High-throughput platform for real-time monitoring of biological processes by multicolor single-molecule fluorescence.

Zero-mode waveguides provide a powerful technology for studying single-molecule real-time dynamics of biological systems at physiological ligand concentrations. We customized a commercial zero-mode waveguide-based DNA sequencer for use as a versatile instrument for single-molecule fluorescence detection and showed that the system provides long fluorophore lifetimes with good signal to noise and low spectral cross-talk. We then used a ribosomal translation assay to show real-time fluidic delivery during data acquisition, showing it is possible to follow the conformation and composition of thousands of single biomolecules simultaneously through four spectral channels. This instrument allows high-throughput multiplexed dynamics of single-molecule biological processes over long timescales. The instrumentation presented here has broad applications to single-molecule studies of biological systems and is easily accessible to the biophysical community.

Fibered confocal microscopy of bladder tumors: an ex vivo study.

BACKGROUND AND PURPOSE: The inadequacy of white-light cystoscopy to detect flat bladder tumors is well recognized. Great interest exists in developing other imaging technologies to augment or supplant conventional cystoscopy. Fibered confocal microscopy offers the promise of providing in vivo histopathologic information to help distinguish malignant from benign bladder lesions. We report the initial use of this technology to visualize tumors in the human bladder. MATERIALS AND METHODS: We performed ex vivo fibered confocal imaging of fresh radical cystectomy specimens using the Mauna Kea Technologies Cellvizio system. The findings were compared with results from standard histopathology. RESULTS: The bladders of four patients were imaged using the fibered confocal microscope. Normal and neoplastic urothelium manifested differences in cellular and vascular density. CONCLUSION: This study demonstrates the feasibility of using fibered confocal microscopy to detect histologic differences between normal and neoplastic urothelium, and establishes a foundation for the use of fiber-based confocal microscopy in clinical studies.

Three-dimensional in vivo imaging by a handheld dual-axes confocal microscope.

We present a handheld dual-axes confocal microscope that is based on a two-dimensional microelectromechanical systems (MEMS) scanner. It performs reflectance and fluorescence imaging at 488 nm wavelength, with three-dimensional imaging capability. The fully packaged microscope has a diameter of 10 mm and acquires images at 4 Hz frame rate with a maximum field of view of 400 microm x 260 microm. The transverse and axial resolutions of the handheld probe are 1.7 microm and 5.8 microm, respectively. Capability to perform real time small animal imaging is demonstrated in vivo in transgenic mice.

Effects of axial resolution improvement on optical coherence tomography (OCT) imaging of gastrointestinal tissues.

Optical coherence tomography (OCT) is an emerging medical imaging technology which generates high resolution, cross-sectional images in situ, without the need for excisional biopsy. Previous clinical studies using endoscopic OCT with standard 10-15 microm axial resolution have demonstrated its capability in diagnosing Barrett's esophagus (BE) and high-grade dysplasia (HGD). Quantitative OCT image analysis has shown promise for detecting HGD in Barrett's esophagus patients. We recently developed an endoscopic OCT system with an improved axial resolution of approximately 5 microm. The goal in this manuscript is to compare standard resolution OCT and ultrahigh resolution OCT (UHR-OCT) for image quality and computer-aided detection using normal and Barrett's esophagus. OCT images of gastrointestinal (GI) tissues were obtained using UHR-OCT (5.5 microm) and standard resolution OCT (13 microm). Image quality including the speckle size and sharpness was compared. Texture features of endoscopic OCT images from normal and Barrett's esophagus were extracted using quantitative metrics including spatial frequency analysis and statistical texture analysis. These features were analyzed using principal component analysis (PCA) to reduce the vector dimension and increase the discriminative power, followed by linear discrimination analysis (LDA). UHR-OCT images of GI tissues improved visualization of fine architectural features compared to standard resolution OCT. In addition, the quantitative image feature analysis showed enhanced discrimination of normal and Barrett's esophagus with UHR-OCT. The ability of UHR-OCT to resolve tissue morphology at improved resolution enables visualization of subtle features in OCT images, which may be useful in disease diagnosis. Enhanced classification of image features using UHR-OCT promises to help in the computer-aided diagnosis of GI diseases.

Detection of colonic dysplasia in vivo using a targeted heptapeptide and confocal microendoscopy.

A combination of targeted probes and new imaging technologies provides a powerful set of tools with the potential to improve the early detection of cancer. To develop a probe for detecting colon cancer, we screened phage display peptide libraries against fresh human colonic adenomas for high-affinity ligands with preferential binding to premalignant tissue. We identified a specific heptapeptide sequence, VRPMPLQ, which we synthesized, conjugated with fluorescein and tested in patients undergoing colonoscopy. We imaged topically administered peptide using a fluorescence confocal microendoscope delivered through the instrument channel of a standard colonoscope. In vivo images were acquired at 12 frames per second with 50-microm working distance and 2.5-microm (transverse) and 20-microm (axial) resolution. The fluorescein-conjugated peptide bound more strongly to dysplastic colonocytes than to adjacent normal cells with 81% sensitivity and 82% specificity. This methodology represents a promising diagnostic imaging approach for the early detection of colorectal cancer and potentially of other epithelial malignancies.

