Atopic dermatitis (AD) and psoriasis are the two most prevalent skin disorders, often assessed through subjective questionnaires or visual evaluations conducted by clinicians, which can be subject to interpersonal variations. This study aims to explore the distinctions between these skin conditions and healthy skin using a portable confocal Raman spectroscopy (CRS) system for objective assessment. Spectral measurements at 671 nm and 785 nm on 9 AD, 6 psoriasis, and 11 healthy subjects reveal lower water content in AD compared to psoriasis and healthy skin. Ceramide subclasses show disease-specific trends, distinguishing AD, and psoriasis. Cholesterol levels further differentiate these conditions, with lower concentrations in lesional AD and significantly higher concentrations in lesional psoriasis compared to healthy skin. These differences contribute to the objective differentiation of skin conditions aiding in thorough assessment and treatment monitoring. Furthermore, it offers valuable insights for developing targeted disease-specific topical treatments.
Vulvar skin, distinct from other areas, undergoes changes due to aging, causing symptoms like dryness and itchiness. While much research focuses on facial or forearm skin, vulva skin properties are underexplored. This study uses an in-house developed fiber-based Diffuse Reflectance Spectroscopy (DRS) system to assess vulva skin changes in 100 women. This objective evaluation includes analyzing tissue chromophores—water, lipid, oxyhemoglobin, and deoxyhemoglobin providing insights into moisture content, lipid levels, oxygen saturation, and blood fraction. DRS, compared to invasive methods, achieved a 65% accuracy in estimating estrogen levels, suggesting its potential for objective diagnosis and monitoring of genitourinary skin conditions.
The growing demand for innovative agricultural technologies drives our research on photonics-based solutions for non-destructive plant analysis. Our work introduces two techniques to overcome the limitations of conventional methods. The first part presents a portable VIS-NIR handheld spectrometer, showcasing strong correlations (0.84 and 0.77) for quantifying anthocyanin and chlorophyll content. In the second part, a customized fiber optic probe using ATR-FTIR enables measurements of plant parameters (water content, antioxidant activity, lignin, and cellulose). Statistical analysis of high-resolution spectra introduces the novel index NDMRI for effective phytochemical differentiation. These non-destructive, rapid, and objective methods promise to optimize agriculture and drive advancements in this field.
We report on fluorescence enhancement using a suspended core photonic crystal fiber (PCF) as an optofluidic platform. By employing metallic nanoparticles and an organic spacer, we achieved a thirty-fold signal enhancement of Cy5 dye at picomolar concentrations. The combination of fluorescence enhancement and PCF offers robustness, ease of use, and high sensitivity. This comprehensive study explores fluorescence enhancement using PCF, highlighting the significant enhancement achieved through metallic nanoparticles and organic spacers associated by the long length of light-analyte interactions offered by the PCF. These findings might contribute to the development of highly sensitive optical fiber-platforms for biomedical applications.
Non-melanoma skin cancers (NMSC) pose challenges with current clinical approaches. In this context, Multispectral Optoacoustic Tomography (MSOT) offers a promising non-invasive imaging solution. With high isotropic resolution and contrast-enhanced capabilities, MSOT provides a 3D tumor map by resolving melanin and haemoglobin signals. Our feature extraction and automated level-set image segmentation algorithm enables tumor profiling and precise boundary delineation of width, depth, and volume. Validated against histology, these metrics aid preoperative tumor mapping and surgical planning as it fosters a comprehensive understanding of tumor morphology and metabolic activities. Thus, clinicians can optimize NMSC management, revolutionizing diagnostics and treatment for improved patient outcomes.
We introduce a compact, non-contact multimodal imaging platform that integrates ultrasound (US) imaging, inclusive of photoacoustic (PA) detection, with optical coherence tomography (OCT). This integration is achieved through a novel virtual acoustic detector array (VADA) technique for all-optical US sensing, utilizing the temporal and spatial resolving capabilities of swept-source optical interferometry. The technique extracts US signals from the phase time evolution of a swept-source OCT's spectral sweep. It enables the virtual construction of the VADA along both lateral and depth directions on the imaging target for non-contact detection of acoustic waves from surrounding US sources. The platform's high-speed scanning (MHz OCT A-scan rate) and ultra-sensitive phase detection (nm displacement sensitivity) allow for the customization of the spatial density of the VADA and the collection of wideband acoustic signals, which are essential for the reconstruction of US images. In our pilot study, we successfully demonstrated the feasibility of this technique. We used a conventional US transducer as an acoustic source. The acoustic field distribution within the imaging target and the morphology and position of the piezoelectric layer were successfully reconstructed, which is based on US waveforms obtained from the VADA.
Auretek is the first of its kind portable Confocal Raman Spectroscopy (CRS) system equipped with fiber based handheld probe, that cascades dual-wavelength lasers. With its flexible probe, Auretek can acquire CRS data of skin from various parts of the human body with depth profiling. A clinical study was conducted to investigate the effects of ceramide-based moisturizer (CBM) on skin physiology and biochemistry. Quantitative skin component characterization in different epidermal layers was done with spectral unmixing analysis of the CRS data. Even though the analysis revealed a significant increase in ceramide content on the side of CBM application, the increase was in the layers within the stratum corneum.
Breast cancer is the most common cancer worldwide. Mammography screening and biopsy procedures for abnormal mammograms are the gold standard detection method, however, it is invasive, time-consuming, and labor-intensive. RNA biomarkers in circulating blood may be an alternative to the gold standard. We have adopted a multi-modal approach using Fourier transform infrared (FTIR) spectroscopy and Raman spectroscopy that provides a complete characterization of RNA biomarker fingerprints in the full spectrum. We have measured 99 patients’ serum samples and achieved a higher accuracy, specificity, and sensitivity using the multi-modal approach combined with machine learning analysis than using the individual techniques alone.
This conference presentation was prepared for the Optical Diagnostics and Sensing XXIII: Toward Point-of-Care Diagnostics conference at SPIE BiOS, 2023.
