This PDF file contains the front matter associated with SPIE Proceedings Volume 9129, including the Title Page, Copyright information, Table of Contents, Introduction (if any), and Conference Committee listing.

We present a low-cost and rapid fabrication and characterisations of polymer nanotubes based substrates inspired by a Gecko’s foot, and demonstrate its suitability for Surface Enhanced Raman Scattering (SERS) applications. Substrates are fabricated in a simple, scalable and cost efficient way by melt wetting of polystyrene (PS) in an anodised alumina (AAO) template, followed by silver or gold evaporation. Scanning electron microscopy reveals the substrates are composed of a dense array of free-standing polystyrene nanotubes topped by silver nanocaps. The gaps (electromagnetic hot spots) between adjacent nanotubes are measured to be 30nm +/-15nm. SERS characterisation of the substrates, employing a monolayer of 4-aminothiophenol (4-ABT) as a model molecule, exhibits an enhancement factor of ~1.6 × 10⁶. This value is consistent with the one obtained from 3D-Finite Difference Time Domain (3D-FDTD) simulations of a simplified version of the sample. The contact angle of the substrates is measured to be 150°, making them super-hydrophobic. This later property renders the samples compatible to very low sample volumes and highly sensitive detection (down to 408ppt) of the environmental pollutant crystal violet in water is demonstrated.

We propose confocal Raman imaging as a label-free single cell characterization method that can be used as an alternative for conventional cell identification techniques that typically require labels, long incubation times and complex sample preparation. In this study it is investigated whether cancer and blood cells can be distinguished based on their Raman spectra. 2D Raman scans are recorded of 114 single cells, i.e. 60 breast (MCF-7), 5 cervix (HeLa) and 39 prostate (LNCaP) cancer cells and 10 monocytes (from healthy donors). For each cell an average spectrum is calculated and principal component analysis is performed on all average cell spectra. The main features of these principal components indicate that the information for cell identification based on Raman spectra mainly comes from the fatty acid composition in the cell. Based on the second and third principal component, blood cells could be distinguished from cancer cells; and prostate cancer cells could be distinguished from breast and cervix cancer cells. However, it was not possible to distinguish breast and cervix cancer cells. The results obtained in this study, demonstrate the potential of confocal Raman imaging for cell type classification and identification purposes.

Raman spectroscopy is a powerful tool for analyzing the composition of biological samples in terms of biomolecular content. Over the past two decades there has been considerable interest in the application of Raman to measuring the concentration of the various constituents in a multicomponent mixture. This is achieved by first building a database of the Raman spectra of the individual components in a pure form. Following this a least squares algorithms is applied to find a best fit that accounts for the spectrum of the mixture. The weights returned by a partial least squares algorithm indicate the relative concentration of each component. Of particular interest has been application of the method to estimate the concentration of various analytes in blood and urine samples, including glucose. In this paper we briefly review the subject of multicomponent analysis by Raman Spectroscopy in terms of experimental methodology, limits of measurement, and applications

Raman microspectroscopy can be applied to the urinary bladder for highly accurate classification and diagnosis of bladder cancer. This technique can be applied in vitro to bladder epithelial cells obtained from urine cytology or in vivo as an optical biopsy" to provide results in real-time with higher sensitivity and specificity than current clinical methods. However, there exists a high degree of variability across experimental parameters which need to be standardised before this technique can be utilized in an everyday clinical environment. In this study, we investigate different laser wavelengths (473 nm and 532 nm), sample substrates (glass, fused silica and calcium fluoride) and multivariate statistical methods in order to gain insight into how these various experimental parameters impact on the sensitivity and specificity of Raman cytology.

We discuss an advanced variant of the correlation-stability (CS) approach to OCT elastography that is capable of quantifying the stiffness differences. The modified variant is based on natural combination of CS approach with the speckle-variance (SV) approach. It allows one to determine the strain dependence of the normalized speckle intensity variance function for two compared subsets taken from the OCT images corresponding to the initial and deformed states of the tissue. In previous studies we considered the basic dependence of the normalized speckle intensity variance function on the tissue strain under the assumption that the influence of translational displacements can be excluded, so that the residual speckle-intensity variations should be produced only by speckle blinking determined by local strains. In the present report we discuss the corresponding algorithms allowing one to exclude the above-mentioned influence of translational displacements. We demonstrate numerically the efficiency of such processing that allows for quantification of stiffness differences in the elastographic mapping based on the CS approach.

