Using FDA-approved drugs as off-label fluorescent dyes for optical biopsies: from in silico design toex vivoproof-of-concept.

Optical biopsies bring the microscope to the patient rather than the tissue to the microscope, and may complement or replace the tissue-harvesting component of the traditional biopsy process with its associated risks. In general, optical biopsies are limited by the lack of endogenous tissue contrast and the small number of clinically approvedin vivodyes. This study tests multiple FDA-approved drugs that have structural similarity to research dyes as off-labelin situfluorescent alternatives to standardex vivohematoxylin & eosin tissue stain. Numerous drug-dye combinations shown here may facilitate relatively safe and fastin situor possiblyin vivostaining of tissue, enabling real-time optical biopsies and other advanced microscopy technologies, which have implications for the speed and performance of tissue- and cellular-level diagnostics.

Investigation of confocal microscopy for differentiation of renal cell carcinoma versus benign tissue. Can an optical biopsy be performed?

OBJECTIVE: Novel optical imaging modalities are under development with the goal of obtaining an "optical biopsy" to efficiently provide pathologic details. One such modality is confocal microscopy which allows in situ visualization of cells within a layer of tissue and imaging of cellular-level structures. The goal of this study is to validate the ability of confocal microscopy to quickly and accurately differentiate between normal renal tissue and cancer. METHODS: Specimens were obtained from patients who underwent robotic partial nephrectomy for renal mass. Samples of suspected normal and tumor tissue were extracted from the excised portion of the kidney and stained with acridine orange. The stained samples were imaged on a Nikon E600 C1 Confocal Microscope. The samples were then submitted for hematoxylin and eosin processing and read by an expert pathologist to provide a gold-standard diagnosis that can later be compared to the confocal images. RESULTS: This study included 11 patients, 17 tissue samples, and 118 confocal images. Of the 17 tissue samples, 10 had a gold-standard diagnosis of cancer and seven were benign. Of 118 confocal images, 66 had a gold-standard diagnosis of cancer and 52 were benign. Six confocal images were used as a training set to train eight observers. The observers were asked to rate the test images on a six point scale and the results were analyzed using a web based receiver operating characteristic curve calculator. The average accuracy, sensitivity, specificity, and area under the empirical receiver operating characteristic curve for this study were 91%, 98%, 81%, and 0.94 respectively. CONCLUSION: This preliminary study suggest that confocal microscopy can be used to distinguish cancer from normal tissue with high sensitivity and specificity. The observers in this study were trained quickly and on only six images. We expect even higher performance as observers become more familiar with the confocal images.

Improvement in optical fiber bundle-based imaging using synchronized fiber motion.

Image quality in fiber bundle-based imaging systems is inherently limited by the size and spacing of the individual fiber cores. The fiber bundle limits the achievable spatial resolution and superimposes a fixed pattern of signal variability across the image. To overcome these limitations, piezoelectric tubes were used to synchronously dither the fiber bundle on both ends. Experimental results using the dithering approach with a commercial fiber bundle showed a substantial decrease in fixed pattern noise and an increase in spatial resolution.

Characterization of TCP-1 probes for molecular imaging of colon cancer.