Surface enhanced Raman scattering (SERS) is a powerful biosensing technique allowing direct detection of target molecules thanks to their vibrational fingerprint Raman spectra. SERS is usually performed on nano roughened plamonic planar substrates/colloidal nanoparticles. However, the irregularities of plasmonic nanostructures lead to measurement reliability limitations. We have recently demonstrated that SERS-probes based on opto-fluidic photonic crystal fiber (PCF) composed of a silica-core surrounded by large air channels are remarkable sensing platforms leading to tremendous SERS sensitivity and excellent measurement reliability (98% in reproducibility and 95% in repeatability). However, a major limitation occurs when looking for highly reliable and easy-to-use biosensing platform. Actual SERS biosensors (planar substrate or fiber probe) require their alignment under a microscope, which could restrict on-field practical use. Here, we address this issue by developing a tapered opto-fluidic PCF allowing excellent reliability, efficient and easy coupling through a Plug-&-Play type modality. This novel type of SERS probe realized by reducing the fiber diameter, which increases the SERS sensitivity while enabling efficient light coupling to the Raman spectrometer with a relative standard deviation (RSD) of only 3.5% in reproducibility and 3.84% in repeatability. We demonstrate this easy Plug-&-Play type coupling based on a simple bare fiber connector, with a RSD of 4.5% in reproducibility measurements (i.e. removing and reconnecting the fiber SERS-probe to the Raman spectrometer without any additional alignment). We envision that this easy-to-use platform can be translated to - clinically viable SERS probes for liquid biopsy.
Surface enhanced Raman spectroscopy (SERS) allows sensitive detection of analytes, thanks to their vibrational Raman spectra. Planar SERS substrates often lack the reliability in measurement due to larger variations in signal intensity contributed by irregularities in nanostructures. In this context, photonic crystal fibers (PCFs) that combine excellent light guiding properties and the possibility to incorporate nanostructures and liquid or gas analytes into their axially aligned air holes, offer tremendous promise as an opto-fluidic SERS platform. Such sensors possess the enormous signal enhancement inherent to SERS and the flexibility of optical fibers. In addition, PCFs offer improved reproducibility, repeatability and sensitivity in measurement compared to planar substrates, due to the larger volume of interaction between the guided light and the analyte, and to highly reliable light couplings into the fiber core. Here, we present a novel design of PCF called ring core fiber (RCF), which is specifically engineered to further increase the interaction area in order to improve the sensitivity and reliability of the sensor. Preliminary experimental results showed that sensitivity of SERS sensing is improved by 115% compared to the best SuC-PCFs. Ongoing numerical simulations indicate that by further optimizing the dimensions of the ring, sensitivity could be improved by at least one order of magnitude with RCF. We envision that this new design with increased sensitivity and measurement reliability could be the next major step towards a clinically viable liquid biopsy fiber probe.
Atopic dermatitis (AD) is a common inflammatory skin disorder which affects ~20% of children and ~3% adults worldwide. There lacks a direct, non-invasive method of evaluating atopic dermatitis (AD) accurately. Here, the use a multispectral raster-scanning optoacoustic mesoscopy (MS-RSOM) as an objective imaging tool for AD is proposed. MS-RSOM is a novel, non-invasive optoacoustic imaging modality which can provide label-free, high resolution imaging up to 1.5 mm below the skin. It can provide useful information on melanin, oxyhemoglobin (HbO2), deoxyhemoglobin (Hb) and oxygen saturation (sO2) from the skin layers. This preliminary study was conducted on 4 AD patients and 2 healthy volunteers using MS-RSOM system. From the MS-RSOM images, the epidermis thickness and oxygen saturation were computed from the healthy volunteers as well as from the non-lesional and eczema lesional areas of the eczema subjects.
Superferromagnetic iron oxide (SFMIOs) is a new paradigm for Magnetic Particle Imaging (MPI) as it approaches the ideal imaging agent characteristics of a steep, step-like magnetization curve. Other than the expected 10-fold SNR and spatial resolution improvements from the square-like hysteresis curve, we demonstrate other unique qualities of SFMIOs that enable robust "chemical-shift-like" color multiplexing to potentially enable a range of magnetic "dyes" that work for at-depth imaging. We also demonstrate the extremely high sensitivity of SFMIO signal to the proximity of SFMIOs to each other, and explain how this could potentially be used as a FRET-like nanoscale ruler.
Confocal Raman spectroscopy (CRS) technology has wide applications, especially in the biomedical field. CRS provides non-destructive, contrast-free and molecule-specific quantitative measurement of samples, including biological tissues. These advantages make it ideal for human skin characterization. We present a portable dual-wavelength CRS system with a handheld measurement probe which connected to the console by optical fibers. To achieve fast switching between the two wavelengths, a fast 2-to-1 fiber array multiplexing module was used. A large bandwidth could be covered at each measurement point in real time. We performed system characterization for this proposed CRS system and measured different samples.
A comprehensive analysis using three machine-learning models for an AI-aided atopic dermatitis (AD) diagnosis and sub-classifying AD severities with 3D Raster Scanning Optoacoustic Mesoscopy (RSOM) images, extracted features from volumetric vascular structures and clinical information.
Inflammatory skin disorder, eczema, is usually assessed by subjective disease scoring systems such as SCORAD and EASI. These scoring systems are based on clinical observations and questionnaires and hence it is subjected to inter and intra-assessor variability. Here, for the first time, we used optoacoustic imaging to image the structural and morphological changes of the skin in a non-invasive manner. Through a clinical study, we computed specific metrics such as epidermis thickness, total blood volume, vessel diameter in the dermis, ratio of low and high frequency signals. We trained a linear kernel-based support vector machine model for eczema classification using these metrics. We could achieve an accuracy of 86.6% and high sensitivity and specificity of 96.2% and 82.1% respectively. We also formulated a novel Eczema Vascular and Structural Index (EVSI) to objectively assess the severity of eczema.
Blood flow measurement in deep tissue is important because the circulatory system transports oxygen and nutrition to the tissue and removes carbon dioxide out from the tissue. Several non-invasive optical methods were developed for blood flow measurement in deep tissue, such as diffuse correlation spectroscopy (DCS) and diffuse speckle contrast analysis (DSCA). In this paper we will introduce a new speckle-based method for fast blood flow measurement in deep tissue: diffuse speckle pulsatile flowmetry (DSPF). By using a multi-mode fiber for speckle pattern detection, DSPF achieves high blood flow measurement rate of 300 Hz. It has one of the fastest measurement rates of blood flow among non-invasive modalities.
