Label-Free Optical Spectroscopy for Early Detection of Oral Cancer.

Oral cancer is the 16th most common cancer worldwide. It commonly arises from painless white or red plaques within the oral cavity. Clinical outcome is highly related to the stage when diagnosed. However, early diagnosis is complex owing to the impracticality of biopsying every potentially premalignant intraoral lesion. Therefore, there is a need to develop a non-invasive cost-effective diagnostic technique to differentiate non-malignant and early-stage malignant lesions. Optical spectroscopy may provide an appropriate solution to facilitate early detection of these lesions. It has many advantages over traditional approaches including cost, speed, objectivity, sensitivity, painlessness, and ease-of use in clinical setting for real-time diagnosis. This review consists of a comprehensive overview of optical spectroscopy for oral cancer diagnosis, epidemiology, and recent improvements in this field for diagnostic purposes. It summarizes major developments in label-free optical spectroscopy, including Raman, fluorescence, and diffuse reflectance spectroscopy during recent years. Among the wide range of optical techniques available, we chose these three for this review because they have the ability to provide biochemical information and show great potential for real-time deep-tissue point-based in vivo analysis. This review also highlights the importance of saliva-based potential biomarkers for non-invasive early-stage diagnosis. It concludes with the discussion on the scope of development and future demands from a clinical point of view.

Sub-micron free-standing metal slabs with dielectric nano-voids of arbitrary shapes embedded beneath atomically-flat surface.

Thin metal slabs with plasmonic nano-voids buried within the skin depth (< 25 nm) of surface plasmon polaritons have been of theoretical as well as technical interests for many years due to its unique optical properties such as sharp absorbance dips and anti-crossing plasmonic dispersion characteristics. Unfortunately, such interesting plasmonic properties have not been experimentally reproduced, especially in the UV-Vis regime, owing to the involuntary surface roughness occurred in systems fabricated using conventional techniques. Here, we describe a versatile cryogenic-stripping approach for encapsulating a monolayer of nano-voids of virtually any arbitrary shapes underneath an atomically-smooth (δ < 0.55 nm) surface of a free-standing metal slab. By artificially varying the topography of the capping metal surface from ultra-smooth to moderately-rough, we show structural symmetricity in a nano-void-metal system can render the overall plasmonic responses becoming profoundly influenced by the surface smoothness. The current fabrication technique is thus of primary importance to the preparation of any kind of smooth nano-void-passivated metal slabs.

Hyper-spectral confocal nano-imaging with a 2D super-lens.

Achieving sub-100 nm resolution over a broad visible bandwidth has long been an elusive goal in the nano-imaging of cell-surface interfaces. While metamaterial super-lenses and near-field optics have been previously demonstrated, these techniques can operate only at one wavelength, and do not provide accesses to the cell-surface interfaces. Here, we investigate a broadband 2D lens comprised of an oblate spheroidal dielectric cavity embedded just beneath a planar metal surface. The lens operates by adiabatically focusing asymmetric plasmon energies at sub-100 nm scale on the low-index side of the thin metal film formed between the cavity top and the planar metal surface. We then proposed the use of our lens in a high-resolution far-field confocal microscopy setup. Due to the surface-field nature of our lens, the presented system holds potential as an indispensable tool for cell-surface interfacial studies that require sub-100 nm hyper-spectral imaging analysis.

Confocal fluorescence polarization microscopy for linear unmixing of spectrally similar labels.

Studies of biological samples often call for simultaneous identification of multiple molecular or structural components. Multiple labelling fluorescence techniques are a powerful way of achieving this. However, the ability to distinguish a number of fluorescent probes unambiguously can be restricted by the fact that fluorescence spectra are generally broad and overlapping. Recently a technique known as linear unmixing has been combined with spectral imaging to discriminate between multiple fluorophores. In this study a scheme is proposed whereby fluorescence polarization information is used to expand the capability of the linear unmixing technique to accommodate additional fluorescent probes. As a proof-of-concept, it is shown that this polarization-based technique can be used to divide the signals generated by two spectrally similar fluorescent probes into their separate components.

Polymer-based microfluidics with surface-enhanced Raman-spectroscopy-active periodic metal nanostructures for biofluid analysis.

The use of microfluidics for biofluid analysis offers a cheaper alternative to conventional techniques in disease diagnosis. However, traditional microfluidics design may be complicated by the need to incorporate separation elements into the system in order to facilitate specific molecular detection. Alternatively, an optical technique known as surface-enhanced Raman spectroscopy (SERS) may be used to enable identification of analyte molecules directly from a complex sample. This will not only simplify design but also reduce overall cost. The concept of SERS-based microfluidics is however not new and has been demonstrated previously by mixing SERS-active metal nanoparticles with a model sample, in situ, within the microchannel. Although the SERS reproducibility of these systems was shown to be acceptable, it is, however, not stable toward variations in the salt content of the sample, as will be shown in this study. We have proposed a microfluidics design whereby periodic SERS-active metal nanostructures are fabricated directly into the microchannel via a simple method of spin coating. Using artificial as well as human urine samples, we show that the current microfluidics is more stable toward variations in the sample's ionic strength.