In this report, we present a comparative discussion of a recently proposed method of elastographic mapping based on comparison of correlation stability (CS) of different parts of sequentially obtained OCT images of the strained tissue and more conventional correlation approaches to elastographic mapping based on the initial reconstruction of the displacement field. The performed study is based on numerical simulations of speckle patterns generated by reproducing main features of image formation in real OCT scanners. Distortions of such speckle patterns caused by either translational motion of the studied sample or its straining are also incorporated in the developed simulation method. Furthermore, both purely geometrical image distortions and the effects of speckle blinking and boiling are incorporated in the used approach. We demonstrate that properly made correlation processing makes it possible to visualize the differences in the local strains without the necessity of applying error-sensitive procedures of numerical differentiation. Different role of the ratio between the coherence length and the optical wavelength for different variants of correlation based elastographic imaging is discussed. We argue that the proposed CS approach opens attractive possibilities for implementation of elastographic mapping in OCT in free-hand mode.

Saturation artifact is a significant image noise due to the limited detectable maximum light intensity of the CCD camera in the spectrometer module of spectral-domain optical coherence tomography (SD-OCT). This effect makes the OCT image appear with saturation noise in certain areas along with even more obvious vertical lines of noise. The unwanted artifacts cover the real sample structure information and can render the image unclear. This artifact problem is significant when using highly focused objective lenses for improving image transverse resolution. In this study, we developed a parabolic-based reconstruction method to estimate the saturated parts of the measured spectra in order to obtain the corresponding pseudo spectra. The saturation artifact is clearly reduced when the OCT images are processed from the pseudo spectra.

Complex investigation of malignant tumors was performed with combined optical coherence tomography (OCT) and Raman spectroscopy (RS) setup: 22 ex vivo lung tissue samples and 23 in vivo experiments with skin tumors. It was shown that combined RS-OCT unit may be used for precise tissue morphology visualization with simultaneous tumor type determination (BCC, malignant melanoma of skin tissues, adenocarcinoma and squamous cell carcinoma of lung). Fast RS phase method for skin and lung tumors identification was proposed. It is based on alteration of Raman spectral intensity in 1300-1340, 1440-1460 and 1640-1680 cm^-1 bands for healthy and malignant tissue. Complex method could identify: malignant melanoma with 88.9% sensitivity and 87.8% specificity; adenocarcinoma with 100% sensitivity and 81.5% specificity; squamous cell carcinomas with 90.9% sensitivity and 77.8% specificity.

Coating of tablets is a widely applied unit operation in the pharmaceutical industry. Thickness and uniformity of the coating layer are crucial for efficacy as well as for compliance. Not only due to different initiatives it is thus essential to monitor and control the coating process in-line. Optical coherence tomography (OCT) was already shown in previous works to be a suitable candidate for in-line monitoring of coating processes. However, to utilize the full potential of the OCT technology an automatic evaluation of the OCT measurements is essential. The automatic evaluation is currently implemented in MATLAB and includes several steps: (1) extraction of features of each A-scan, (2) classification of Ascan measurements based on their features, (3) detection of interfaces (air/coating and coating/tablet core), (4) correction of distortions due to the curvature of the bi-convex tablets and the oblique orientation of the tablets, and (5) determining the coating thickness. The algorithm is tested on OCT data acquired by moving the sensor head of the OCT system across a static tablet bed. The coating thickness variations of single tablets (i.e., intra-tablet coating variability) can additionally be analyzed as OCT allows the measurement of the coating thickness on multiple displaced positions on one single tablet. Specifically, the information about those parameters emphasizes the high capability of the OCT technology to improve process understanding and to assure a high product quality.

