17-40% reduction) in dysplastic tissue sections, indicative of increased metabolic activity, was observed in one-third of the paired samples. These results provide valuable insight into the biological basis of the differences in fluorescence of normal and precancerous cervical tissue.

Low Temperature Fluorescence Imaging of Freeze-trapped Human Cervical Tissues.

We characterized the fluorescence intensity distribution within the epithelia and stroma of frozen human cervical tissues at the following excitation-emission wavelength pairs: 440, 525 nm and 365, 460 nm. The intensities at both excitation-emission wavelength pairs are significantly lower in the epithelia of severely dysplastic tissues, relative to that in normal and inflammatory tissues. Furthermore, there are small differences in (1) the epithelial intensity of severe dysplasia and mild dysplasia at 440, 525 nm and (2) the stromal intensity of inflammatory and severely dysplastic tissues at 365, 460 nm. A comparison of the ratio of intensities at 440, 525 nm and 365, 460 nm between the epithelia of each tissue type indicates that this ratio is lowest in severely dysplastic tissues. It is interesting to note that the epithelial and stromal intensities are comparable at 365, 460 nm; however, at 440, 525 nm, the epithelial intensity is more than a factor of two less that that of the stroma for all tissue types.

Fiber confocal reflectance microscope (FCRM) for in-vivo imaging.

In-vivo imaging can be achieved with a coherent-fiber-bundle based confocal reflectance microscope. Such a microscope could provide the means to detect pre-cancerous lesions in the cervix by characterizing cells' nuclear-to-cytoplasmic ratio. In this paper we present the design of such a fiber confocal reflectance microscope, with an emphasis on its optical sub-systems. The optical sub-systems consist of a commercially available microscope objective and custom designed telescope, scan lens, and coupling lens systems. The performance of the fiber confocal reflectance microscope was evaluated by imaging a resolution bar target and human cervical biopsy tissues. The results presented in this paper demonstrate a lateral resolution of 2 microm and axial resolution of 6 microm. The sensitivity of the system defined by the smallest refractive-index mismatch that can be detected is approximately Delta n 0.05.