Immune response against angiosarcoma following lower fluence rate clinical photodynamic therapy.

Tumor response to photodynamic therapy (PDT) is dependent on treatment parameters used. In particular, the light fluence rate may be an important determinant of the treatment outcome. In this clinical case report, we describe the response of angiosarcoma to PDT carried out using different fluence rates and drug and light doses. A patient with recurrent multifocal angiosarcoma of the head and neck was recruited for PDT. A new generation chlorin-based photosensitizer, Fotolon, was administered at a dose of 2.0 to 5.7 mg/kg. The lesions were irradiated with 665 nm laser light for a light dose of 65 to 200 J/cm2 delivered at a fluence rate of 80 or 150 mW/cm2. High dose PDT carried out at a high fluence rate resulted in local control of the disease for up to a year; however, the disease recurred and PDT had to be repeated. PDT of new lesions carried out at a lower fluence rate resulted in tumor eradication. More significantly, it also resulted in spontaneous remission of neighboring and distant untreated lesions. Repeat PDT carried out on a recurrent lesion at a lower fluence rate resulted in eradication of both treated and untreated lesions despite the lower total light dose delivered. Immunohistochemical examination of biopsy samples implies that PDT could have activated a cell-mediated immune response against untreated lesions. Subsequent histopathological examination of the lesion sites showed negative for disease. Our clinical observations show that lower fluence rate PDT results in better outcome and also indicate that the fluence rate, rather than the total light dose, is a more crucial determinant of the treatment outcome. Specifically, lower fluence rate PDT appears to activate the body's immune response against untreated lesions.

Laser confocal endomicroscopy as a novel technique for fluorescence diagnostic imaging of the oral cavity.

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.

Effects of N-methyl pyrrolidone on the uptake of hypericin in human bladder carcinoma and co-staining with DAPI investigated by confocal microscopy.

Photodynamic diagnosis (PDD) using hypericin (HY), a natural photosensitizer, detects bladder cancer significantly better than white light endoscopy. However, the lipophilicity of HY complicates its administration for clinical applications. Currently, pharmaceutical preparations for HY without plasma protein are being developed. Formulations containing a biocompatible solvent, N-methyl pyrrolidone (NMP) have been shown to enhance the photodynamic therapeutic effects of HY. It was recently reported that, NMP formulations of HY were able to produce significantly higher contrast for fluorescence detection of tumors than albumin-containing HY formulations. This present work hypothesizes that NMP acts both as a solvent and penetration enhancer to improve the delivery of HY into cells by increasing the permeability of cell membranes. This paper reports the use of 3-D confocal microscopy to monitor real-time uptake of HY in human carcinoma. 3-D confocal microscopy was used to investigate the possibility of nuclear localization of HY in MGH cells. The fluorescence of HY was confirmed to be emitted from HY containing cells using spectrometry. The localization of a DNA fluorescent probe 4', 6-diamidino-2-phenylindole dihydrochloride (DAPI) was used to confirm the possibility of colocalization of DAPI and HY. The colocalization analysis in the present study suggests that it was very unlikely that HY colocalized in the nucleus that was stained by DAPI. Fluorescein leakage tests showed that 1% NMP changes the permeability of cell membranes, and enhanced the delivery of HY into cells resulting in lower cell survival ratios. Thus, NMP was able to enhance the photodynamic therapeutic effects of HY on cancer cells.

Growth of optical-quality anthracene crystals doped with dibenzoterrylene for controlled single photon production.

Dibenzoterrylene (DBT) molecules within a crystalline anthracene matrix show promise as quantum emitters for controlled, single photon production. We present the design and construction of a chamber in which we reproducibly grow doped anthracene crystals of optical quality that are several mm across and a few µm thick. We demonstrate control of the DBT concentration over the range 6-300 parts per trillion and show that these DBT molecules are stable single-photon emitters. We interpret our data with a simple model that provides some information on the vapour pressure of DBT.

Confocal endomicroscopic imaging of normal and neoplastic human tongue tissue using ALA-induced-PPIX fluorescence: a preliminary study.

