Imaging molecular expression on vascular endothelial cells by in vivo immunofluorescence microscopy

Massachusetts General Hospital, Boston 02114, USA.
Molecular Imaging (Impact Factor: 1.96). 01/2006; 5(1):31-40. DOI: 10.2310/7290.2006.00004
Source: PubMed


Molecular expression on the vascular endothelium is critical in regulating the interaction of circulating cells with the blood vessel wall. Leukocytes as well as circulating cancer cells gain entry into tissue by interacting with adhesion molecules on the endothelial cells (EC). Molecular targets on the EC are increasingly being explored for tissue-specific delivery of therapeutic and imaging agents. Here we use in vivo immunofluorescence microscopy to visualize the endothelial molecular expression in the vasculature of live animals. High-resolution images are obtained by optical sectioning through the intact skin using in vivo confocal and multiphoton microscopy after in situ labeling of EC surface markers with fluorescent antibodies. Other vascular beds such as the bone marrow and ocular blood vessels can be imaged with little or no tissue manipulation. Live imaging is particularly useful for following the dynamic expression of inducible molecules such as E-selectin during an inflammatory response.

Download full-text


Available from: Judith Runnels, Mar 13, 2014
  • Source
    • "In particular, near infrared fluorescence (NIR) imaging has found a greater potential for clinical application because of its long wavelength (650–900 nm), where light absorbance and scattering are significantly lower, and autofluorescence of normal tissues is also greatly reduced [25]. In view of this approach, various target-specific NIR conjugates have been reported for targeting tumor imaging, e.g., tumor integrin, αvβ3 [4], tumor growth factors or their receptors [18], [26]–[30], glycoprotein [31], or tumor specific protease [21]. Successful applications in various preclinical tumor models have been reported, though most of these studies were performed on surface tumors. "
    [Show abstract] [Hide abstract]
    ABSTRACT: It is recognized that cancer cells exhibit highly elevated glucose metabolism compared to non-tumor cells. We have applied in vivo optical imaging to study dynamic uptake of a near-infrared dye-labeled glucose analogue, 2-deoxyglucose (2-DG) by orthotopic glioma in a mouse model. The orthotopic glioma model was established by surgically implanting U87-luc glioma cells into the right caudal nuclear area of nude mice. Intracranial tumor growth was monitored longitudinally by bioluminescence imaging and MRI. When tumor size reached >4 mm diameter, dynamic fluorescence imaging was performed after an injection of the NIR labeled 2-DG, IRDye800CW 2-DG. Real-time whole body images acquired immediately after i.v. infusion clearly visualized the near-infrared dye circulating into various internal organs sequentially. Dynamic fluorescence imaging revealed significantly higher signal intensity in the tumor side of the brain than the contralateral normal brain 24 h after injection (tumor/normal ratio, TNR = 2.8+/-0.7). Even stronger contrast was achieved by removing the scalp (TNR = 3.7+/-1.1) and skull (TNR = 4.2+/-1.1) of the mice. In contrast, a control dye, IRDye800CW carboxylate, showed little difference (1.1+/-0.2). Ex vivo fluorescence imaging performed on ultrathin cryosections (20 microm) of tumor bearing whole brain revealed distinct tumor margins. Microscopic imaging identified cytoplasmic locations of the 2-DG dye in tumor cells. Our results suggest that the near-infrared dye labeled 2-DG may serve as a useful fluorescence imaging probe to noninvasively assess intracranial tumor burden in preclinical animal models.
    Preview · Article · Nov 2009 · PLoS ONE
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The development of endothelium-specific imaging agents capable of specific binding to human cells under the conditions of flow for the needs of regenerative medicine and cancer research. The goal of the study was testing the feasibility of optical imaging of human endothelial cells implanted in mice. Mouse model of adoptive human endothelial cell transfer was obtained by implanting cells in Matrigel matrix in subcutaneous space (Kang, Torres, Wald, Weissleder, and Bogdanov, Jr., Targeted imaging of human endothelial-specific marker in a model of adoptive cell transfer. Lab. Invest. 86: 599-609, 2006). Several endothelium-specific proteins were labeled with near-infrared fluorochrome (Cy5.5) and tested in vitro. Fluorescence imaging using anti-human CD31 antibody was performed in vivo. The obtained results were corroborated by using fluorescence microscopy of tissue sections. We determined that monoclonal anti-human CD31 antibodies labeled with Cy5.5 were efficiently binding to human endothelial cells and were not subject to rapid endocytosis. We further demonstrated that specific near-infrared optical imaging signal was present only in Matrigel implants seeded with human endothelium cells and was absent from control Matrigel implants. Histology showed staining of cells lining vessels and revealed the formation of branched networks of CD31-positive cells. Anti-human CD31 antibodies tagged with near-infrared fluorochromes can be used for detection of perfused blood vessels harboring human endothelial cells in animal models of adoptive transfer.
    Preview · Article · Jul 2007 · Pharmaceutical Research
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: We report an optical molecular imaging technique that exploits local administration of fluorophore-conjugated antibodies and confocal fluorescence microscopy to achieve high-contrast imaging of host cell populations in normal and tumor tissue in living mice. The method achieves micron-scale spatial resolution to depths greater than 100 mum. We illustrate the capabilities of this approach by imaging two dendritic cell populations in the skin and normal and tumor vasculature in vivo.
    Full-text · Article · Jan 2008 · Journal of Biomedical Optics
Show more