In vivo biomarkers for targeting colorectal neoplasms.

Colorectal carcinoma continues to be a leading cause of cancer morbidity and mortality despite widespread adoption of screening methods. Targeted detection and therapy using recent advances in our knowledge of in vivo cancer biomarkers promise to significantly improve methods for early detection, risk stratification, and therapeutic intervention. The behavior of molecular targets in transformed tissues is being comprehensively assessed using new techniques of gene expression profiling and high throughput analyses. The identification of promising targets is stimulating the development of novel molecular probes, including significant progress in the field of activatable and peptide probes. These probes are being evaluated in small animal models of colorectal neoplasia and recently in the clinic. Furthermore, innovations in optical imaging instrumentation are resulting in the scaling down of size for endoscope compatibility. Advances in target identification, probe development, and novel instruments are progressing rapidly, and the integration of these technologies has a promising future in molecular medicine.

Functional imaging of colonic mucosa with a fibered confocal microscope for real-time in vivo pathology.

BACKGROUND & AIMS: Histologic interpretation of disease currently is performed with static images of excised tissues, and is limited by processing artifact, sampling error, and interpretive variability. The aim of this study was to show the use of functional optical imaging of viable mucosa for quantitative evaluation of colonic neoplasia in real time. METHODS: Fluorescein (5 mg/mL) was administered topically in 54 human subjects undergoing screening colonoscopy. Fluorescence images were collected with 488-nm excitation at 12 frames/s with the confocal microendoscopy system. Movement of fluorescein in the transient period (<5 s) and the lamina propria:crypt contrast ratio in the steady-state phase (>5 s) were quantified. RESULTS: Normal mucosa showed circular crypts with uniform size, hyperplasia revealed proliferative glands with serrated lumens, and adenomas displayed distorted elongated glands. For t less than 5 seconds, fluorescein passed through normal epithelium with a peak speed of 1.14 +/- 0.09 microm/s at t = 0.5 seconds, and accumulated into lamina propria as points of fluorescence that moved through the interglandular space with an average speed of 41.7 +/- 3.4 microm/s. Passage of fluorescein through adenomatous mucosa was delayed substantially. For t greater than 5 seconds, high sensitivity, specificity, and accuracy was achieved using a discriminant function to evaluate the contrast ratio to distinguish normal from lesional mucosa (91%, 87%, and 89%, respectively; P < .001), hyperplasia from adenoma (97%, 96%, and 96%, respectively; P < .001), and tubular from villous adenoma (100%, 92%, and 93%, respectively; P < .001). CONCLUSIONS: Confocal imaging can be performed in vivo to assess the functional behavior of tissue in real time for providing pathologic interpretation, representing a new method for histologic evaluation.

Benign and malignant lesions in the human breast depicted with ultrahigh resolution and three-dimensional optical coherence tomography.

Institutional review board approval at the participating institutions was obtained. Informed consent was waived for this HIPAA-compliant study. The study purpose was to establish the correspondence of optical coherence tomographic (OCT) image findings with histopathologic findings to understand which features characteristic of breast lesions can be visualized with OCT. Imaging was performed in 119 specimens from 35 women aged 29-81 years with 3.5-microm axial resolution and 6-microm transverse resolution at 1.1-microm wavelength on freshly excised specimens of human breast tissue. Three-dimensional imaging was performed in 43 specimens from 23 patients. Microstructure of normal breast parenchyma, including glands, lobules, and lactiferous ducts, and stromal changes associated with infiltrating cancer were visible. Fibrocystic changes and benign fibroadenomas were identified. Imaging of ductal carcinoma in situ, infiltrating cancer, and microcalcifications correlated with corresponding histopathologic findings. OCT is potentially useful for visualization of breast lesions at a resolution greater than that of currently available clinical imaging methods.

Ultrahigh-resolution and 3-dimensional optical coherence tomography ex vivo imaging of the large and small intestines.