A flexible membrane based Surface-Enhanced Raman Spectroscopy (SERS) sensor was developed as a viable point-of-care platform to monitor changes of these surrogate indicators of healing status in chronic wounds, such as tumor necrosis factor alpha (TNFα) and matrix metalloproteinase (MMPs). In terms of performance, SERS approach is superior to enzyme-based assays, which are resource intensive. We demonstrated the efficiency of this flexible SERS platform for the sensitive detection of TNFα and MMP9 in the nM to pM range. These substrates may be incorporated into wound dressings to permit routine monitoring of wound status.
We report a label-free Surface Enhanced Raman Spectroscopy (SERS) for pleural fluid analysis to distinguish Lung cancer from controls patients. Herein, we have used a novel silver coated silicon Nanopillar (SCSNP) as SERS substrate to acquire multiple SERS spectra for each pleural fluid sample and advanced chemometrics methods. We report a classification accuracy of 85% along with sensitivity and specificity of 87% and 83% respectively for the detection of Lung cancer over control pleural fluid samples with a receiver operating characteristics (ROC) area under curve value of 0.92 using PLS-DA binary classifier to distinguish between lung cancer over control subjects.
Significance: Noninvasive in vivo fast pulsatile blood flow measurement in deep tissue is important because the blood flow waveform is correlated with physiological parameters, such as blood pressure and elasticity of blood vessels. Compromised blood flow may cause diseases, such as stroke, foot ulcer, and myocardial ischemia. There is great clinical demand for a portable and cost-effective device for noninvasive pulsatile blood flow measurement.
Aim: A diffuse-optics-based method, diffuse speckle pulsatile flowmetry (DSPF), was developed for fast measurement (∼300 Hz) of deep tissue blood flow noninvasively. To validate its performance, both a phantom experiment and in vivo demonstration were conducted.
Approach: Over the past two decades, single-mode fibers have been used as detection fibers in most diffuse-optics-based deep tissue blood flow measurement modalities. We used a multimode (MM) detection fiber with a core size of 200 μm for diffused speckle pattern detection. A background intensity correction algorithm was implemented for speckle contrast calculation. The MM detection fiber helped to achieve a level of deep tissue blood flow measurement similar to that of conventional modalities, such as diffuse correlation spectroscopy and diffuse speckle contrast analysis, but it increases the measurement rate of blood flow to 300 Hz.
Results: The design and implementation of the DSPF system were introduced. The theory of the background intensity correction for the diffused speckle pattern detected by the MM fiber was explained. A flow phantom was built for validation of the performance of the DSPF system. An in vivo cuff-induced occlusion experiment was performed to demonstrate the capability of the proposed DSPF system.
Conclusions: An MM detection fiber can help to achieve fast (∼300 Hz) pulsatile blood flow measurement in the proposed DSPF method. The cost-effective device and the fiber-based flexible probe increase the usability of the DSPF system significantly.
We present a dual-wavelength fast mechanical scanning optical-resolution photoacoustic microscopy system (OR-PAM) in this paper. Conventional mechanical scanning OR-PAM system takes ~ 30 min for one frame of single wavelength imaging. The acquisition of two wavelengths will double the total imaging time. By using a fast-linear stage, a high signal-to-noise (SNR) optical and acoustic combiner and 2 lasers at different wavelengths, we achieved a B-scan rate of 12 Hz and reduced the acquisition time to ~1.5 min for dual-wavelength imaging. This system can be used in large field optical resolution imaging. Contrast-free vascular and functional imaging can be achieved using this system. Both ex vivo and in vivo imaging results are demonstrated.
Photoacoustic microscopy (PAM) is a fast-growing biomedical imaging technique that provides high-resolution in vivo imaging beyond the optical diffusion limit. Depending on the scalable lateral resolution and achievable penetration depth, PAM can be classified into optical resolution PAM (OR-PAM) and acoustic resolution PAM (AR-PAM). The use of a microelectromechanical systems (MEMS) scanner has improved OR-PAM imaging speed significantly and is highly beneficial in the development of miniaturized handheld devices. The shallow penetration depth of OR-PAM limits the use of such devices for a wide range of clinical applications. We report the use of a high-speed MEMS scanner for both OR-PAM and AR-PAM. A high-speed, wide-area scanning integrated OR-AR-PAM system combining MEMS scanner and raster mechanical movement was developed. A lateral resolution of 5 μm and penetration depth ∼0.9-mm in vivo was achieved using OR-PAM at 586 nm, whereas a lateral resolution of 84 μm and penetration depth of ∼2-mm in vivo was achieved using AR-PAM at 532 nm.
The hemodynamics and oxygen saturation status of vascular are very important biomarkers for disease, such as brain glioma tumor and ischemia-reperfusion ulcer. Therefore, a high spatial resolution imaging tool for vascular imaging is demanded. Conventional optical imaging modalities, including confocal microscopy and two-photon microscopy, require external contrast agent to image blood vessels and are not sensitive to oxygen saturation. The development of photoacoustic microscopy provides a contrast-free, high-spatial resolution and functional vascular imaging tool. It’s gaining more and more popularity in biomedical research. In this paper, we introduce a dual-wavelength opticalresolution photoacoustic microscopy (OR-PAM) system for functional imaging of vasculature. This system has demonstrated its application in brain glioma tumor imaging, as well as skin ischemia-reperfusion imaging.
The International Photoacoustic Standardisation Consortium (IPASC) emerged from SPIE 2018, established to drive consensus on photoacoustic system testing. As photoacoustic imaging (PAI) matures from research laboratories into clinical trials, it is essential to establish best-practice guidelines for photoacoustic image acquisition, analysis and reporting, and a standardised approach for technical system validation. The primary goal of the IPASC is to create widely accepted phantoms for testing preclinical and clinical PAI systems. To achieve this, the IPASC has formed five working groups (WGs). The first and second WGs have defined optical and acoustic properties, suitable materials, and configurations of photoacoustic image quality phantoms. These phantoms consist of a bulk material embedded with targets to enable quantitative assessment of image quality characteristics including resolution and sensitivity across depth. The third WG has recorded details such as illumination and detection configurations of PAI instruments available within the consortium, leading to proposals for system-specific phantom geometries. This PAI system inventory was also used by WG4 in identifying approaches to data collection and sharing. Finally, WG5 investigated means for phantom fabrication, material characterisation and PAI of phantoms. Following a pilot multi-centre phantom imaging study within the consortium, the IPASC settled on an internationally agreed set of standardised recommendations and imaging procedures. This leads to advances in: (1) quantitative comparison of PAI data acquired with different data acquisition and analysis methods; (2) provision of a publicly available reference data set for testing new algorithms; and (3) technical validation of new and existing PAI devices across multiple centres.