Confocal endomicroscopy is a novel and non-invasive microscopic technique that enables surface and subsurface imaging of living tissues or cells in vivo. The purpose of this study was to assess the possibility of utilizing a rigid confocal endomicroscope (RCE) system developed for detecting morphological changes in living normal and neoplastic human tongue tissue in combination with 5-aminolevulinic acid (ALA)-induced endogenous protoporphyrin IX (PPIX) fluorescence. Three patients with squamous cell carcinoma (SCC) of the tongue were examined using the novel RCE system with the excitation wavelength at 488 nm from an argon-ion laser and the detection wavelengths of the tissue fluorescence above 515 nm. Patients were topically applied with 0.4% of 5-ALA rinsing solution to the oral mucosa for approximately 15 min, and then the confocal endomicroscopic imaging of tissue PPIX fluorescence was performed on the lesion sites of the tongue after an optimal incubation period of 90-120 min. For comparison purposes, ALA-PPIX fluorescence confocal endomicroscopic imaging was also carried out on the normal sites of the tongue in vivo from two healthy volunteers. Image distortions due to tissue motion can be minimized using a specially designed tissue stabilizer attached to the RCE probe. Good quality ALA-mediated confocal fluorescence images of the tongue can be acquired in real-time, providing well-defined micro-morphological structures (e.g., filiform papillae, keratinized epithelium and fungiform papillae) of the tongue in vivo. Changes of tissue structures in oral tissue associated with cancer transformation can also be clearly identified using the RCE imaging. Preliminary results obtained in this study suggest that ALA-mediated rigid confocal endomicroscopy may have a significant potential for the rapid, non-invasive diagnosis and evaluation of early oral cancers in vivo.

Frequency shifts in SERS for biosensing.

We report an observation of a peculiar effect in which the vibrational frequencies of antibody-conjugated SERS-active reporter molecules are shifted in quantitative correlation with the concentration of the targeted antigen. We attribute the frequency shifts to mechanical perturbations in the antibody-reporter complex, as a result of antibody-antigen interaction forces. Our observation thus demonstrates the potentiality of an antibody-conjugated SERS-active reporter complex as a SERS-active nanomechanical sensor for biodetection. Remarkably, our sensing scheme, despite employing only one antibody, was found to be able to achieve detection sensitivity comparable to that of a conventional sandwich immunoassay. Additionally, we have carried out a proof-of-concept study into using multiple "stress-sensitive" SERS reporters for multiplexed detection of antigen-antibody bindings at the subdiffraction limit. The current work could therefore pave the way to realizing a label-free high-density protein nanoarray.

Clinical SERS: are we there yet?

Surface Enhanced Raman Spectroscopy or SERS has witnessed many successes over the past 3 decades, owing particularly to its simplicity of use as well as its highly-multiplexing capability. This article provides an overview of SERS and its applicability in the field of bio-medicine. We will preview recent developments in SERS substrate designs, and the various sensing technologies that are based on the SERS phenomenon. An overview of the clinical applications of SERS is also included. Finally, we provide an opinion on the future trends of this unique spectroscopic technique.

Chlorin e6-polyvinylpyrrolidone mediated photodynamic therapy--A potential bladder sparing option for high risk non-muscle invasive bladder cancer.

BACKGROUND: Bladder sparing treatment options for high risk non-muscle invasive blader cancer (NMIBC) after intravesical Bacillus Calmette-Guerin (BCG) failure are limited. OBJECTIVE: To evaluate photodynamic therapy (PDT) using chlorin e6-polyvinylpyrrolidone (Ce6-PVP) as a bladder sparing therapy for NMIBC refractory to intravesical BCG therapy. MATERIALS AND METHODS: Between July 2004 and June 2009, patients with recurrent NMIBC after induction intravesical BCG therapy were treated with PDT performed with a 665nm laser and light dosimetry of 10-24J/cm(2). The patients underwent cystoscopic surveillance for tumour recurrence post PDT. Post treatment lower urinary tract symptoms and bladder capacity were also monitored. Serum and urine samples were collected for spectrometric quantification of photosensitizer levels. RESULTS: Five patients underwent PDT, with a total of seven treatments performed. One patient received intravenous Ce6-PVP, while the rest received intravesical Ce6-PVP.The median age was 80 years (mean 79 years, range 72-88 years). There were three patients with primary CIS of the bladder and two with T1 high grade TCC and CIS of the bladder. At a median follow-up of 29 months (mean 25 months, range 6-36 months), two patients were disease free, two patients developed recurrence and one patient progressed to muscle invasive disease. There were no immediate adverse effects. The patient receiving intravenous Ce6-PVP developed an enterovesical fistula 16 months post PDT. CONCLUSIONS: Despite being a small pilot study, intravesical Ce6-PVP mediated PDT is a feasible bladder sparing treatment option for recurrent high risk non-muscle invasive bladder carcinoma in selected individuals.

Single-crystalline MFe(2)O(4) nanotubes/nanorings synthesized by thermal transformation process for biological applications.

