Zhongyao Liu

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 endoscopes 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.

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.

Quantifying Human Eosinophils Using Three-Dimensional Volumetric Images Collected With Multiphoton Fluorescence Microscopy

Eosinophilic esophagitis (EoE) is a relatively new disease with ~10-fold increase in prevalence over the past 20 years,1,2 and has been found in ~6.5% of the population undergoing upper endoscopy.3 This disease has become one of the leading causes of dysphagia and food impaction in adults. For diagnosis, an endoscopy is performed where multiple biopsies are collected at random throughout the length of the esophagus, including the proximal and distal regions. On histopathology, the primary feature of EoE is infiltration of eosinophils into the mucosa. These mediators of inflammation may contribute to the development of structural abnormalities of the esophagus, including edema, rings, furrows, and strictures.4 Clinical symptoms do not improve with high-dose proton pump inhibitor therapy, and the pH in distal esophagus is usually normal.5 However, the diagnostic criteria for this disease appear to lack clarity. EoE may be difficult to distinguish from GERD,6 which is also associated with increased eosinophilia but to a lesser extent, and the two diseases may be present at the same time. Eosinophils can also trigger allergic symptoms in other parts of the gastrointestinal tract.7 The degree of mucosal hypereosinophilia that defines EoE is controversial. While a diagnostic criteria of ≥15 eosinophils per high-power-field (hpf) on histology has been proposed,5 values as high as 30 eosinophils per hpf have been used, and no single number is widely accepted.8–10 Diagnostic uncertainty for this disease may be attributed in part to its patchy and focal nature. In addition, there is little known about the density or spatial distribution of eosinophils throughout the mucosa. Marked variability has been found within and between biopsy specimens of individual patients, resulting in a low sensitivity for detection. Currently, biopsy specimens are sectioned along a plane whose orientation to the mucosal surface is unknown. A non-uniform distribution of infiltrating eosinophils within the mucosa could result in a highly variable cell count that depends on the angle of sectioning, resulting in an inaccurate result. A novel method that can quickly and reliably quantify the number of cells over a 3-dimensional (3D) volume could be used to overcome this tissue processing limitation. Human eosinophils contain granules that produce an intense autofluorescence in comparison to surrounding squamous epithelium.11–13 There is evidence to support flavin adenine dinucleotide (FAD) as the source of this endogenous fluorescence.14 FAD is a coenzyme in the mitochondrial electron transport chain that has a maximum absorption at 445 nm, resulting in a peak fluorescence emission of 525 nm.15 Multi-photon microscopy (MPM) is a powerful method for collecting fluorescence images from cells and tissues,16 and has been used to perform in vivo imaging of FAD from squamous epithelium in animals.17,18 MPM imaging has inherent 3D resolution, uses near-infrared excitation for superior tissue penetration, has lower photobleaching effects, and is capable of providing quantitative information.19 We have previously demonstrated the use of MPM imaging as a highly accurate method for identifying and quantifying human eosinophils from mucosal smears of patients with allergic rhinitis.20 In this study, we aim to demonstrate the use of MPM to detect eosinophils within squamous epithelium, characterize the distribution of eosinophils with depth below the mucosal surface, and quantify the number of eosinophils within a 3D volume.

Amplifying transmission and compact suspension for a low-profile, large-displacement piezoelectric actuator

A low-profile, piezoelectrically-driven microactuator is presented that achieves very large stroke lengths within size constraints suitable for certain endoscopic microscopy applications. The actuator utilizes a transmission consisting of lever arm and chevron-beam structures to amplify high-force, low-displacement motion of a ceramic lead-zirconate-titanate (PZT) brick into large displacement of a translational platform. For ±120 V input, a full range of 486 μm of motion is achieved, with natural frequency greater than 500 Hz. This corresponds to an anticipated In addition, the lateral translational platform is supported by a redesign of common folded silicon flexures to provide large transverse and vertical stiffness when the width of the actuator is limited.

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.

Wide-field near-infrared fluorescence endoscope for real-time in vivo imaging

A diode-pumped solid state laser is used to deliver excitation at λex = 671 nm. The beam is expanded by a pair of relay lenses (f1 = 30 and f2 = 50 mm) to 3 mm diameter, filling the aperture of a fluid light cable that is coupled to a Hopkins II rigid endoscope. Near-infrared fluorescence images are collected by the endoscope and transmitted by another set of relay lenses onto a CCD detector that has dimensions of 8.7x6.9 mm2 (1388x1040 pixels). A zoom lens system (F#1.6-16 aperture) with a tunable focal length (20-100 mm) magnifies the image to fill the dimensions of the CCD. A band pass filter allows fluorescence with spectral range λem = 696 to 736 nm to be collected. The system achieves a resolution of 9.8 μm and field-of-view of 3.6 mm at a distance of 2.5 mm between the distal end of the endoscope and the tissue. Images are collected at a rate of 10 frames per second. A filter wheel is incorporated into the handle of the instrument housing to rapidly switch between reflectance and fluorescence images. Cy5.5-labeled peptides were delivered through the 1 mm diameter instrument channel in the endoscope. Near-infrared fluorescence images demonstrated specific peptide binding to spontaneous adenomas that developed beginning at 2 months of age in a genetically-engineered mouse with mutation of one allele in the APC gene. This integrated methodology represents a powerful tool that can achieve real time detection of disease in the colon and other hollow organs.