A Microelectromechanical Systems (MEMS)-based rapid scanning photoacoustic microscopy (PAM) is available to help life science research in neuroscience, cell biology, and in vivo imaging. MicroPhotoAcoustics (MPA; Ronkonkoma, NY), the only manufacturer and vendor of Optical Resolution (OR)-PAM systems, has developed a commercial PAM system with switchable optical and acoustic resolution (OR- and AR-PAM). To achieve real-time imaging capability without sacrificing high signal-to-noise ratios (SNRs), a 2-axis water-proofing MEMS scanner made of flexible polydimethylsiloxane (PDMS) was demonstrated by collaboration with Pohang University of Science and Technology (South Korea) that promises to dramatically increase the system’s imaging speed. This flexible scanner results in a wide scanning range and a fast imaging speed (5 B-scan images per second). Equipped with different excitation sources, in vivo PA images of microvasculatures in a mouse ear was obtained. The lateral and axial resolutions of the OR-PAM system are 4.38 μm. It is expected that this MEMS-based fast OR-PAM system can be significantly useful in both preclinical and clinical applications. With the continuation of new technological advancements and discoveries, MPA plans to further advance PAM to achieve faster imaging speed, higher spatial resolution at deeper tissue layer, and address a broader range of biomedical applications.
Nanoparticle contrast agents for targeted imaging have widespread diagnostic applications with cellular resolution, specificity and selectivity for visualization and assessment of various disease processes. Of particular interest are gold nanoparticles owing to the tunability of the localised surface plasmon resonance (LSPR) and its relative inertness. Synthesizing gold nanoprobes in the near infrared (NIR) region is of particular interest in developing nanosensors due to the minimal light attenuation from biomolecules. The ability of plasmonic gold nanostars (GNS), with novel shape-dependent dual LSPR, to elicit signal contrast at NIR wavelengths is described here for multiple biomedical modalities. First, the surface enhanced Raman scattering (SERS) capability of these dual plasmonic GNS has been demonstrated to elicit high SERS enhancement factor (EF) of 2 x 10e7 with 785 nm excitation and the potential to elicit the highest SERS EF ever reported for gold nanoparticles, with further longer wavelength excitations at and beyond 1064 nm.
We have also demonstrated the longer wavelength contrast imaging capability of GNS with photoacoustic imaging (PAI) and for photothermal therapy (PTT). GNS possess unique structural characteristics that impart superior optical properties resulting in higher photothermal efficiency. The photothermal capability of GNS was demonstrated in vivo with localized temperature rise of 9℃ in tumors when irradiated with a 1064 nm CW laser that resulted in significant tumor cell death. Since photothermal conversion is the optical process responsible for eliciting PA contrast and for PTT, this development represents a novel theranostic substrate to be used at 1064 nm excitation, a longer wavelength than the conventional clinical range. The ability of GNS to elicit signal contrast at NIR wavelengths has also been demonstrated for photothermal optical coherence tomography (PT-OCT). When irradiated with a 1064 nm continuous wave laser, GNS elicited photothermal contrast well beyond 2 mm, displaying great potential for deep tissue imaging. We have also recently obtained a European Commission grant worth €5.98M on developing, demonstrating and validating a novel GNS enhanced photoacoustic imaging platform which will be capable of tracking mesenchymal stem cells (MSC) and MSC-derived exosomes, at unprecedented depth and sensitivity.
We present a water-proof Microelectromechanical systems (MEMS) based scanning optical resolution Photoacoustic Microscopy (OR-PAM) system and its application in glioma tumor mouse model study. The presented OR-PAM system has high optical resolution (~3 μm) and high scanning speed (up to 50 kHz A-scan rate), which is ideal for cerebral vascular imaging. In this study, the mice with glioma tumor are treated with vascular disrupting agent (VDA). OR-PAM system is utilized to image the cerebral with the whole skull intact before and after the injection of VDA. By image registration, the response of every single blood vessel can be traced. This will provide us deeper understanding of the drug effect.
A focused-scanning photoacoustic microscopy (PAM) is available to help advancing life science research in neuroscience, cell biology, and in vivo imaging. At this early stage, the only one manufacturer of PAM systems, MicroPhotoAcoustics (MPA; Ronkonkoma, NY), MPA has developed a commercial PAM system with switchable optical and acoustic resolution (OR- and AR-PAM), using multiple patents licensed from the lab of Lihong Wang, who pioneered photoacoustics. The system includes different excitation sources. Two kilohertz-tunable, Q-switched, Diode Pumped Solid-State (DPSS) lasers offering a up to 30kHz pulse repetition rate and 9 ns pulse duration with 532 and 559 nm to achieve functional photoacoustic tomography for sO2 (oxygen saturation of hemoglobin) imaging in OR-PAM. A Ti:sapphire laser from 700 to 900 nm to achieve deep-tissue imaging. OR-PAM provides up to 1 mm penetration depth and 5 μm lateral resolution. while AR-PAM offers up to 3 mm imaging depth and 45 μm lateral resolution. The scanning step sizes for OR- and AR-PAM are 0.625 and 6.25 μm, respectively. Researchers have used the system for a range of applications, including preclinical neural imaging; imaging of cell nuclei in intestine, ear, and leg; and preclinical human imaging of finger cuticle. With the continuation of new technological advancements and discoveries, MPA plans to further advance PAM to achieve faster imaging speed, higher spatial resolution at deeper tissue layer, and address a broader range of biomedical applications.
Nanoparticle contrast agents for molecular targeted imaging have widespread interest in diagnostic applications with cellular resolution, specificity and selectivity for visualization and assessment of various disease processes. Of particular interest is gold nanoparticle owing to its tunability of the surface plasmon resonance (SPR) and its relative inertness. Here we present the synthesis of anisotropic multi-branched star shaped gold nanoparticles exhibiting dual-band plasmon absorption peaks and its application as a contrast agent for multispectral photoacoustic imaging. The transverse plasmon absorption peak of the synthesised dual plasmonic gold nanostar (DPGNS) was around 700 nm and that of longitudinal plasmon absorption in the longer wavelength region around 1050-1150 nm. Unlike most reported PA contrast agent with surface plasmon absorption in the range of 700 to 800 nm showing moderate tissue penetration, 1050-1200 nm range lies in the farther region of the optical window of biological tissue where scattering and the intrinsic optical extinction of endogenous chromophores is at its minimum. We also present a proof of principle demonstration of DPGNS as contrast agent for multispectral photoacoustic animal imaging. Our results show that DPGNS are promising for PA imaging with extended-depth imaging applications.