We report a general thermal transformation approach to synthesize single-crystalline magnetic transition metal oxides nanotubes/nanorings including magnetite Fe(3)O(4), maghematite gamma-Fe(2)O(3), and ferrites MFe(2)O(4) (M = Co, Mn, Ni, Cu) using hematite alpha-Fe(2)O(3) nanotubes/nanorings template. While the straightforward reduction or reduction-oxides process was employed to produce Fe(3)O(4) and gamma-Fe(2)O(3), the alpha-Fe(2)O(3)/M(OH)(2) core/shell nanostructure was used as precursor to prepare MFe(2)O(4) nanotubes via MFe(2)O(4-x) (0 < x < 1) intermediate. The transformed ferrites nanocrystals retain the hollow structure and single-crystalline nature of the original templates. However, the crystallographic orientation-relationships of cubic spinel ferrites and trigonal hematite show strong correlation with their morpologies. The hollow-structured MFe(2)O(4) nanocrystals with tunable size, shape, and composition have exhibited unique magnetic properties. Moreover, they have been demonstrated as a highly effective peroxidase mimic catalysts for laboratory immunoassays or as a universal nanocapsules hybridized with luminescent QDs for magnetic separation and optical probe of lung cancer cells, suggesting that these biocompatible magnetic nanotubes/nanorings have great potential in biomedicine and biomagnetic applications.

Investigation into a surface plasmon related heating effect in surface enhanced Raman spectroscopy.

The irreversible loss of surface enhanced Raman spectroscopy (SERS) intensity derived from electrostatically immobilized Au colloidal substrate is studied. The total intensity of the SERS scattering from crystal violet molecules adsorbed onto the substrate was monitored for up to 240 s under continuous laser irradiation. The rate of signal decay was found to depend upon the thickness of the liquid layer over the coated substrate. On the basis of this observation, we propose a plausible mechanism in which surface plasmon related heating induced a small but significant lateral particle diffusion in closely spaced Au particles. The result of the shifting is an increase in the average interparticle distance, which subsequently reduces the electromagnetic coupling between the Au nanoparticles, and in turn causes a reduction in the SERS activity. Finally, a three-state model is proposed and shown to satisfactorily describe the observed decays.

Early diagnosis of oral cancer based on the surface plasmon resonance of gold nanoparticles.

The high mortality rate in cancer such as oral squamous cell carcinoma is commonly attributed to the difficulties in detecting the disease at an early treatable stage. In this study, we exploited the ability of gold nanoparticles to undergo coupled surface plasmon resonance and set up strong electric fields when closely-spaced to improve the molecular contrast signal in reflectance-based imaging and also to enhance the Raman signal of bioanalytes in cancer. Colloidal gold nanoparticles were synthesized and conjugated to anti-epidermal growth factor receptor (EGFR) for imaging. A self-assembled surface enhanced Raman scattering (SERS)-active gold nanoparticle monolayer film was also developed as a biosensing surface using a simple drop-dry approach. We have shown that gold nanoparticles could elicit an optical contrast to discriminate between cancerous and normal cells and their conjugation with antibodies allowed them to map the expression of relevant biomarkers for molecular imaging under confocal reflectance microscopy. We have also shown that the SERS spectra of saliva from the closely-packed gold nanoparticles films was differentiable between those acquired from normal individuals and oral cancer patients, thus showing promise of a simple SERS-based saliva assay for early diagnosis of oral cancer.

Deposition method for preparing SERS-active gold nanoparticle substrates.

Surface-enhanced Raman scattering or SERS, discovered some 20 years ago, has recently become a promising tool for routine biofluid assays in a clinical setting. Many attempts have been made to produce cheap and reproducible SERS-active substrates. In this study, we report on the fabrication of SERS-active substrates through the convective assembly of gold (Au) particles on electrostatically charged glass slides. We show that, by a proper control of the initial particle concentration in an evaporating Au suspension droplet, it is possible to obtain a closely packed colloidal film capable of generating SERS activity. Finally, AFM and SERS measurements of the resulting films reveal comparability in performance with previous silane-immobilized Au colloidal films. The minimum electromagnetic enhancement factor of our films is estimated to be about 2 x 10(4).

Reduction of polarization-induced artifacts in grating-based spectrometers.

An optical device that converts unpolarized light into a single polarization state is described. The device is based on a polarizing beam splitter that separates the two polarization directions. The beam splitter is combined with two pairs of equilateral prisms that are used to collimate the two beams in terms of both propagation and polarization directions. When it is used in combination with a blazed diffraction grating, this device is shown to effectively remove the polarization dependence of the first-order diffracted power. The device has an insertion loss of approximately 14% for purely s-polarized light. However, for unpolarized light incident upon the two gratings studied here, the increased throughput of the p-polarized component leads to an average relative gain in overall efficiency of 13%-19%, depending on the grating. In collimating the two polarization directions, the device may cause a reduction in spectral resolution for a rectangular entrance slit. As a result, the device is more likely to find use in spectrometers that have a circular aperture, such as that provided by an optical fiber.