Oral lesions are conventionally diagnosed using white light endoscopy and histopathology. This can pose a challenge because the lesions may be difficult to visualise under white light illumination. Confocal laser endomicroscopy can be used for confocal fluorescence imaging of surface and subsurface cellular and tissue structures. To move toward real-time "virtual" biopsy of oral lesions, we interfaced an embedded computing system to a confocal laser endomicroscope to achieve a prototype three-dimensional (3-D) fluorescence imaging system. A field-programmable gated array computing platform was programmed to enable synchronization of cross-sectional image grabbing and Z-depth scanning, automate the acquisition of confocal image stacks and perform volume rendering. Fluorescence imaging of the human and murine oral cavities was carried out using the fluorescent dyes fluorescein sodium and hypericin. Volume rendering of cellular and tissue structures from the oral cavity demonstrate the potential of the system for 3-D fluorescence visualization of the oral cavity in real-time. We aim toward achieving a real-time virtual biopsy technique that can complement current diagnostic techniques and aid in targeted biopsy for better clinical outcomes.
We report novel bioconjugated nanosensitizers as optical and therapeutic probes for the detection, monitoring and
treatment of cancer. These nanosensitisers, consisting of hypericin loaded bioconjugated gold nanoparticles, can act as
tumor cell specific therapeutic photosensitizers for photodynamic therapy coupled with additional photothermal effects
rendered by plasmonic heating effects of gold nanoparticles. In addition to the therapeutic effects, the nanosensitizer can
be developed as optical probes for state-of-the-art multi-modality in-vivo optical imaging technology such as in-vivo 3D
confocal fluorescence endomicroscopic imaging, optical coherence tomography (OCT) with improved optical contrast
using nano-gold and Surface Enhanced Raman Scattering (SERS) based imaging and bio-sensing. These techniques can
be used in tandem or independently as in-vivo optical biopsy techniques to specifically detect and monitor specific
cancer cells in-vivo. Such novel nanosensitizer based optical biopsy imaging technique has the potential to provide an
alternative to tissue biopsy and will enable clinicians to make real-time diagnosis, determine surgical margins during
operative procedures and perform targeted treatment of cancers.
Oral lesions are conventionally diagnosed using white light endoscopy and histopathology of biopsy samples. Oral
lesions are often flat and difficult to visualize under white light illumination. Moreover, histopathology is timeconsuming
and there is a need to develop minimally invasive optical biopsy techniques to complement current
techniques. Confocal laser endomicroscopy holds promise for virtual biopsy in disease diagnosis. This technique enables
fluorescence imaging of tissue structures at microscopic resolution. We have developed a prototype real-time 3-
dimensional (3D) imaging system using a laser endomicroscope interfaced with embedded computing. A Field-
Programmable Gate Array computing platform has been programmed to synchronize cross-sectional image grabbing and
Z-depth scanning, as well as automate acquisition of confocal image stacks. A PC was used for real-time volume
rendering of the confocal image stacks. We conducted pre-clinical and pilot clinical studies to image the murine and
human oral cavity. High quality volume renderings of the confocal image stacks were generated using 3D texture slicing.
Tissue morphology and 3D structures could be visualized. The results demonstrate the potential of the system for
diagnostic imaging of the oral cavity. This paves the way toward real-time virtual biopsy of oral lesions, with the aim to
achieve same-day diagnosis in a clinical setting.
Photodynamic therapy (PDT) is an alternative cancer treatment modality that offers localized treatment using a photosensitizer and light. However, tumor angiogenesis is a major concern following PDT-induced hypoxia as it promotes recurrence. Bevacizumab is a monoclonal antibody that targets vascular endothelial growth factor (VEGF), thus preventing angiogenesis. The combination of PDT with antiangiogenic agents such as bevacizumab has shown promise in preclinical studies. We use confocal endomicroscopy to study the antiangiogenic effects of PDT in combination with bevacizumab. This technique offers in vivo surface and subsurface fluorescence imaging of tissue. Mice bearing xenograft bladder carcinoma tumors were treated with PDT, bevacizumab, or PDT and bevacizumab combination therapy. In tumor regression experiments, combination therapy treated tumors show the most regression. Confocal fluorescence endomicroscopy enables visualization of tumor blood vessels following treatment. Combination therapy treated tumors show the most posttreatment damage with reduced cross-sectional area of vessels. Immunohistochemistry and immunofluorescence studies show that VEGF expression is significantly downregulated in the tumors treated by combination therapy. Overall, combining PDT and bevacizumab is a promising cancer treatment approach. We also demonstrate that confocal endomicroscopy is useful for visualization of vasculature and evaluation of angiogenic response following therapeutic intervention.