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.
Gold-coated array patterned with tightly-packed nanospheres was developed as a substrate base for constructing SERSenriched
nanogaps with Au-nanoparticles (GNPs). Using 1,2-ethanedithiol as a linker, Au-NPs (=17-40nm) were
anchored covalently on the sphere-array. Thin Au layer was sputtered on the substrate to mask the citrate coating of
GNPs that could demote the sensing mechanism. The negatively-charged GNP surface warrants the colloidal stability,
but the resulting repulsive force keeps the immobilized NPs apart by about 40nm. The attained gap size is inadequately
narrow to sustain any intense enhancement owing to the near-field nature of SERS. Minimal amount of NaCl was then
added to slightly perturb the colloidal stability by reducing their surface charge. Notably, the interparticle-gap reduces at
increasing amount of salt, giving rise to increased packing density of GNPs. The SERS enhancement is also found to
exponentially increase at decreasing gap size. Nevertheless, the minimum gap achieved is limited to merely 7nm.
Excessive addition of salt would eventually induce complete aggregation of particles, forming clustered NPs on the
array. A simple sputtering-growth approach is therefore proposed to further minimize the interparticle gap by enlarging
the seeded NPs based on mild sputtering. The SEM images confirm that the gap below 7nm is achievable. With advent
of the colloidal chemistry, the combined salt-induced aggregation and sputtering-growth techniques can be applied to
engineer interparticle gap that is crucial to realize an ultrasensitive SERS biosensor. The proposed two-step preparation
can be potentially adopted to fabricate the SERS-enriched nanogaps on the microfluidics platform.
Nanostructure substrates are effective biosensor to spectrally differentiate multiple compounds by Surface-enhanced
Raman scattering (SERS). Metal film over nanosphere (MFON) has been demonstrated to exhibit reproducible and
predictable Raman enhancement. MFON can be fabricated using an economical process in which polystyrene (PS)
nanospheres are self-assembled on a planar solid supports and then followed by metal coating. In this work, we
investigate the MFON substrates with bimetallic coating to combine the optical-enhancing and stability features from Ag
and Au layers. The SERS responses are then quantified from the resultant bimetallic structures with 2-Naphthalenethiol.
We show that the bimetallic substrate of optimal Au/Ag thickness ratio renders SERS enhancement and stability
exceeding those of the Au-coated MFON. Compared to Au array, the bimetallic substrate exhibits quasi-bimetallic
nanoparticles of surpassing SERS (2.5 times) with enhancement factor determined to be 2×107. As a proof-of-concept for
biosensing in microfluidics, SERS nanotag was prepared and tested on the optimized BMFON. In addition, we propose a
fabrication scheme to construct MFON with alternating sizes (100nm and 400nm) of nanosphere. At optimal
proportional amount, the 100nm-spheres were packed within the gaps between the 400nm-spheres. The resultant
morphology renders additional nanogaps that could possibly lead to increment in SERS enhancement.
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.
Red blood cells (RBCs) have been found to undergo "programmed cell death," or eryptosis, and understanding this process can provide more information about apoptosis of nucleated cells. Photothermal (PT) response, a label-free photothermal noninvasive technique, is proposed as a tool to monitor the cell death process of living human RBCs upon glucose depletion. Since the physiological status of the dying cells is highly sensitive to photothermal parameters (e.g., thermal diffusivity, absorption, etc.), we applied linear PT response to continuously monitor the death mechanism of RBC when depleted of glucose. The kinetics of the assay where the cell's PT response transforms from linear to nonlinear regime is reported. In addition, quantitative monitoring was performed by extracting the relevant photothermal parameters from the PT response. Twofold increases in thermal diffusivity and size reduction were found in the linear PT response during cell death. Our results reveal that photothermal parameters change earlier than phosphatidylserine externalization (used for fluorescent studies), allowing us to detect the initial stage of eryptosis in a quantitative manner. Hence, the proposed tool, in addition to detection of eryptosis earlier than fluorescence, could also reveal physiological status of the cells through quantitative photothermal parameter extraction.
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.
Temporal changes in signal intensity of Surface Enhanced Raman Scattering (SERS) upon laser excitation is an
interesting phenomenon in plasmonics. In-depth understanding of the phenomena is highly important especially when
developing a SERS sensor based on the intensity variation of particular Raman peak/band. One of the main challenges in
such a technique is the intensity reduction at a given location upon consecutive measurements. Previously, signal loss in
SERS measurement was attributed to the electric-field induced roughness relaxations in the SERS active surface. In such
cases, as the surface is smoothened out, signals are completely lost. In our observation, the reduction in the spectral
intensity is irreversible but never completely lost and a major part of it can be attributed to the plasmon induced heating
effect. Here, we experimentally demonstrate this effect by studying the SERS signal from four different Raman active
molecules adsorbed onto substrates that contain uniform nano-roughened bi-metallic silver/gold coating. Possible
mechanism that leads to irreversible signal loss is explained. Moreover, solutions for minimising such plasmonic heating
when developing a biosensor are also discussed.
The control of image contrast is essential toward optimizing a contrast enhancement procedure in optical coherence tomography (OCT). In this study, the in vivo control of optical contrast in a mouse tumor model with gold nanoshells as a contrast agent is examined. Gold nanoshells are administered into mice, with the injected dosage and particle surface parameters varied and its concentration in the tumor under each condition is determined using a noninvasive theoretical OCT modeling technique. The results show that too high a concentration of gold nanoshells in the tumor only enhances the OCT signal near the tissue surface, while significantly attenuating the signal deeper into the tissue. With an appropriate dosage, IV delivery of gold nanoshells allows a moderate concentration of 6.2×109 particles/ml in tumor to achieve a good OCT signal enhancement with minimal signal attenuation with depth. An increase in the IV dosage of gold nanoshells reveals a corresponding nonlinear increase in their tumor concentration, as well as a nonlinear reduction in the fractional concentration of injected gold nanoshells. Furthermore, this fractional concentration is improved with the use of antiepodermal growth factor receptor (EGFR) surface functionalization, which also reduces the time required for tumor delivery from 6 to 2 h.