Molecular probes capable of detecting colorectal cancer (CRC) are needed for early CRC diagnosis. The objective of this study was to characterize c[CTPSPFSHC]OH (TCP-1), a small peptide derived from phage display selection, for targeting human CRC xenografts using technetium-99m ((99m)Tc)-labeled TCP-1 and fluorescent cyanine-7 (Cy7)-labeled form of the peptide (Cy7-TCP-1). (99m)Tc-TCP-1 was generated by modifying TCP-1 with succinimidyl-6-hydrazino-nicotinamide (S-HYNIC) followed by radiolabeling. In vitro saturation binding experiments were performed for (99m)Tc-TCP-1 in human HCT116 colon cancer cells. SCID mice with human HCT116 cancer xenografts were imaged with (99m)Tc-TCP-1 or control peptide using a small-animal SPECT imager: Group I (n=5) received no blockade; Group II (n=5) received a blocking dose of non-radiolabeled TCP-1. Group III (n=5) were imaged with (99m)Tc-labeled control peptide (inactive peptide). SCID mice with human PC3 prostate cancer xenografts (Group IV, n=5) were also imaged with (99m)Tc-TCP-1. Eight additional SCID mice bearing HCT116 xenografts in dorsal skinfold window chambers (DSWC) were imaged by direct positron imaging of (18)F-fluorodeoxyglucose ((18)F-FDG) and fluorescence microscopy of Cy7-TCP-1. In vitro(99m)Tc-HYNIC-TCP-1 binding assays on HCT 116 cells indicated a mean Kd of 3.04±0.52nM. In cancer xenografts, (99m)Tc-TCP-1 radioactivity (%ID/g) was 1.01±0.15 in the absence of blockade and was reduced to 0.26±0.04 (P<0.01) with blockade. No radioactive uptake was observed in the PC3 tumors with (99m)Tc-TCP-1 or HCT116 tumors with inactive peptide. Cy7-TCP-1 activity localized not only in metabolically active tumors, as defined by (18)F-FDG imaging, but also in peritumoral microvasculature. In conclusion, TCP-1 probes may have a distinct targeting mechanism with high selectivity for CRC and tumor-associated vasculature. Molecular imaging with TCP-1 probes appears promising to detect malignant colorectal lesions.

Pilot Clinical Evaluation of a Confocal Microlaparoscope for Ovarian Cancer Detection.

OBJECTIVE: The aim of this study is to evaluate the performance of a confocal fluorescence microlaparoscope for in vivo detection of ovarian cancer. METHODS/MATERIALS: Seventy-one patients scheduled for open or laparoscopic oophorectomy were consented for the imaging study. High-resolution confocal microlaparoscopic images of the epithelial surface of the ovary were acquired in vivo or ex vivo after tissue staining using acridine orange. Standard histologic evaluation of extracted tissue samples was performed and used as the gold standard of disease diagnosis. Trained human observers from different specialties viewed the microlaparoscopic images, rating each image on a 6-point scale ranging from "definitely not cancer" to "definitely cancer." Receiver operating characteristic curves were generated using these scores and the gold standard histopathologic diagnosis. Area under the receiver operating characteristic curve (AUC) was calculated as a performance metric. RESULTS: Forty-five of the consented patients were used in the final evaluation study. From these 45 patients, 63 tissue locations or samples were identified and imaged with the confocal microlaparoscope. Twenty of the samples were high-grade cancers, and the remaining 43 samples were normal or noncancerous. Twenty-three of the samples were imaged in vivo, and the remaining 40 samples were imaged ex vivo. The average AUC score and standard error (SE) for detection of cancer in all images were 0.88 and 0.02, respectively. An independent-samples t test was conducted to compare AUC scores for in vivo and ex vivo conditions. No statistically significant difference in the AUC score for in vivo (AUC, 0.850; SE, 0.049) and ex vivo (AUC, 0.888; SE, 0.027) conditions was observed, t(6) = 1.318, P = 0.2355. CONCLUSIONS: Area under the receiver operating characteristic curve scores indicate that high-resolution in vivo images obtained by the confocal laparoscope can distinguish between normal and malignant ovarian surface epithelium. In addition, in vivo performance is similar to that which can be obtained from ex vivo tissue.

Fluorescence and reflectance spectral imaging system for a murine mammary window chamber model.

A spectral imaging system was developed to study the development of breast cancer xenografts in a murine mammary window chamber model. The instrument is configured to work with either a laser to excite fluorescence or a broadband light source for diffuse reflectance imaging. Two applications were demonstrated. First, spectral imaging of fluorescence signals was demonstrated with a GFP-breast cancer tumor and fluorescein injection. Second, based on the principles of broadband reflectance spectroscopy, the instrument was used to monitor dynamic changes of tissue absorbance to yield tissue oxygenation maps at different time points during tumor progression.

