In vivo fluorescence microscopic imaging of biological systems in human disease states and animal models is possible
with high optical resolution and mega pixel point-scanning performance using optimised off-the-shelf turn-key devices.
There are however various trade-offs between tissue access and instrument performance when miniaturising in vivo
A miniature confocal scanning technology that was developed for clinical human endoscopy has been configured into a
portable device for direct hand-held interrogation of living tissue in whole animal models (Optiscan FIVE-1 system).
Scanning probes of 6.3mm diameter with a distal tip diameter of 5.0mm were constructed either in a 150mm length for
accessible tissue, or a 300mm probe for laparoscopic interrogation of internal tissues in larger animal models. Both
devices collect fluorescence confocal images (excitation 488 nm; emission >505 or >550 nm) comprised of 1024 x 1204
sampling points/image frame, with lateral resolution 0.7um; axial resolution 7um; FOV 475 x 475um. The operator can
dynamically control imaging depth from the tissue surface to approx 250um in 4um steps via an internally integrated zaxis
actuator. Further miniaturisation is achieved using an imaging contact probe based on scanning the proximal end of
a high-density optical fibre bundle (~30,000 fibres) of <1mm diameter to transfer the confocal imaging plane to tissue in
intact small animal organs, albeit at lower resolution (30,000 sampling points/image). In rodent models, imaging was
performed using various fluorescent staining protocols including fluorescently labelled receptor ligands, labelled
antibodies, FITC-dextrans, vital dyes and labelled cells administered topically or intravenously. Abdominal organs of
large animals were accessed laparoscopically and contrasted using i.v. fluorescein-sodium. Articular cartilage of sheep
and pigs was fluorescently stained with calcein-AM or fluorescein. Surface and sub-surface cellular and sub-cellular
details could be readily visualised in vivo at high resolution. In rodent disease models, in vivo endomicroscopy with
appropriate fluorescent agents allowed examination of thrombosis formation, tumour microvasculature and liver
metastases, diagnosis and staging of ulcerative colitis, liver necrosis and glomerulonephritis. Miniaturised confocal
endomicroscopy allows rapid in vivo molecular and subsurface microscopy of normal and pathologic tissue at high
resolution in small and large whole animal models. Fluorescein endomicroscopy has recently been introduced into the
medical device market as a clinical imaging tool in GI endoscopy and is undergoing clinical evaluation in laparoscopic
surgery. This medical usage is encouraging in-situ endomicroscopy as an important pre-clinical research tool to observe
microscopic and molecular system biologic events in vivo in animal models for various human diseases.
Malignancies of the oral cavity are conventionally diagnosed by white light endoscopy, biopsy, and histopathology. However, it is often difficult to distinguish between benign and premalignant or early lesions. A laser confocal endomicroscope (LCE) offers noninvasive, in vivo surface and subsurface fluorescence imaging of tissue. We investigate the use of an LCE with a rigid probe for diagnostic imaging of the oral cavity. Fluorescein and 5-aminolevulinic acid (ALA) were used to carry out fluorescence imaging in vivo and on resected tissue samples of the oral cavity. In human subjects, ALA-induced protoporphyrin IX (PpIX) fluorescence images from the normal tongue were compared to images obtained from patients with squamous cell carcinoma (SCC) of the tongue. Using rat models, images from normal rat tongues were compared to those from carcinogen-induced models of SCC. Good structural images of the oral cavity were obtained using ALA and fluorescein, and morphological differences between normal and lesion tissue can be distinguished. The use of a pharmaceutical-grade solvent Pharmasolve® enhanced the subsurface depth from which images can be obtained. Our initial results show that laser confocal fluorescence endomicroscopy has potential as a noninvasive optical imaging method for the diagnosis of oral cavity malignancies.
Confocal endomicroscopy is a novel, noninvasive microscopic technique that enables surface and subsurface imaging of living tissues or cells in vivo. This study was to explore the possibility of utilizing a novel rigid confocal endomicroscope (RCE) system for detecting morphological changes in living normal and neoplastic human and murine tongue tissue in combination with different photosensitizers, i.e. hypericin and 5-aminolevulinic acid (ALA) induced endogenous protoporphyrin IX (PPIX) fluorescence. Subjects were topically or systemically applied photosensitizer to the oral mucosa, and then fluorescence confocal endomicroscopy was performed on the tongue using the RCE system with the laser excitation wavelength at 488 nm. The preliminary results showed that confocal fluorescence images of the tongue can be acquired in real-time with well-defined micro-morphological structures, and changes of tissue structures associated with cancer transformation can also be identified. This study suggests that photosensitizer-mediated confocal endomicroscopy have a significant potential for rapid, non-invasive detection of early oral cancers in vivo.
Conference Committee Involvement (4)
Endoscopic Microscopy V
25 January 2010 | San Francisco, California, United States
Endoscopic Microscopy III
20 January 2008 | San Jose, California, United States
Endoscopic Microscopy II
21 January 2007 | San Jose, California, United States
22 January 2006 | San Jose, California, United States