Oral cancers are currently diagnosed using white light endoscopy and histopathology. However, oral tumours are mostly
superficial and can be difficult to visualise. Here we present the use of hypericin with fluorescence endoscopy and laser
confocal fluorescence endomicroscopy interfaced with embedded computing for the diagnosis of oral cancers.
Fluorescence imaging of oral lesions was carried out in the clinic using a fluorescence endoscope. The images were
analyzed to extract the red to blue (R/B) ratios to discriminate between tissue types. The results showed that the R/B
ratio is a good image parameter to discriminate between normal, hyperplastic and malignant oral tissue. We are also
developing an embedded, real-time computing system interfaced to a fluorescence endomicroscope for 3D visualization
of tumors, where synchronization of cross-sectional image grabbing and Z-depth scanning is realized through
programming a Field-Programmable Gate Array. In addition to the programming task, a proprietary control circuit has
been developed for the automated 3D reconstruction of fluorescence sections; and preliminary results from fluorescent
samples have demonstrated the potential of this system for real-time in vivo 3D visualization of tumours. This will
ultimately enable same-day clinical diagnosis to be achieved and further enhance the clinical usefulness of fluorescence
diagnostic imaging.
Photodynamic therapy (PDT) is a standard treatment for various malignant and non-malignant conditions. Though
therapeutic responses are encouraging, recurrences have been noted, as one of the limitations of PDT is treatment-induced
hypoxia that triggers angiogenesis. The present study evaluates the use of angiogenic inhibitors Avastin, that
targets vascular endothelial growth factor (VEGF) and Erbitux that targets epidermal growth factor receptor (EGFR)
with PDT in an in vivo bladder carcinoma xenograft. Tumor bearing mice were assigned to 6 different categories:
control, PDT only, Avastin + Erbitux, PDT + Avastin, PDT + Erbitux and PDT + Avastin and Erbitux. Treated and
control tumors were monitored for recurrence for up to 90 days. VEGF and EGFR expression was detected in the tumor
tissue. Migratory assay was performed to establish the inhibitory effect of the angiogenesis agents. Using confocal laser
endomicroscopy, the tumor microvasculature was assessed. Tumors treated with the combination therapy of PDT +
inhibitors showed significantly greater response compared to control and PDT only treated group. Combination therapy
treated tumors also showed the most post-treatment damage with reduced tumor vasculature. These results demonstrate
that the combination of PDT with inhibitors that target different angiogenesis pathways can improve tumor control.
Photodynamic diagnosis (PDD) exploits the photoactive nature of certain compounds, namely photosensitizers, in order
to enhance the visual demarcation between normal and neoplastic tissue. Hypericin is one such potent photosensitizer
that preferentially accumulate in neoplastic tissue, and fluoresce in the visible spectrum when illuminated with light of an
appropriate wavelength. In our study, we investigated the role of E-cadherin in the selective permeation of hypericin in
bladder cancer tissues. Clinical studies were done on a series of 43 histologically graded bladder cancer biopsy
specimens, obtained from 28 patients who received intravesical instillations with 8μM hypericin solution for at least 2
hours. Immunohistochemical staining was used to assess the expression of E-cadherin, in the cryosectioned tissues.
Hypericin uptake was examined by fluorescence microscopy. Immunohistochemical staining showed a clear expression
of E-cadherin along the urothelial lining of the normal and pre-malignant tissues. Partial expression of these cell
adhesion molecules were still observed in malignant tissues, however there was a loss of expression to variable extends
along the urothelium. Thus, loss of intercellular adhesion can be associated with enhanced hypericin permeation through
paracellular diffusion.
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