A successful detection of inherently weak Raman signal from molecules is possible with giant enhancement of signal by the process of surface-enhanced Raman scattering (SERS). The SERS-induced enhancement is typically achieved when the molecules adsorbed onto the surface of a noble-metal substrate with nanometric roughness. Such SERS-substrate could be economically fabricated by convective assembly of polystyrene beads followed by metal deposition. The
characterization of mono-metallic substrate showed that the SERS enhancement factor increases with increasing
thickness of Ag or Au, with Ag-substrate giving the greatest SERS enhancement. However, the formation of silver oxide
layer could reduce the shelf-life of the Ag-substrate. Alternatively, Au is also used as the coating material owing to its
chemical inertness and biocompatibility. Despite the decent enhancement of the Au-substrate, Au-layer was found to be
unstable after prolonged incubation in crystal violet solution. The inherent deficiency in adhesiveness of Au to the glass
limits its use as a reliable and cost-effective substrate. In an attempt to improve the SERS-substrate, bimetallic substrate
was fabricated by depositing the Au-film, as a protective layer, on the Ag-substrate. In this case, the top layer of Au of
the bimetallic substrate remained intact after chemical treatment. Furthermore, the bimetallic substrate was shown to
give comparable level of enhancement as an Ag-substrate by choosing a proper thickness ratio of the bimetallic layers.
The result suggests that the design of bimetallic substrate could be optimized to maximize the SERS enhancement while
retaining a decent stability after laser illumination and chemical treatment. Our findings suggest that bimetallic substrates
are potentially useful for a reliable SERS-based biosensing.
Surface Enhanced Raman Scattering (SERS) technique is used as an indispensable and sensitive modality for bio-sensing
due to its ability to distinguish the analyte molecules based on their distinct 'fingerprint' spectra. One of the most
promising SERS substrates for biosensing was fabricated by coating noble metal film over orderly packed nanospheres.
However, the major challenge in developing such a sensor is to achieve reproducible SERS substrate. Here, we report a
new class of SERS substrate with ordered 3D nanostructures fabricated on silicon wafer by deep UV lithography
technique followed by bi-metallic coating of silver and gold. Compared to the substrate fabricated by conventional
nanosphere lithography, this approach allows better control of the nanostructures, which in turn gives uniform surface
roughness for the metal film to provide adequate SERS enhancement with high reproducibility. Significance of this
substrate for biomedical application was demonstrated by glucose sensing under physiologically relevant conditions.
Partitioning and localization of glucose molecules within the first few nanometers of active SERS substrate was achieved
by a self assembled monolayer (SAM) on the surface of substrate.
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.
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.
We report the detection of interactions between a photosensitizer, hypericin (HY), and its solvent system prepared with a formulation additive, polyvinylpyrrolidone (PVP), a commonly used pharmaceutical excipient. Fluorescence correlation spectroscopy (FCS) and fluorescence lifetime imaging microscopy (FLIM) were used to study aggregation and binding of HY in the presence of PVP. Digitized fluorescence endoscopic imaging (DFEI) was used to study the effect of the pharmaceutical formulation in the in vivo tumor implanted chick chorioallantoic membrane (CAM) model. The results presented reveal the coordination of HY-PVP binding, HY disaggregation in the presence of PVP, and strengthened HY tumor uptake selectivity. PVP is thus suggested as a potential adjuvant to previously investigated N-methyl pyrrolidone (NMP) in the HY delivery system as well as a replacement for the conventionally used albumin in the HY bladder instillation fluids preparation for clinical use.
Our aim is to apply image analysis on photosensitizer fluorescence and compare the relationship between histopathology and endoscopic fluorescence imaging. The correlation between hypericin fluorescence and histopathology of diseased tissue was explored in a clinical study involving 58 fluorescence cystoscopic images from 23 patients. Based on quantification of fluorescence colorimetric parameters extracted from the image analysis, diagnostic functions were developed to pathologically classify the bladder cancer. Our preliminary results show that the differences in fluorescence intensity ratios among the three different grades of bladder cancer are statistically significant. The results also show a decrease in macroscopic fluorescence intensity that correlated with higher cancer grades. By combining both the red-to-green and red-to-blue fluorescence intensity ratios into a 2-D scatter plot and defining diagnostic linear discrimination functions on the data points, this technique is able to yield an average sensitivity and specificity of around 68.6% and 86.1%, respectively, for pathological cancer grading of the three different grades of bladder cancer in our study. We conclude that our proposed approach in applying colorimetric intensity ratio analysis on hypericin fluorescence shows potential to optically grade bladder cancer in situ.
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.
The application of gold nanoparticles as a contrast agent in optical bioimaging is well appreciated, but limited to a
narrow excitation range due to its rather invariable optical resonance typically at 520 nm. Compared to gold
nanoparticles, the optical response of gold nanoshells can be tuned to match the higher excitation wavelength of many
promising clinical reflectance-based imaging modalities such as the optical coherence tomography (OCT). In this study,
we demonstrate the tunability of gold nanoshells to improve the optical contrast of backscattering signal under confocal
reflectance microscopy and OCT. The gold nanoshells were synthesized and conjugated to antibodies for in vitro
demonstration of their selective optical contrast in cancer cells over normal cells under the confocal reflectance
microscopy. The OCT signals from these gold nanoshells were compared to that from bare silica cores and intrinsic
tissue scattering using 1% Intralipid. We have shown that gold nanoshells are able to elicit an optical contrast to
discriminate between cancerous and normal cells under the confocal reflectance microscopy based on differences in
molecular markers expression. Compared to bare silica core, the presence of the gold shell is able to effect a higher
backscattered OCT signal with an apparent contrast over 1% Intralipid. This contrast can be made to be dependent on
the molecular marker expression with antibody specificity.