Full-field optical coherence microscopy (FF-OCM) is an established optical technology based on low-coherence interference microscopy for high-resolution non-invasive three-dimensional imaging of semi-transparent samples. We present an extension of the technique setting up an achromatic imaging system over a spectral range extending from 530 nm to 1700 nm, to provide tomographic images in three distinct bands centered at 635 nm, 870 nm and 1170 nm. Image contrast enhancement as well as sample characterization is performed using the conventional RGB color channel representation. Light is emitted by a halogen lamp and then separates into two arms of a Linnik-type interferometer with microscope objectives placed in each arm. The images are projected onto a visible to short-wavelength infrared detector based on an InGaAs photodiode array. Enface oriented tomographic images are obtained by arithmetic combination of four phase-shifted interferometric images. Great care was taken to reach similar performances in the three bands. An axial resolution of ~1.9μm and a transverse resolution of ~2.4μm are achieved in the three bands. A dynamic dispersion compensation system is set up to preserve axial resolution and signal intensity level when the imaging depth is varied. Images of biological samples revealing their spectral properties are shown as illustration of improved detection capability with enhanced contrast.

Using Doppler optical coherence tomography (DOCT) we study stress-related intracranial hemorrhages (ICHs) in newborn rats. We investigate a masked stage of ICH development that corresponds to the first 4 h after the stress. We show that this period is characterized by significant changes in the diameter of the sagittal vein and the velocity of the cerebral venous blood flow (CVBF). We discuss diagnostic abilities of wavelet-based methods and consider an adaptive technique allowing us to reveal clearest distinctions in the dynamics of CVBF between normal and stressed newborn rats. Finally, we conclude that the venous insufficiency in newborns and a reduced response of the sagittal vein to adrenaline are related to important prognostic markers of the risk of ICH development.

The method of intraoperative analysis of tumor markers such as structural changes, concentrations of 5- ALA induced protoporphyrin IX and hemoglobin in the area of tissue resection was developed. A device for performing this method is a neurosurgical aspiration cannulae coupled with the fiber optic probe. The configuration of fibers at the end of cannulae was developed according to the results of numerical modeling of light distribution in biological tissues. The optimal distance between the illuminating and receiving fiber was found for biologically relevant interval of optical properties. On this particular distance the detected diffuse reflectance depends on scattering coefficient almost linearly. Array of optical phantoms containing hemoglobin, protoporphyrin IX and fat emulsion (as scattering media) in various concentrations was prepared to verify the method. The recovery of hemoglobin and protoporphyrin IX concentrations in the scattering media with an error less than 10% has been demonstrated. The fat emulsion concentration estimation accuracy was less than 12%. The first clinical test was carried out during glioblastoma multiforme resection in Burdenko Neurosurgery Institute and confirmed that sensitivity of this method is enough to detect investigated tumor markers in vivo. This method will allow intraoperative analysis of the structural and metabolical tumor markers directly in the zone of destruction of tumor tissue, thereby increasing the degree of radical removal and preservation of healthy tissue.

Differential interference contrast images (DIC) are the direct representation of the refractive index fluctuations of human cervical tissues. These refractive index fluctuations are known to follow self-similar behaviour and in general are multifractal in nature. In this present study, multifractal detrended fluctuation analysis (MFDFA) on refractive index fluctuations from DIC images has been performed by unfolding the tissue-images horizontally and vertically. Our analysis clearly shows that refractive index fluctuations of human cervical tissues are anisotropic-fractal in nature and anisotropy reduced as cancer progress.

In order to study, for example, the influence of pharmaceuticals or pathogens on different cell types under identical measurement conditions and to analyze interactions between different cellular specimens a minimally-invasive quantitative observation of mixed cell cultures is of particular interest. Quantitative phase microscopy (QPM) provides high resolution detection of optical path length changes that is suitable for stain-free minimally-invasive live cell analysis. Due to low light intensities for object illumination, QPM minimizes the interaction with the sample and is in particular suitable for long term time-lapse investigations, e.g., for the detection of cell morphology alterations due to drugs and toxins. Furthermore, QPM has been demonstrated to be a versatile tool for the quantification of cellular growth, the extraction morphological parameters and cell motility. We studied the feasibility of QPM for the analysis of mixed cell cultures. It was explored if quantitative phase images provide sufficient information to distinguish between different cell types and to extract cell specific parameters. For the experiments quantitative phase imaging with digital holographic microscopy (DHM) was utilized. Mixed cell cultures with different types of human pancreatic tumor cells were observed with quantitative DHM phase contrast up to 35 h. The obtained series of quantitative phase images were evaluated by adapted algorithms for image segmentation. From the segmented images the cellular dry mass and the mean cell thickness were calculated and used in the further analysis as parameters to quantify the reliability the measurement principle. The obtained results demonstrate that it is possible to characterize the growth of cell types with different morphologies in a mixed cell culture separately by consideration of specimen size and cell thickness in the evaluation of quantitative DHM phase images.