BACKGROUND: Ultrahigh-resolution optical coherence tomography (OCT) has an axial resolution of <5 microm, 2 to 3 times finer than standard OCT. This study investigates ultrahigh-resolution and three-dimensional OCT for ex vivo imaging of the large and small intestines and correlates images with histology. METHODS: Ultrahigh-resolution OCT imaging was performed on fresh surgical specimens from the large and small intestines in the pathology laboratory, and images were correlated with histology. OCT was performed at 1.3-microm wavelength with 4.5-microm axial x 11-microm transverse resolution and at 1.1-microm wavelength with 3.5-microm axial x 6-microm transverse resolution. Three-dimensional OCT also was investigated. RESULTS: Normal and pathologic areas from 23 surgical specimens of the large and small intestines were imaged. Ultrahigh-resolution OCT distinguished the epithelial layer of the mucosa and visualized individual villi, glands, and crypts. Finer transverse resolutions improved visualization of features, e.g., the epithelium, but reduced the depth of field. Architectural distortion of glands from inflammatory and neoplastic processes was observed. Three-dimensional rendering enabled visualization of surface pit pattern and mucosal folds as well as subsurface crypt microstructure. CONCLUSIONS: This study evaluates new OCT technology and can provide a baseline for interpreting future ultrahigh-resolution endoscopic OCT studies.

Effect of tissue preservation on imaging using ultrahigh resolution optical coherence tomography.

Ultrahigh resolution optical coherence tomography (OCT) is an emerging imaging modality that enables noninvasive imaging of tissue with 1- to 3-microm resolutions. Initial OCT studies have typically been performed using harvested tissue specimens (ex vivo). No reports have investigated postexcision tissue degradation on OCT image quality. We investigate the effects of formalin fixation and commonly used cell culture media on tissue optical scattering characteristics in OCT images at different times postexcision compared to in vivo conditions. OCT imaging at 800-nm wavelength with 1.5-mum axial resolution is used to image the hamster cheek pouch in vivo, followed by excision and imaging during preservation in phosphate-buffered saline (PBS), Dulbecco's Modified Eagle's Media (DMEM), and 10% neutral-buffered formalin. Imaging is performed in vivo and at sequential time points postexcision from 15 min to 10 to 18 h. Formalin fixation results in increases in scattering intensity from the muscle layers, as well as shrinkage of the epithelium, muscle, and connective tissue of approximately 50%. PBS preservation shows loss of optical contrast within two hours, occurring predominantly in deep muscle and connective tissue. DMEM maintains tissue structure and optical scattering characteristics close to in vivo conditions up to 4 to 6 h after excision and best preserved tissue optical properties when compared to in vivo imaging.

High-resolution imaging of the thyroid gland using optical coherence tomography.

BACKGROUND: Current diagnostic imaging modalities of the thyroid gland cannot reliably distinguish benign from malignant lesions, primarily because of their inability to visualize microscopic structure. A high-resolution imaging technique capable of examining thyroid tissue architectural morphology in real time is needed. Optical coherence tomography (OCT) has been shown to achieve high resolutions approaching the cellular range (1-15 microm). The feasibility of optical coherence tomography for imaging thyroid tissue was explored ex vivo on the human thyroid gland. METHODS: High-resolution OCT was performed in real time at 2 to 4 frames per second on three postmortem and 15 surgically excised thyroid glands containing normal, hyperplastic, and neoplastic tissue. OCT images acquired were compared with those obtained using standard histopathologic methods. RESULTS: The microstructure of the normal thyroid gland, including colloid-filled follicles as small as 15 microm and their supporting stroma, was clearly identified. OCT images of degenerative, hyperplastic, adenomatous, and malignant change within the thyroid gland were shown to correlate well with corresponding histopathologic findings. CONCLUSIONS: The ability of OCT to image thyroid tissue microarchitecture makes it a potentially powerful technology that can be used to assess the thyroid gland at a resolution greater than currently available clinical imaging modalities.

Optical coherence tomography using a continuous-wave, high-power, Raman continuum light source.

High performance, short coherence length light sources with broad bandwidths and high output powers are critical for high-speed, ultrahigh resolution OCT imaging. We demonstrate a new, high performance light source for ultrahigh resolution OCT. Bandwidths of 140 nm at 1300 nm center wavelength with high output powers of 330 mW are generated by an all-fiber Raman light source based on a continuous-wave Yb-fiber laser-pumped microstructure fiber. The light source is compact, robust, turnkey and requires no optical alignment. In vivo, ultrahigh resolution, high-speed, time domain OCT imaging with <5 microm axial resolution is demonstrated.

High-speed path-length scanning with a multiple-pass cavity delay line.

Techniques for high-speed delay scanning are important for low-coherence interferometry, optical coherence tomography, pump probe measurements, and other applications. We demonstrate a novel scanning delay line using a multiple-pass cavity. Differential delays are accumulated with each pass so that millimeter delays can be generated with tens of micrometer mirror displacements. With special design criteria, misalignment sensitivity can be dramatically reduced. The system is demonstrated to scan 6 m/s at 2-kHz repetition rates. Real-time optical coherence tomography imaging with 500 pixel images at four frames/s is performed. Using a Cr:forsterite laser source, we obtained axial image resolutions of 6 microm with 92-dB sensitivity.