In this paper, we present a non-rotatory circumferential scanning optical probe integrated with a MEMS scanner for in
vivo endoscopic optical coherence tomography (OCT). OCT is an emerging optical imaging technique that allows high
resolution cross-sectional imaging of tissue microstructure. To extend its usage to endoscopic applications, a
miniaturized optical probe based on Microelectromechanical Systems (MEMS) fabrication techniques is currently
desired. A 3D electrothermally actuated micromirror realized using micromachining single crystal silicon (SCS) process
highlights its very large angular deflection, about 45 degree, with low driving voltage for safety consideration. The
micromirror is integrated with a GRIN lens into a waterproof package which is compatible with requirements for
minimally invasive endoscopic procedures. To implement circumferential scanning substantially for diagnosis on certain
pathological conditions, such as Barret's esophagus, the micromirror is mounted on 90 degree to optical axis of GRIN
lens. 4 Bimorph actuators that are connected to the mirror on one end via supporting beams and springs are selected in
this micromirror design. When actuators of the micromirror are driven by 4 channels of sinusoidal waveforms with 90
degree phase differences, beam focused by a GRIN is redirected out of the endoscope by 45 degree tilting mirror plate
and achieve circumferential scanning pattern. This novel driving method making full use of very large angular deflection
capability of our micromirror is totally different from previously developed or developing micromotor-like rotatory
MEMS device for circumferential scanning.
Design and development of a 3D scanning MEMS micromirror integrated miniaturized optical probe has been presented
in this article. The probe is designed to be less than 2 mm in diameter and has dynamic scanning modality for larger field
of view. Scanning is achieved using 3D micromirror device, which has 16º out of plane and 360º beam rotation
capability. Initial target of 45º out of plane deflection is yet to be achieved. The probe being developed currently would
have scanning capability in one quarter of 360º full rotation. The field of view would still be very large and multiple
optical biopsies would be possible for planned cancer model diagnostics. The feasibility of using scanning mirror into an
optical probe was demonstrated using scanning repeatability and OCT imaging tests. Geometrical optics and package
design using silicon optical bench have been established. Miniaturized 3D scanning micromirror have been designed and
developed with 16º out of plane deflection demonstrated. Probe package integration and optical testing are carried out.
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.
Advanced optical technologies for in vivo imaging e.g. OCT and confocal reflectance endomicroscopy while being able to image stromal morphology, are unable to image biomolecular changes associated with carcinogenesis. Furthermore, the contrast between neoplastic and normal tissues from such advanced optical technologies is often too low to be of any clinical value. Due to their favorable optical properties including their ability to resonantly scatter light at surface plasmon resonance to present potentially good contrast for reflectance-mode imaging, we aim to develop gold nanoparticles as optical contrast agents coupled with these optical imaging systems to perform cancer targeting bioimaging for early diagnosis of epithelial carcinoma. In this study, 20 nm gold nanoparticles were synthesized and conjugated with anti-EGFR (Epidermal Growth Factor Receptor). EGFR is a cell surface receptor biomarker that is highly expressed in majority of epithelial cancer compared to normal cells. The resulting anti-EGFR conjugated gold nanoparticles were allowed to interact with the nasopharyngeal carcinoma CNE2 cells in vitro. The exact localization of the gold bioconjugates on the cell surface EGFR receptors was investigated using confocal immunofluorescence microscopy. We have demonstrated that the binding and localization of the gold bioconjugates on the cell surface increased the reflectance and scattering properties of the CNE2 cells and provide good optical contrast for the cancer cells under confocal reflectance microscopy. Thus our study has demonstrated the potential of gold nanoparticles to
target and illuminate cancer cells for bioimaging.
Surface enhanced Raman spectroscopy (SERS) discovered some 30 years ago has gained popularity as a powerful analytical tool for developing chemo- and bio-sensing. The combination of SERS with the microfluidics technology can provide a miniaturized and portable device for bio-fluid analysis. However, as will be pointed out in this study, heat generated in a SERS-active substrate as a result of laser-induced plasmon resonance can unfavorably affect the sensitivity of a SERS-based microfluidic device. We will show that the plasmon-induced heat associated with SERS can significantly reduce the signal strength from the analyte under certain circumstances, and show heat-induced morphological changes in the SERS-active substrate as a primary cause of the observed signal changes. This study indicates that sufficient heat dissipation is crucial for the proper working of a SERS-based microfluidic device.
Bladder cancer is the fourth common malignant disease worldwide, accounting for 4% of all cancer cases. In Singapore, it is the ninth most common form of cancer. The high mortality rate can be reduced by early treatment following precancerous screening. Currently, the gold standard for screening bladder tumors is histological examination of biopsy specimen, which is both invasive and time-consuming. In this study ex vivo urine fluorescence cytology is investigated to offer a timely and biopsy-free means for detecting bladder cancers. Sediments in patients' urine samples were extracted and incubated with a novel photosensitizer, hypericin. Laser confocal microscopy was used to capture the fluorescence images at an excitation wavelength of 488 nm. Images were subsequently processed to single out the exfoliated bladder cells from the other cells based on the cellular size. Intensity histogram of each targeted cell was plotted and feature vectors, derived from the histogram moments, were used to represent each sample. A difference in the distribution of the feature vectors of normal and low-grade cancerous bladder cells was observed. Diagnostic algorithm for discriminating between normal and low-grade cancerous cells is elucidated in this paper. This study suggests that the fluorescence intensity profiles of hypericin in bladder cells can potentially provide an automated quantitative means of early bladder
cancer diagnosis.
Photodynamic diagnosis of bladder carcinoma based on hypericin fluorescence cystoscopy has shown to have a higher degree of sensitivity for the detection of flat bladder carcinoma compared to white light cystoscopy. The potential of the photosensitizer hypericin-induced fluorescence in performing non-invasive optical biopsy to grade bladder cancer in vivo using fluorescence cystoscopic image analysis without surgical resection for tissue biopsy is investigated in this study. The correlation between tissue fluorescence and histopathology of diseased tissue was explored and a diagnostic algorithm based on fluorescence image analysis was developed to classify the bladder cancer without surgical resection for tissue biopsy. Preliminary results suggest a correlation between tissue fluorescence and bladder cancer grade. By combining both the red-to-blue and red-to-green intensity ratios into a 2D scatter plot yields an average sensitivity and specificity of around 70% and 85% respectively for pathological cancer grading of the three different grades of bladder cancer. Therefore, the diagnostic algorithm based on colorimetric intensity ratio analysis of hypericin fluorescence
cystoscopic images developed in this preliminary study shows promising potential to optically diagnose and grade bladder cancer in vivo.
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.