The Laser Doppler flowmetry (LDF) is a non-invasive method for estimating the tissular blood flow and speed at a microscopic scale (microcirculation). It is used for medical research as well as for the diagnosis of diseases related to circulatory system tissues and organs including the issues of microvascular flow (perfusion). It is based on the Doppler effect, created by the interaction between the laser light and tissues. LDF measures the mean blood flow in a volume formed by the single laser beam, that penetrate into the skin. The size of this measurement volume is crucial and depends on skin absorption, and is not directly reachable. Therefore, current developments of the LDF are focused on the use of always more complex and sophisticated signal processing methods. On the other hand, laser Double Doppler Flowmeter (FL2D) proposes to use two laser beams to generate the measurement volume. This volume would be perfectly stable and localized at the intersection of the two laser beams. With FL2D we will be able to determine the absolute blood flow of a specific artery. One aimed application would be to help clinical physicians in health care units.

In light of the population aging in many developed countries, there is a great economical interest in improving the speed and cost-efficiency of healthcare. Clinical diagnosis tools are key to these improvements, with biophotonics providing a means to achieve them. Standard optical microscopy of in vitro biological samples has been an important diagnosis tool since the invention of the microscope, with well known resolution limits. Nonlinear optical imaging improves on the resolution limits of linear microscopy, while providing higher contrast images and a greater penetration depth due to the red-shifted incident light compared to standard optical microscopy. It also provides information on molecular orientation and chirality. Adaptive optics can improve the quality of nonlinear optical images. We analyzed the effect of sensorless adaptive optics on the quality of the nonlinear optical images of biological samples. We demonstrate that care needs to be taken when using a large field of view. Our findings provide information on how to improve the quality of nonlinear optical imaging, and can be generalized to other in vitro biological samples. The image quality improvements achieved by adaptive optics should help speed up clinical diagnostics in vitro, while increasing their accuracy and helping decrease detection limits. The same principles apply to in vivo biological samples, and in the future it may be possible to extend these findings to other nonlinear optical effects used in biological imaging.

DIC (Differential Interference Contrast Image) images of cervical pre-cancer tissues are taken from epithelium region, on which wavelet transform and multi-fractal analysis are applied. Discrete wavelet transform (DWT) through Daubechies basis are done for identifying fluctuations over polynomial trends for clear characterization and differentiation of tissues. A systematic investigation of denoised images is carried out through the continuous Morlet wavelet. The scalogram reveals the changes in coefficient peak values from grade-I to grade-III. Wavelet normalized energy plots are computed in order to show the difference of periodicity among different grades of cancerous tissues. Using the multi-fractal de-trended fluctuation analysis (MFDFA), it is observed that the values of Hurst exponent and width of singularity spectrum decrease as cancer progresses from grade-I to grade-III tissue.