Fluorescence Detection of Colonic Neoplasia Using a Novel Peptide

Background. Overexpression of AnnexinA2 (ANXA2) has been reported in several epithelial cancers, including colorectal cancers (CRC) (Cancer Letters, 2007). Increasing expression of gastrin gene, and hence progastrin (PG), has also been reported in CRCs. We recently discovered that ANXA2 serves as a non-conventional receptor for PG (Oncogene, 2007). We additionally reported that binding of PGwith extracellular, membrane associated, ANXA2 results in endocytosis and co-localization of PG/ANXA2 intracellularly (Gastro, 2010), and that ANXA2 is required for mediating mitogenic and anti-apoptotic effects of PG on colon cancer cells. ANXA2 is normally involved with intracellular trafficking. During the course of our studies we discovered that ANXA2 is also present on outer membranes of CRC cells, bound to a 29KDa transmembrane protein, and also released into the medium. To further understand physiological significance of these unexpected findings, we examined if ANXA2 is present in serum of patients positive for colorectal growth and if ANXA2 co-localizes with PG in tumor sections, in relation to disease status. Methods, Results and Conclusions. Secretion of ANXA2 (10-50ng/106 cells) was confirmed in conditioned medium (CM) of CRC cells using quantitative Western Blot assay using standard curve with rhANXA2; nontransformed cells were negative. Serum from CRC tumor bearing mice were positive for ANXA2, while control mice were negative. Next, serum samples were obtained from consented patients at time of endoscopy or as discarded samples from CRC patients on day of surgery, as per our IRB protocols. Serum from patients, negative for colonic growths (Normal, N) had low levels of ANXA2 (<1ng/ml), while serum from patients with hyperproliferative growths (Hp), Adenomas (Ad) and adenocarcinomas (AdCA) were on an average positive for 42, 80, and 490 ngs/ml, respectively, suggesting elevation of serum ANXA2 in relation to disease status. Paraffin blocks of Hp, Ads, AdCAs and adjoining N mucosa were obtained as discarded specimens from Department of Pathology and stained immunofluorescently for PG/ANXA2. Hp, Ads and AdCAs were increasingly positive for ANXA2 and PG expression compared to N specimens; ~ 10, 25 and 60 % of respective specimens were positive for intracellular co-localization of PG/ANXA2, suggesting presence of functional PG/ANXA2 axis, confirmed by progressive elevation of nuclear β-catenin and stem cell markers (DCAMKL+1, Lgr5) in relation to disease status. It remains to be determined if, 1) presence of co-localized PG/ANXA2 intracellularly represents a prognostic marker for predicting treatment/recurrence, and 2) if serum ANXA2 levels can be used as a non-invasive diagnostic marker for predicting presence of colorectal growths, stage of disease and/or relapse. Supported by NCI grants CA97959 and CA114264 to PS.

132 Improved Detection of Murine Colonic Dysplasia by Fluorescent-Labeled Multimer Peptide Using NIR Wide-Field Endoscopy

G A A b st ra ct s round-shaped ones in tumor glands (stromal Twist1+cells, A/R = 2.69, glandular Twist1+ cells, A/R= 1.67, A/R p<0.001). TWIST1 expression in the stromal compartment was associated with chromosomal rather than microsatellite instability (84% vs. 68%, respectively; p= 0.05), advanced TNM stage (91% in stage IV vs 72% in stage I/II, p=0.001), and worse disease specific survival (p=0.007). None of 15 adenoma expressed TWIST1 in the neoplastic or stromal compartment (p<0.001 vs CRC). We assessed the neoplastic origin of TWIST1+ stromal cells in humanCRC by combining fluorescent in situ hybridization and immunohistochemistry. Chromosome 7 trisomy was traced in the nuclei of TWIST1+ stromal cells in 60% of CRC specimens harboring this chromosomal abnormality in tumor cells. In an orthotopic In Vivo murine model of rectal cancer, the Twist1+ cells CT26 with a mensenchymal signature, grew round-shaped in syngenic Balb/c mice, but switched to fibroblast shape once invading the stroma. Conclusion: TWIST1 plays a crucial role in maintaining EMT in human CRC. TWIST1-dependent up-regulation of MMP-1 is a newly identified mechanism activating matrix-degradation and cell invasiveness in EMT. By using TWIST1 as marker of invasive EMT cells, we first demonstrated that a fraction of peri-tumoral stromal cells, disguised as fibroblasts, are actually neoplastic. Based on our findings, CRC stroma should no longer be considered a mere recipient of non-neoplastic elements but rather a true incubator for cancer EMT-cells. Targeting TWIST1+ stromal cells might open new scenarios for interfering with cancer invasiveness, and with the metastatic cascade of CRC.