Worldwide, oral cancer is the sixth most common cancer for both sexes. In Singapore, the 5-year survival rate of oral cancer is about 50%. The high mortality rate has been attributed to the difficulties in detecting the disease in an early treatable stage. Currently, the standard screening procedures for oral cancer are histopathology examination of biopsied tissues and exfoliative cytological assessment. These techniques, unfortunately, are low in sensitivity. In this study, we exploit the high amplification factor of SERS to investigate on the possibility of utilising molecular vibrational information from saliva samples to detect oral cancer early. All raw saliva samples were centrifuged at 13,000 krpm for 5 minutes to remove unwanted particles prior to SERS measurements. The purified saliva samples were then applied directly on gold particle films, followed by excitation with a 633 nm HeNe laser. SERS spectrum can be obtained in less than 2 minutes for each sample. We have studied the saliva spectra acquired from 5 normal individuals and 5 patients with oral cancer. In addition, we also observe new peaks at 1097 cm-1 and 1627 cm-1 in some of the abnormal samples. These peaks are not present in the spectra acquired from the normal samples. Preliminary measurements will be presented. This study may lead to the development of a sensitive and portable diagnostics system for oral cancer.
A digitized fluorescence endoscopic imaging (DFEI) system combined with 5-Aminolevulinic Acid (5-ALA) induced protoporphyrin IX (PPIX) was developed for the detection of neoplasms in the oral cavity and uterine-cervix. The system has the capability of producing both the digital and video fluorescence images in real time, and also quantifying the fluorescence images. The results show that using the DFEI system associated with the fluorescence image quantification method, both high sensitivity and specificity can be achieved during the head and neck, and obstetric and gynaecology clinical trials. The red fluorescence intensity distribution in the lesion area can also be obtained after digital image processing to better understand the situation of PPIX accumulation in the tissues. Furthermore, applying the intensity ratio IR/IB at red and blue wavelength regions of the tissue targeted, where IR includes the intensity of PPIX fluorescence and red tissue autofluorescence, and IB is the intensity of diffusely back-scattered excitation blue light, different histopathological grades of lesions can be classified by the DEFI system, suggesting a significant potential of the noninvasive optical biopsy for the early cancer detection.
A digitized fluorescence endoscopy imaging system combined with 5-Aminolevulinic Acid (5-ALA) induced Protoporphyrin IX (PPIX) has been developed for the detection of neoplasms in oral cavity. It mainly consists of the illumination console, fluorescence detection unit, computer system for image acquisition, processing and analysis, and online image display system as well. The developed system can produce both the digital and video fluorescence images in real time, and can be used to quantify fluorescence images acquired. Preliminary results from the Head and Neck clinic show that high sensitivity and high specificity can be achieved. Furthermore, applying the intensity ratios at two different wavelength regions, the developed system shows the capability of differentiating between different histopathological stages of oral lesions, suggesting a significant potential for realizing the non-invasive optical biopsy for early cancer diagnosis.
Fluorescence excitation-emission matrices (EEMs) were measured from normal and tumourous oral tissues in vitro using excitation wavelengths ranging from 220 to 500 nm. The prominent fluorescence excitation-emission peaks: (228 nm; 260/338/475 nm), (282 nm; 337/475/660 nm), (335 nm; 472 nm) and (465 nm, 523 nm) for normal tissues; (228 nm; 335/475/660 nm), (282 nm; 330/660 nm) and (350 nm; 460 nm) for tumour tissues, can be found in EEM maps, indicating the dominant fluorophores responsible for tissue autofluorescence are tryptophan, NADH, FAD, and porphyrin compounds in oral tissues. The optimal excitation wavelengths were found to be 280, 335, and 465 nm. The empirical discrimination algorithms based on I475/I660, and I520/I660 were developed. These algorithms yield an average sensitivity and specificity of 95% and 88%, respectively.
This paper reports further studies of Radiation Induced Fibrosarcoma (RIF-1) tumor cells which have been made resistant to Photodynamic Therapy by multiple treatment and regrowth in vitro using the hematoporphyrin derivative photosensitizer Photofrin. Previous work has shown both structural and functional changes in the mitochondria of the resistant (RIF-8A) cells. Colocalization of Photofrin and the mitochondrial localizer Rhodamine-123 was assessed by double-label confocal fluorescence microscopy (CFM). At 18h Photofrin incubation, there was strong correlation in discrete subcellular sites between Photofrin and Rhodamine fluorescence. However, in RIF-8A cells there were also discrete regions of Rhodamine localization which showed weak or no Photofrin fluorescence. This was not observed in RIF-1 cells. CFM measurements also showed that the Photofrin fluorescence after 18h incubation was reduced by increasing concentration of Rhodamine (30 min. incubation), and that this dependence was different for the two cell types. The RIF-8A cells were also shown to be cross-resistant to cisplatin and to have an associated reduced level of Pt-DNA adducts, suggesting the possibility of increased repair capacity. Cross-resistance was not observed, however, with a ruthenium phthalocyanine photosensitizer nor, as previously reported, with other chemotherapeutic agents such as Adriamcyin. Thus, there is a complex pattern of cross-resistance with these cells. Preliminary observations of the effects of a respiratory chain inhibitor (oligomycin) and an uncoupler of oxidative phosphorylation (FCCP) indicate differences between RIF-8A and RIF-1 which may be related to the condensed mitochondrial structure of the RIF-8A cells.
The validity of a threshold model in photodynamic therapy (PDT) has been demonstrated in various normal animal tissues with porphyrin and phthalocyanine photosensitizers. This model states that, given a specific tissue and photosensitizer (PS), necrosis will result if the number of photons absorbed by the PS per unit volume of tissue exceeds a threshold value. The purpose of this study was to determine the threshold value for normal brain tissue and for intracranially implanted VX2 tumors using a rabbit model. Additionally, the dependence of the threshold value on other factors currently not included in the threshold model, such as oxygenation of the target tissue, was investigated. Calculation of the threshold requires knowledge of three parameters: (1) the radius of necrosis (for interstitial irradiation), (2) the light fluence at the necrotic boundary and (3) the photosensitizer concentration in the tissue. The animals were sensitized using PhotofrinTM at various time-delays between injection of the sensitizer and illumination. The output from an argon laser was coupled into an optical fiber, which was implanted in the brain under stereotactic guidance. The animals were sacrificed 24 to 48 hours after PDT and the radius of necrosis was determined by serial microscopy. These data were used to calculate the photodynamic thresholds for normal brain and tumor tissue.
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