Nonlinear optical phenomena cover a broad research area. The emphasis is mostly on the generation of higher harmonics to be used in laser designs or on the characterization capabilities of nonlinear optics. The latter ability of nonlinear optics is important when combined with a microscope to detect simultaneously multiphoton fluorescence and second-harmonic generation. Submicron size features can then be investigated separately and information on their structure can be revealed by second-harmonic generation. For example, the point group symmetry can be determined in situ and in vivo in complex media. Moreover, nonlinear optical microscopy has several additional advantages: the generation and detection of nonlinear signals is intrinsically confocal and degradation, if present, only occurs at a localized places in the structure. In biological structures, multiphoton fluorescence and second-harmonic generation do not necessarily occur in the same type of the structure. This can be exploited to visualize different structures in one sample by simultaneous detection of two-photon fluorescence and second-harmonic generation. Also, the incident beam can be tuned to fit in the biological window of biological structures, which gives second-harmonic generation microscopy a significant advantage over linear microscopy due to absorbance issues in the visible wavelength range. We exploit these advantages to characterize collagen-bearing biological structures. Collagen is the dominant structural protein in connective tissue in mammals. Being the most abundant protein in the mammal clade, it is essential for the very existence of it. Collagen is a protein with a very strict quaternary structure. The most simple Ramachandran model states that an amino-acid sequence of Glycine-prolin-hydroxyprolin leads to a right-handed helical structure. The inherent stability is such that a sole helix cannot exist for a prolonged period of time, it will therefore combine with 2 near identical helices, resulting in the formation of the superhelical structure tropocollagen. Subsequently tropocollagen will align in a linear direction forming the fibers composing collagen-tissue. Due to the superhelical nature of collagen, this structure is ideal to be probed by second-harmonic generation.

The paper describes results of development of fundus-camera with non-glare optical scheme. The scheme is based on multiple lenses with a light gathering power (D/F ≤ 1) substantially less than one. Illumination of fundus can be provided through eye’s pupil less than 3 mm. And much attention was directed to method of calculation of the no-glare optical scheme. The key idea is that geometry of optic elements of the system provides that glare in the form of ghost reflection from optical surface of one element focuses on a small-size absorbing screen located on another optical surface. Shows the possibility of implementation and the experimental results. During experiments with B/W camera we got Full HD color image of fundus having the eye’s pupil diameter of 1.5 mm and illumination of fundus tissue in accordance with sanitary rules.

Microfractures in bone, secondary to repetitive stress, particularly in the lower extremities, are an important problem for military recruits and for athletes. They also may occur in those with brittle bones, such as the elderly, or in patients taking bisphosphonates for osteoporosis. Microfractures can be early predictors of major bone fracture and may be as important as changes in bone density in predicting where and how likely a major fracture will occur. Unlike major bone fractures, microfractures can be difficult to detect by conventional methods. We explored a second NIR spectral window from 1,100 nm to 1,350 nm, and a third spectral window from 1,600 nm to 1,870 nm to image microfractures through tissue media. Due to a reduction in scattering at longer NIR wavelengths, employment of the second and third NIR windows may allow for deeper penetration into tissue and higher contrast images of microfractures underneath the skin.

The SPADnet FP7 European project is aimed at a new generation of fully digital, scalable and networked photonic components to enable large area image sensors, with primary target gamma-ray and coincidence detection in (Time-of- Flight) Positron Emission Tomography (PET). SPADnet relies on standard CMOS technology, therefore allowing for MRI compatibility. SPADnet innovates in several areas of PET systems, from optical coupling to single-photon sensor architectures, from intelligent ring networks to reconstruction algorithms. It is built around a natively digital, intelligent SPAD (Single-Photon Avalanche Diode)-based sensor device which comprises an array of 8×16 pixels, each composed of 4 mini-SiPMs with in situ time-to-digital conversion, a multi-ring network to filter, carry, and process data produced by the sensors at 2Gbps, and a 130nm CMOS process enabling mass-production of photonic modules that are optically interfaced to scintillator crystals. A few tens of sensor devices are tightly abutted on a single PCB to form a so-called sensor tile, thanks to TSV (Through Silicon Via) connections to their backside (replacing conventional wire bonding). The sensor tile is in turn interfaced to an FPGA-based PCB on its back. The resulting photonic module acts as an autonomous sensing and computing unit, individually detecting gamma photons as well as thermal and Compton events. It determines in real time basic information for each scintillation event, such as exact time of arrival, position and energy, and communicates it to its peers in the field of view. Coincidence detection does therefore occur directly in the ring itself, in a differed and distributed manner to ensure scalability. The selected true coincidence events are then collected by a snooper module, from which they are transferred to an external reconstruction computer using Gigabit Ethernet.

