Targeted vertical cross-sectional imaging with handheld near-infrared dual axes confocal fluorescence endomicroscope.

We demonstrate vertical cross-sectional (XZ-plane) images of near-infrared (NIR) fluorescence with a handheld dual axes confocal endomicroscope that reveals specific binding of a Cy5.5-labeled peptide to pre-malignant colonic mucosa. This view is perpendicular to the tissue surface, and is similar to that used by pathologists. The scan head is 10 mm in outer diameter (OD), and integrates a one dimensional (1-D) microelectromechanical systems (MEMS) X-axis scanner and a bulky lead zirconate titanate (PZT) based Z-axis actuator. The microscope images in a raster-scanning pattern with a ±6 degrees (mechanical) scan angle at ~3 kHz in the X-axis (fast) and up to 10 Hz (0-400 µm) in the Z-axis (slow). Vertical cross-sectional fluorescence images are collected with a transverse and axial resolution of 4 and 5 µm, respectively, over a field-of-view of 800 µm (width) × 400 µm (depth). NIR vertical cross-sectional fluorescence images of fresh mouse colonic mucosa demonstrate histology-like imaging performance with this miniature instrument.

In vivo targeting of colonic dysplasia on fluorescence endoscopy with near-infrared octapeptide.

OBJECTIVE: To demonstrate a near-infrared (NIR) peptide that is highly specific for colonic adenomas on fluorescence endoscopy in vivo. DESIGN: A 3 mm diameter endoscope was adapted to deliver 671 nm illumination and collect NIR fluorescence (696-736 nm). Target (QPIHPNNM) and control (YTTNKH) peptides were labelled with Cy5.5, a NIR dye, and characterised by mass spectra. The peptides were topically administered separately (100 µM) through the endoscope's instrument channel into the distal colon of CPC;Apc mice, genetically engineered to spontaneously develop adenomas. After 5 min for incubation, the unbound peptides were rinsed off, and images were collected at a rate of 10 frames/s. Regions of interest were identified around the adenoma and adjacent normal-appearing mucosa on white light. Intensity measurements were made from these same regions on fluorescence, and the target-to-background ratio (TBR) was calculated. RESULTS: An image resolution of 9.8 µm and field of view of 3.6 mm was achieved at a distance of 2.5 mm between the distal end of the instrument and the tissue surface. On mass spectra, the experimental mass-to-charge ratio for the Cy5.5-labelled target and control peptides agreed with expected values. The NIR fluorescence images of adenomas revealed individual dysplastic crypts with distorted morphology. By comparison, only amorphous surface features could be visualised from reflected NIR light. The average TBR for adenomas was found to be 3.42 ± 1.30 and 1.88 ± 0.38 for the target and control peptides, respectively, p=0.007. CONCLUSION: A NIR peptide was shown to be highly specific for colonic adenomas on fluorescence endoscopy in vivo and to achieve sub-cellular resolution images.

Near-infrared-labeled peptide multimer functions as phage mimic for high affinity, specific targeting of colonic adenomas in vivo (with videos).

BACKGROUND: Fluorescent-labeled peptides are being developed to improve the endoscopic detection of colonic dysplasia. OBJECTIVE: To demonstrate a near-infrared peptide multimer that functions as a phage mimic for in vivo detection of colonic adenomas. DESIGN: A peptide multimer was synthesized by using trilysine as a dendritic wedge to mimic the presentation of peptides on phage, and all peptides, including the multimer, were fluorescent-labeled with Cy5.5. SETTING: Small-animal imaging facility. ANIMAL SUBJECTS: Genetically engineered CPC;Apc mice that spontaneously develop colonic adenomas. INTERVENTION: Near-infrared-labeled AKPGYLS peptide multimer was administered topically into the distal colons of the mice, and endoscopic images of adenomas were captured. Fluorescence intensities were quantified by target-to-background (T/B) ratios, and adenoma dimensions were measured with calipers after imaging. Validation of specific peptide binding was performed on cryosectioned specimens and cells by using confocal microscopy and flow cytometry. MAIN OUTCOME MEASUREMENTS: Fluorescence T/B ratios from colonic adenomas and adjacent normal-appearing mucosa. RESULTS: AKP-multimer, monomer, trilysine core, and Cy5.5 resulted in mean (± SD) T/B ratios of 3.85 ± 0.25, 2.21 ± 0.13, 1.56 ± 0.12, and 1.19 ± 0.11, respectively, P < .01 on in vivo imaging. Peptide multimer showed higher contrast and greater specificity for dysplastic crypts as compared with other probes. Peptide multimer demonstrated significantly greater binding to HT29 cells on flow cytometry and fluorescence microscopy in comparison to monomer and trilysine core. A binding affinity of 6.4 nm/L and time constant of 0.1136 minutes(-1) (8.8 minutes) was measured for multimer. LIMITATIONS: Only distal colonic adenomas were imaged. CONCLUSION: Peptide multimers combine strengths of multiple individual peptides to enhance binding interactions and demonstrate significantly higher specificity and affinity for tumor targets.