Analysis of multimode fiber bundles for endoscopic spectral-domain optical coherence tomography.

A theoretical analysis of the use of a fiber bundle in spectral-domain optical coherence tomography (OCT) systems is presented. The fiber bundle enables a flexible endoscopic design and provides fast, parallelized acquisition of the OCT data. However, the multimode characteristic of the fibers in the fiber bundle affects the depth sensitivity of the imaging system. A description of light interference in a multimode fiber is presented along with numerical simulations and experimental studies to illustrate the theoretical analysis.

Dynamic oxygenation measurements using a phosphorescent coating within a mammary window chamber mouse model.

Phosphorescent lifetime imaging was employed to measure the spatial and temporal distribution of oxygen partial pressure in tissue under the coverslip of a mammary window chamber breast cancer mouse model. A thin platinum-porphyrin coating, whose phosphorescent lifetime varies monotonically with oxygen partial pressure, was applied to the coverslip surface. Dynamic temporal responses to induced modulations in oxygenation levels were measured using this approach.

Confocal microlaparoscope for imaging the fallopian tube.

Recent evidence suggests that ovarian cancer can originate in the fallopian tube. Unlike many other cancers, poor access to the ovary and fallopian tubes has limited the ability to study the progression of this deadly disease and to diagnosis it during the early stage when it is most amenable to therapy. A rigid confocal microlaparoscope system designed to image the epithelial surface of the ovary in vivo was previously reported. A new confocal microlaparoscope with an articulating distal tip has been developed to enable in vivo access to human fallopian tubes. The new microlaparoscope is compatible with 5-mm trocars and includes a 2.2-mm-diameter articulating distal tip consisting of a bare fiber bundle and an automated dye delivery system for fluorescence confocal imaging. This small articulating device should enable the confocal microlaparoscope to image early stage ovarian cancer arising inside the fallopian tube. Ex vivo images of animal tissue and human fallopian tube using the new articulating device are presented along with in vivo imaging results using the rigid confocal microlaparoscope system.

Multi-modality imaging of a murine mammary window chamber for breast cancer research.

Window chamber models have been developed and utilized as a means to study the complex microenvironment in which cancers develop, proliferate, and metastasize in small animals. Here we utilize rapid prototyping printer technology to construct a new plastic orthotopic mammary window chamber that is compatible with magnetic resonance imaging, nuclear imaging, and optical imaging. Optical imaging allows for high-resolution cellular and molecular level analysis of tissues; magnetic resonance imaging provides quantitative measures of tumor size, perfusion, diffusion, fat/water content relaxation parameters; and a nuclear imaging technique, called the Beta Imager, supports functional and metabolic imaging. Our demonstration of the multiple imaging capabilities of this model suggests that it can be used as a powerful platform for studying basic cancer biology and developing new cancer therapies.

Design and Performance of a Multi-Point Scan Confocal Microendoscope.

Confocal fluorescence microendoscopy provides high-resolution cellular-level imaging via a minimally invasive procedure, but requires fast scanning to achieve real-time imaging in vivo. Ideal confocal imaging performance is obtained with a point scanning system, but the scan rates required for in vivo biomedical imaging can be difficult to achieve. By scanning a line of illumination in one direction in conjunction with a stationary confocal slit aperture, very high image acquisition speeds can be achieved, but at the cost of a reduction in image quality. Here, the design, implementation, and experimental verification of a custom multi-point aperture modification to a line-scanning multi-spectral confocal microendoscope is presented. This new design improves the axial resolution of a line-scan system while maintaining high imaging rates. In addition, compared to the line-scanning configuration, previously reported simulations predicted that the multi-point aperture geometry greatly reduces the effects of tissue scatter on image quality. Experimental results confirming this prediction are presented.

Ultrathin single-channel fiberscopes for biomedical imaging.