Biosensing is important for detection and characterization of microorganisms. When the detection and characterization of targeted microorganisms require micron-scale resolutions, optical biosensing techniques are especially beneficial. Optical biosensing can be applied through direct or indirect optical sensing techniques. The latter have demonstrated especially high sensitivities for the detection of targeted microorganisms with labeling. Unfortunately, such systems rely on high-resolution microscopy with microscopic sampling areas to image the labeled target microorganisms. This leads to long characterization times for applications such as pathogen detection in water quality monitoring where users must scan the micron-scale sampling areas across millimeter- or even centimeter-scale samples. This work introduces retroreflector labels for the detection and characterization of microorganisms for macroscopic sample sizes. The demonstrated retroreflective imaging system uses a laser source to illuminate the sample, in lieu of the fluorescent excitation source, and micron-scale retroreflector labels, in lieu of fluorescent stains/proteins. Antibodies are used to bind retroreflectors to targeted microorganisms. The presence of these microscopic retroreflector-microorganism pairs is monitored in a retroreflected image that is captured by a distant image sensor which shows a well-localized retroreflected beamspot for each pair. Characteristics of an appropriately-designed retroreflective imaging system which provide a quantifiable record of microorganism-coupled retroreflectors across macroscopic sample sizes are presented. Retroreflection directionality, collimation, and contrast are investigated for both corner-cube retroreflectors and spherical retroreflectors (of varying refractive indices). It is ultimately found that such a system is an effective tool for the detection and characterization of microorganism targets, down to a single-target detection limit.

This paper presents the design, simulation, fabrication, and characterization of a thin-film Fabry-Perot resonator composed of titanium dioxide (TiO₂) and silicon dioxide (SiO₂) thin-films. The optical filter is developed to be integrated with a light emitting diode (LED) for enabling narrow-band imaging (NBI) in endoscopy. The NBI is a high resolution imaging technique that uses spectrally centered blue light (415 nm) and green light (540 nm) to illuminate the target tissue. The light at 415 nm enhances the imaging of superficial veins due to their hemoglobin absorption, while the light at 540 nm penetrates deeper into the mucosa, thus enhances the sub-epithelial vessels imaging. Typically the endoscopes and endoscopic capsules use white light for acquiring images of the gastrointestinal (GI) tract. However, implementing the NBI technique in endoscopic capsules enhances their capabilities for the clinical applications. A commercially available blue LED with a maximum peak intensity at 404 nm and Full Width Half Maximum (FWHM) of 20 nm is integrated with a narrow band blue filter as the NBI light source. The thin film simulations show a maximum spectral transmittance of 36 %, that is centered at 415 nm with FWHM of 13 nm for combined the blue LED and a Fabry Perot resonator system. A custom made deposition scheme was developed for the fabrication of the blue optical filter by RF sputtering. RF powered reactive sputtering at 200 W with the gas flows of argon and oxygen that are controlled for a 5:1 ratio gives the optimum optical conditions for TiO₂ thin films. For SiO₂ thin films, a non-reactive RF sputtering at 150 W with argon gas flow at 15 sccm results in the best optical performance. The TiO₂ and SiO₂ thin films were fully characterized by an ellipsometer in the wavelength range between 250 nm to 1600 nm. Finally, the optical performance of the blue optical filter is measured and presented.

An organic solvent-free sugar-based transparency nanopatterning material which had specific desired properties such as nanostructures of subwavelength grating and moth-eye antireflection, acceptable thermal stability of 160 °C, and low imaginary refractive index of less than 0.005 at 350-800 nm was proposed using electron beam lithography. The organic solvent-free sugar-based transparency nanopatterning material is expected for non-petroleum resources, environmental affair, safety, easiness of handling, and health of the working people, instead of the common developable process of tetramethylammonium hydroxide. 120 nm moth-eye antireflection nanopatterns images with exposure dose of 10 μC/cm² were provided by specific process conditions of electron beam lithography. The developed sugar derivatives with hydroxyl groups and EB sensitive groups in the organic solvent-free sugar-based transparency nanopatterning material were applicable to future development of optical interface films of biology and electronics as a novel chemical design.