Identification of nasal eosinophils using two-photon excited fluorescence.

BACKGROUND: Eosinophils trigger symptoms in allergic rhinitis. New diagnostic methods for identifying nasal eosinophils based on autofluorescence of flavin adenine dinucleotide in eosinophil granules could offer rapid monitoring without fixation or staining. Two-photon excitation is a powerful method for detecting this intrinsic fluorescence. OBJECTIVES: To demonstrate the use of 2-photon excited fluorescence (TPEF) to detect eosinophils from nasal mucosa in a proof-of-concept study for a future miniature in vivo imaging instrument. METHODS: Thirty subjects with rhinitis were recruited. Results of our standard environmental panel were recorded. Fluorescence images were collected from nasal cytology smears with a 2-photon microscope. Cells were evaluated for intensity and size, and compared with Hansel stains. Correlation of cell count was made by linear regression, diagnostic performance was evaluated at various intensity thresholds, and correlation of nasal eosinophil count to allergic status was done through the Wilcoxon rank-sum test. RESULTS: The fluorescence intensity of eosinophils compared with epithelial cells was 13.8 ± 4.3 versus 3.7 ± 1.8 (P < .01), and the size was 27.0 ± 10.2 versus 392.0 ± 214.6 µm2 (P < .01), respectively. Using both fluorescence intensity and size, a total accuracy of 100% is achieved. Eosinophil count on TPEF correlates with Hansel stain, R(2) = 0.91. Nasal eosinophil count correlates with allergic status on both TPEF (P = .008) and Hansel stain images (P = .027). CONCLUSIONS: TPEF is a promising novel technique for identifying and quantifying nasal eosinophils on nasal cytology specimens without the need for fixation or staining. Future development of a rhinoscope-compatible 2-photon microscope could be used as a clinical adjunct for the diagnosis and management of rhinitis patients in vivo.

Longitudinal molecular imaging with single cell resolution of disseminated ovarian cancer in mice with a LED-based confocal microendoscope.

PURPOSE: We engineered a flexible fiber-optic microendoscope for longitudinal optical imaging studies in a mouse model of disseminated ovarian cancer. PROCEDURES: The microendoscope delivers 470 nm excitation light from a light-emitting diode through a fiber-optic bundle with outer diameter of 680 µm. Optics were optimized to maximize power and lateral resolution. We used this instrument to repetitively monitor intraperitoneal growth of HeyA8 ovarian cancer cells stably transduced with green fluorescent protein over 4 weeks. RESULTS: The microendoscope achieves 0.7 mW power and lateral resolution of 4 µm. Initial in vivo imaging studies visualized single cells and small clusters of malignant cells with subsequent studies showing tumor masses and vasculature. We also resolved single cells within intraperitoneal tumor masses. CONCLUSIONS: These studies establish microendoscope technology with single cell resolution for minimally-invasive, longitudinal imaging in living animals. This technology will advance future molecular imaging studies of ovarian cancer and other diseases.

Step-height measurement by means of a dual-frequency interferometric confocal microscope.

A novel instrument, the dual-frequency interferometric confocal microscope (DICM), which facilitates the measurement of step features, is investigated. It combines the advantages of the high resolution (subnanometer) of heterodyne interferometry and the relatively large measurement range (approximately 5 microm) of confocal microscopy. The axial response curves of the confocal microscopy system are compared in experiments in which microscopic objects with various numerical apertures and magnifications are used. The results prove that the variation in light intensity is enough to permit discrimination of different orders of interference fringes. The DICM has been successfully utilized to measure the step height of a standard mask, and the experimental results agree well with those measured by scanning probe microscopes. The results also show that the system has good repeatability, with a maximum deviation of 5 nm.

Nondestructive measurement of an optical fiber refractive-index profile by a transmitted-light differential interference contact microscope.

A novel transmitted-light differential interference contrast (DIC) system is used for nondestructive measurement of the refractive-index profile (RIP) of an optical fiber. By means of this system the phase of a measured light beam can be modulated with an analyzer, and the phase distribution of a fiber is obtained by calculation of the various interference patterns. The measurement theory and structure and some typical applications of this system are demonstrated. The results of measuring RIPs in graded-index fiber are presented. Both the experimental results and theoretical analysis show that the system takes the advantage of high index resolution and of sufficient measurement accuracy for measuring the refractive index of the optical fiber. The system has strong ability to overcome environmental disturbance because of its common-path design. Moreover, one can use the system to measure the RIP along the fiber axis and acquire an image of the three-dimensional RIP of the fiber.