Ultrathin flexible fiberscopes typically have separate illumination and imaging channels and are available in diameters ranging from 0.5 to 2.5 mm. Diameters can potentially be reduced by combining the illumination and imaging paths into a single fiberoptic channel. Single-channel fiberscopes must incorporate a system to minimize Fresnel reflections from air-glass interfaces within the common illumination and detection path. The Fresnel reflection at the proximal surface of the fiber bundle is particularly problematic. This paper describes and compares methods to reduce the background signal from the proximal surface of the fiber bundle. Three techniques are evaluated: (1) antireflective (AR)-coating the proximal face of the fiber, (2) incorporating crossed polarizers into the light path, and (3) a novel technique called numerical aperture sharing, whereby a portion of the image numerical aperture is devoted to illumination and a portion to detection.

Multimodality pH imaging in a mouse dorsal skin fold window chamber model.

Upregulate levels of expression and activity of membrane H+ ion pumps in cancer cells drives the extracellular pH (pHe,) to values lower than normal. Furthermore, disregulated pH is indicative of the changes in glycolytic metabolism in tumor cells and has been shown to facilitate extracellular tissue remodeling during metastasis Therefore, measurement of pHe could be a useful cancer biomarker for diagnostic and therapy monitoring evaluation. Multimodality in-vivo imaging of pHe in tumorous tissue in a mouse dorsal skin fold window chamber (DSFWC) model is described. A custom-made plastic window chamber structure was developed that is compatible with both imaging optical and MR imaging modalities and provides a model system for continuous study of the same tissue microenvironment on multiple imaging platforms over a 3-week period. For optical imaging of pHe, SNARF-1 carboxylic acid is injected intravenously into a SCID mouse with an implanted tumor. A ratiometric measurement of the fluorescence signal captured on a confocal microscope reveals the pHe of the tissue visible within the window chamber. This imaging method was used in a preliminary study to evaluate sodium bicarbonate as a potential drug treatment to reverse tissue acidosis. For MR imaging of pHe the chemical exchange saturation transfer (CEST) was used as an alternative way of measuring pHe in a DSFWC model. ULTRAVIST®, a FDA approved x-ray/CT contrast agent has been shown to have a CEST effect that is pH dependent. A ratiometric analysis of water saturation at 5.6 and 4.2 ppm chemical shift provides a means to estimate the local pHe.

Error analysis of ratiometric imaging of extracellular pH in a window chamber model.

Ratiometric fluorescence-imaging technique is commonly used to measure extracellular pH in tumors and surrounding tissue within a dorsal skin-fold window chamber. Using a pH-sensitive fluorophore such as carboxy SNARF-1 one can measure pH distributions with high precision. However, it is often observed that the measured pH is lower than expected, with a bias that varies from one image to another. A comprehensive analysis of possible error sources is presented. These error sources include photon noise, estimator bias, instrument errors, temperature, and calibration errors from biological factors.

Dual modality fluorescence confocal and spectral-domain optical coherence tomography microendoscope.

Optical biopsy facilitates in vivo disease diagnoses by providing a real-time in situ view of tissue in a clinical setting. Fluorescence confocal microendoscopy and optical coherence tomography (OCT) are two methods that have demonstrated significant potential in this context. These techniques provide complementary viewpoints. The high resolution and contrast associated with confocal systems allow en face visualization of sub-cellular details and cellular organization within a thin layer of biological tissue. OCT provides cross-sectional images showing the tissue micro-architecture to a depth beyond the reach of confocal systems. We present a novel design for a bench-top imaging system that incorporates both confocal and OCT modalities in the same optical train allowing the potential for rapid switching between the two imaging techniques. Preliminary results using simple phantoms show that it is possible to realize both confocal microendoscopy and OCT through a fiber bundle based imaging system.

Errors in confocal fluorescence ratiometric imaging microscopy due to chromatic aberration.