The misfolding and aggregation of amyloid proteins has been associated with incurable diseases such as Alzheimer's or Parkinson's disease. In the specific case of Alzheimer's disease, recent studies have shown that cell toxicity is caused by soluble oligomeric forms of aggregates appearing in the early stages of aggregation, rather than by insoluble fibrils. Research on new strategies of diagnosis is imperative to detect the disease prior to the onset of clinical symptoms. Here, we propose the use of an optical method for protein aggregation dynamic studies using a Bloch surface wave based approach. A one dimension photonic crystal made of a periodic stack of silicon oxide and silicon nitride layers is used to excite a Bloch surface wave, which is sensitive to variation of the refractive index of an aqueous solution. The aim is to detect the early dynamic events of protein aggregation and fibrillogenesis of the amyloid-beta peptide Aβ42, which plays a central role in the onset of the Alzheimer’s disease. The detection principle relies on the refractive index changes caused by the depletion of the Aβ42 monomer concentration during oligomerization and fibrillization. We demonstrate the efficacy of the Bloch surface wave approach by monitoring in real-time the first crucial steps of Aβ42 oligomerization.

The angular light scattering profile of microscopic particles significantly depends on their morphological parameters, such as size and shape. This dependency is widely used in state-of-the-art flow cytometry methods for particle classification. We recently introduced the spectrally encoded angular light scattering (SEALS) method, with potential application in scanning flow cytometry (SFC). We show that a one-to-one wavelength-to-angle mapping enables the measurement of the angular dependence of scattered light from microscopic particles over a wide dynamic range. Improvement in dynamic range is obtained by equalizing the angular scattering dependence via spectral equalization. The resulting continuous angular spectrum is obtained without mechanical scanning, enabling single-shot measurement. Using this information, particle morphology can be determined with improved accuracy. We derive and experimentally verify an analytic wavelength-to-angle mapping model, facilitating rapid data processing. As a proof of concept, we demonstrate the method’s capability of distinguishing differently sized polystyrene beads. The combination of SEALS with time-stretch dispersive Fourier transform (TS-DFT) offers real-time and high-throughput (high frame rate) measurements and renders the method suitable for integration in standard flow cytometers: By transforming the spectrum into time and slowing the time scale, using group velocity dispersion (GVD), single-shot spectra can be obtained at high throughput, using a photodiode and a real-time digitizer. The amount of group velocity dispersion is chosen to time-stretch the optical pulses, that is, to slow them down, such that they do not overlap and may be digitized in real-time.

Flow cytometry relies on the detection of cells selectively stained with fluorescence markers. Optically they can be detected as fluorescence particles. The use of microuidics offers a wide range of benefits over traditional flow cytometer designs but when replacing expensive components with inexpensive counterparts the sensitivity of the instrument suffers. To increase the sensitivity of the detection system, spatial modulation has been proposed. Spatial modulation is implemented via _ne pitched shadow masks close to the microuidic channel which generate a signal pattern when a fluorescent particle passes by. Using a _ne pitch and long total length for the pattern a high spatial resolution and long total exposure time are combined. Particle detection from local maxima is not directly possible with spatially modulated signals due to the jagged pulse shape. We compare the performance of different approaches for particle detection from local maxima. Matched filtering and the derivative of the correlation signal provide either a good peak-signal-to-noise ratio (PSNR) or a high spatial resolution. But both approaches suffer from low dynamic range due to side maxima. We derive the solution for a minimum- mean-square-error (MMSE) filter which transforms the modulated pulse shape into a target pulse shape with a single strong maximum. We investigate the performance of the MMSE filter and find that it provides tunable suppression of noise and side maxima along with a high spatial resolution. The use of the MMSE filter therefore is an ideal choice for particle detection from spatially modulated signals.

The aim of this study was to evaluate the porphyrin involvement in the red fluorescence observed in dental caries with Soprolife^® light-induced fluorescence camera in treatments mode (SOPRO, ACTEON Group, La Ciotat, France) and Vistacam^® camera (DÜRR DENTAL AG, Bietigheim-Bissingen, Germany). The International Caries Detection and Assessment System (ICDAS) was used to rand the samples. Human teeth cross-sections, ranked from ICDAS score 0 to 6, were examined by epi-fluorescence microscopy and Confocal Raman microscopy. Comparable studies were done with Protoporphyrin IX, Porphyrin I and Pentosidine solutions. An RGB analysis of Soprolife^® images was performed using ImageJ Software (1.46r, National Institutes of Health, USA). Fluorescence spectroscopy and MicroRaman spectroscopy revealed the presence of Protoporphyrin IX, in carious enamel, dentin and dental plaque. However, the presence of porphyrin I and pentosidine cannot be excluded. The results indicated that not only porphyrin were implicated in the red fluorescence, Advanced Glygation Endproducts (AGEs) of the Maillard reaction also contributed to this phenomenon.

