Gaoming Li

In vivo photoacoustic tomography of EGFR overexpressed in hepatocellular carcinoma mouse xenograft.

EGFR is a promising cell surface target for in vivo imaging that is highly overexpressed in hepatocellular carcinoma (HCC), a common cancer worldwide. Peptides penetrate easily into tumors for deep imaging, and clear rapidly from the circulation to minimize background. We aim to demonstrate use of an EGFR specific peptide to detect HCC xenograft tumors in mice with photoacoustic imaging. Nude mice implanted with human HCC cells that overexpress EGFR were injected intravenously with Cy5.5-labeled EGFR and scrambled control peptides respectively. Photoacoustic images collected from 0 to 24 h. Photoacoustic signal peaked in tumors at 3 h post-injection. Images from 0 to 1.8 cm beneath the skin revealed increased target-to-background (T/B) ratio from tumors. The T/B ratio was significantly greater for the EGFR versus control peptide. Clearance of signal was observed by ∼24 h. EGFR overexpression was validated with immunofluorescence and immunohistochemistry. A peptide specific for EGFR delivered systemically can detect HCC xenograft tumors in vivo with photoacoustic imaging.

Multiplexed Targeting of Barrett's Neoplasia with a Heterobivalent Ligand: Imaging Study on Mouse Xenograft in Vivo and Human Specimens ex Vivo.

Esophageal adenocarcinoma (EAC) is a molecularly heterogeneous disease that is rising rapidly in incidence and has poor prognosis. We developed a heterobivalent peptide to target detection of early Barretts neoplasia by combining monomer heptapeptides specific for either EGFR or ErbB2 in a heterodimer configuration. The structure of a triethylene glycol linker was optimized to maximize binding interactions to the surface receptors on cells. The Cy5.5-labeled heterodimer QRH*-KSP*-E3-Cy5.5 demonstrated specific binding to each target and showed 3-fold greater fluorescence intensity and 2-fold higher affinity compared with those of either monomer alone. Peak uptake in xenograft tumors was observed at 2 h postinjection with systemic clearance by ∼24 h in vivo. Furthermore, ligand binding was evaluated on human esophageal specimens ex vivo, and 88% sensitivity and 87% specificity were found for the detection of either high-grade dysplasia (HGD) or EAC. This peptide heterodimer shows promise for targeted detection of early Barretts neoplasia in clinical study.

An Electrostatic MEMS Translational Scanner with Large Out-of-Plane Stroke for Remote Axial-Scanning in Multi-Photon Microscopy

We present an electrostatic microelectromechanical systems (MEMS) resonant scanner with large out-of-plane translational stroke for fast axial-scanning in a multi-photon microscope system for real-time vertical cross-sectional imaging. The scanner has a compact footprint with dimensions of 2.1 mm × 2.1 mm × 0.44 mm, and employs a novel lever-based compliant mechanism to enable large vertical displacements of a reflective mirror with slight tilt angles. Test results show that by using parametrical resonance, the scanner can provide a fast out-of-plane translational motion with ≥400 μm displacement and ≤0.14° tilt angle over a wide frequency range of ~390 Hz at ambient pressure. By employing this MEMS translational scanner and a biaxial MEMS mirror for lateral scanning, vertical cross-sectional imaging with a beam axial-scanning range of 200 μm and a frame rate of ~5–10 Hz is enabled in a remote scan multi-photon fluorescence imaging system.

In vivo near-infrared imaging of ErbB2 expressing breast tumors with dual-axes confocal endomicroscopy using a targeted peptide

ErbB2 expression in early breast cancer can predict tumor aggressiveness and clinical outcomes in large patient populations. Accurate assessment with physical biopsy and conventional pathology can be limited by tumor heterogeneity. We aim to demonstrate real-time optical sectioning using a near-infrared labeled ErbB2 peptide that generates tumor-specific contrast in human xenograft breast tumors in vivo. We used IRDye800CW as the fluorophore, validated performance characteristics for specific peptide binding to cells in vitro, and investigated peak peptide uptake in tumors using photoacoustic tomography. We performed real-time optical imaging using a handheld dual-axes confocal fluorescence endomicroscope that collects light off-axis to reduce tissue scattering for greater imaging depths. Optical sections in either the vertical or horizontal plane were collected with sub-cellular resolution. Also, we found significantly greater peptide binding to pre-clinical xenograft breast cancer in vivo and to human specimens of invasive ductal carcinoma that express ErbB2 ex vivo. We used a scrambled peptide for control. Peptide biodistribution showed high tumor uptake by comparison with other organs to support safety. This novel integrated imaging strategy is promising for visualizing ErbB2 expression in breast tumors and serve as an adjunct during surgery to improve diagnostic accuracy, identify tumor margins, and stage early cancers.