Confocal fluorescence ratiometric imaging is an optical technique used to measure a variety of important biological parameters. A small amount of chromatic aberration in the microscope system can introduce a variation in the signal ratio dependent on the fluorophore concentration gradient along the optical axis and lead to bias in the measurement. We present a theoretical model of this effect. Experimental results and simulations clearly demonstrate that this error can be significant and should not be ignored.

Statistical analysis and optimization of frequency-domain fluorescence lifetime imaging microscopy using homodyne lock-in detection.

Fluorescence lifetime imaging microscopy is used widely in biological research, but the accuracy and precision of lifetime measurements are limited. Photon noise is an inherent error source that cannot be eliminated. In this paper, we present a general approach to compute the probability density of the estimated lifetime for frequency-domain fluorescence lifetime imaging microscopy using homodyne lock-in detection. The analysis for commonly used excitation methods, including sinusoidal modulation, square-wave modulation, and a periodically pulsed light source, are given and compared to the results of Monte Carlo simulations. The optimum parameters of the excitation waveforms to minimize the variance of the estimated lifetimes are also derived and compared to previously published results.

In vivo imaging of ovarian tissue using a novel confocal microlaparoscope.

OBJECTIVE: The objective of the study was to develop a clinical confocal microlaparoscope for imaging ovary epithelium in vivo with the long-term objective of diagnosing cancer in vivo. STUDY DESIGN: A confocal microlaparoscope was developed and used to image the ovaries of 21 patients in vivo using fluorescein sodium and acridine orange as the fluorescent contrast agents. RESULTS: The device was tested in vivo and demonstrated to be safe and function as designed. Real-time cellular visualization of ovary epithelium was demonstrated. CONCLUSION: The confocal microlaparoscope represents a new type of in vivo imaging device. With its ability to image cellular details in real time, it has the potential to aid in the early diagnosis of cancer. Initially the device may be used to locate unusual regions for guided biopsies. In the long term, the device may be able to supplant traditional biopsies and allow the surgeon to identify early-stage ovarian cancer.

Monte Carlo characterization of parallelized fluorescence confocal systems imaging in turbid media.

We characterize and compare the axial and lateral performance of fluorescence confocal systems imaging in turbid media. The aperture configurations studied are a single pinhole, a slit, a Nipkow disk, and a linear array of pinholes. Systems with parallelized apertures are used clinically because they enable high-speed and real-time imaging. Understanding how they perform in highly scattering tissue is important. A Monte Carlo model was developed to characterize parallelized system performance in a scattering media representative of human tissues. The results indicate that a slit aperture has degraded performance, both laterally and axially. In contrast, the analysis reveals that multipinhole apertures such as a Nipkow disk or a linear pinhole array can achieve performance nearly equivalent to a single pinhole aperture. The optimal aperture spacing for the multipinhole apertures was determined for a specific tissue model. In addition to comparing aperture configurations, the effects of tissue nonradiative absorption, scattering anisotropy, and fluorophore concentration on lateral and axial performance of confocal systems were studied.

Clinical confocal microlaparoscope for real-time in vivo optical biopsies.

Successful treatment of cancer is highly dependent on the stage at which it is diagnosed. Early diagnosis, when the disease is still localized at its origin, results in very high cure rates-even for cancers that typically have poor prognosis. Biopsies are often used for diagnosis of disease. However, because biopsies are destructive, only a limited number can be taken. This leads to reduced sensitivity for detection due to sampling error. A real-time fluorescence confocal microlaparoscope has been developed that provides instant in vivo cellular images, comparable to those provided by histology, through a nondestructive procedure. The device includes an integrated contrast agent delivery mechanism and a computerized depth scan system. The instrument uses a fiber bundle to relay the image plane of a slit-scan confocal microlaparoscope into tissue. It has a 3-mum lateral resolution and a 25-mum axial resolution. Initial in vivo clinical testing using the device to image human ovaries has been done in 21 patients. Results indicate that the device can successfully image organs in vivo without complications. Results with excised tissue demonstrate that the instrument can resolve sufficient cellular detail to visualize the cellular changes associated with the onset of cancer.