Targeted sections in either XY or XZ plane with dual-axes confocal endomicroscope (Conference Presentation)

We demonstrate a dual axes confocal architecture, which can be used to collect horizontal(XY-plane) or vertical cross-sectional(XZ-plane) images for tissue. This scanner head is 5.5mm in outer diameter(OD), and integrates a 3D MEMS scanner with a compact chip size of 3.2×2.9mm2. To realize the miniaturization, there are some obstacles of the small size of 3D MEMS scanner, MEMS wire bundle, the air pressure effect for MEMS motion, the processing of parabolic mirror, and optical alignment to come over. In our probe, separation mechanical structure for optical alignment was adopted and a step shape MEMS holder was designed to deal with the difficult of MEMS wire bundle. Peptides have been demonstrated tremendous potential for in vivo use to detect colonic dysplasia. This class of in vivo molecular probe can be labeled with near-infrared (NIR) dyes for visualizing the full depth of the epithelium in small animals. To confirm our probe performance, we take use of USAF 1951 resolution target to test its lateral and axial resolution. It has lateral and axial resolution of 2.49um and 4.98um, respectively. When we collect the fluorescence imaging of colon, it shows that the field of view are 1000um×1000um (horizontal) and 1000um×430um (vertical). The horizontal and vertical cross-sectional images of fresh mouse colonic mucosa demonstrate imaging performance with this miniature instrument.

MEMS-based side-view endomicroscope for in vivo small animal imaging(Conference Presentation)

Tremendous advances have been made in technological development of whole body molecular imaging, including PET, SPECT, MRI, bioluminescence, and ultrasound. However, a great unmet need still exists for high resolution imaging of biological processes that occur in the epithelium, the thin layer of tissue where many important cancers originate. Confocal endomicroscopes designed with a fiber bundle are used in the clinic, but they can only create images in the horizontal plane. Imaging in the plane perpendicular to the tissue surface is also important because epithelial cells differentiate in the vertical direction. Subtle changes in normal tissue differentiation patterns can reveal the early expression of cancer biomarkers. In this work, we present a side-viewing confocal endomicroscope that can collect images in either horizontal or oblique plane using an integrated monolithic electrostatic 3D MEMS scanner. The endomicroscope can perform sub-cellular resolution imaging in both the horizontal plane and the oblique plane with FOVs of 500 x 700 µm2 and 500 x 200 µm2. A side-viewing probe will allow optimal contact between the imaging window and the luminal wall, which makes it easy to navigate in the hollow organ. The endomicroscope is packaged into a stainless steel tube with outer diameter of 4.2 mm, which can be used for both small animal and human GI tract imaging. We demonstrate in vivo imaging of colonic dysplasia in mice, showing the endomicroscope can potentially be used for early detection and staging of colon cancer.

A MEMS scanner with lateral and axial scanning capability for dual axes confocal endomicroscopic in-vivo imaging (Conference Presentation)

Aimed to build a dual-axes confocal endomicroscope with an outer diameter of 5.5mm for in-vivo imaging applications, an electrostatic MEMS scanner has been developed to enable two dimensional (2D) light scanning in either horizontal plane or vertical cross-sectional plane. The device has a compact structure design to match the dual axes confocal architecture in the probe without blocking the collimated light beams of excitation and collection, and a cutting-free silicon-on-insulator(SOI) micromachining process is used for the fabrication. A novel lever-based gimbal-like mechanism is employed to enable three degrees of freedom motions for lateral and axial light scanning, and its geometry is optimized for achieving large deflection with high scanning speed. Based on parametric excitation, the device can work in resonant modes. Testing result shows that, up to ±27° optical deflection angle for inner axis torsion motion with a frequency of ~4.9kHz, up to ±28.5° optical deflection angle for outer axis torsion motion with a frequency of~0.65kHz and ~360μm stroke for out-of-plane translation motion with a frequency of ~0.53kHz are achieved with <60V driving voltage. Based on these results, 2D imaging with frame rate of 5~10Hz and large field of view (1000μm x 1000μm in horizontal plane and 1000μm x 400μm in vertical plane) can be enabled by this scanner.