The architecture of collagen is important in maintenance and regeneration of higher vertebrates’ tissues. We had been studying the changes to this architecture with in situ multi-photon optical microscopy that combines nonlinear optical phenomena of second harmonic generation (SHG) and two-photon fluorescence (TPF) signals from collagen hydrogels prepared from different collagen solid content, polymerized at different temperatures, with different ions as well as modified with reducing sugars. We incubated 2 g/l collagen hydrogels with 0.1 M fructose at 37 °C and after about 20 days observed a significant induction of in situ fluorescence. The twophoton fluorescence emission was centered at about 460 nm for 730 nm excitation wavelength and shifted to 480 nm when we changed the excitation wavelength to 790 nm. The one-photon fluorescence emission was centered at about 416 nm when excitation was 330 nm. It red shifted and split into two peaks centered at about 430 nm and 460 nm for 370 nm excitation; 460 nm peak became predominant for 385 nm excitation and further shifted to 470 nm for 390 nm excitation. SHG and TPF imaging showed restructuring of hydrogels upon this modification. We will discuss these findings within the context of our ongoing dermal wound repair research.

T-tubules (TT) are invaginations of the surface sarcolemma (SS) that mediate the rapid propagation of the action potential (AP) to the cardiomyocyte core. We employed the advantages of an ultrafast random access multi-photon (RAMP) microscope (Sacconi et al., PNAS 2012) with a double staining approach to optically record t-tubular AP and, simultaneously, the corresponding local Ca2+-release in different positions across the cardiomyocytes. Despite a uniform AP between SS and TT at steady-state stimulation, in control cardiomyocytes we observed a non-negligible be variability of local Ca2+-transient amplitude and kinetics. This variability was significantly reduced by applying 0.1μM Isoproterenol, which increases the opening probability of Ca2+-release units. In the rat heart failure model (HF), we previously demonstrated that some tubular elements fail to propagate AP. We found that the tubules unable to propagate AP, displayed a reduced correspondent Ca2+-transient amplitude as well as a slower Ca2+ rise compared to electrically coupled tubules. Moreover variability of Ca2+-transient kinetics were increased in HF. Finally, TT that did not show AP, occasionally exhibited spontaneous depolarizations that were never accompanied by local Ca2+-release in the absence of any pro-arrhythmogenic stimulation. Simultaneous recording of AP and Ca2+-transient allows us to probe the spatio-temporal variability of Ca2+-release, whereas the investigation of Ca2+-transient in HF discloses an unexpected uncoupling between t-tubular depolarization and Ca2+-release in remodeled tubules. This work was funded by the European Union 7th Framework Program (FP7/2007- 2013) under grant agreement n° 284464, 241526, by the Italian Ministry of University and Research (NANOMAX), and by Telethon-Italy (GGP13162).

Alterations in intracellular cardiomyocyte calcium handling have a key role in initiating and sustaining arrhythmias. Arrhythmogenic calcium leak from sarcoplasmic reticulum (SR) can be attributed to all means by which calcium exits the SR store in an abnormal fashion. Abnormal SR calcium exit maymanifest as intracellular Ca2+ sparks and/or Ca2+ waves. Ca2+ signaling in arrhythmogenesis has been mainly studied in isolated cardiomyocytes and given that the extracellular matrix influences both Ca2+ and membrane potential dynamics in the intact heart and underlies environmentally mediated changes, understanding how Ca2+ and voltage are regulated in the intact heart will represent a tremendous advancement in the understanding of arrhythmogenic mechanisms. Using novel high-speed multiphoton microscopy techinques, such as multispot and random access, we investigated animal models with inherited and acquired arrhythmias to assess the role of Ca2+ and voltage signals as arrhythmia triggers in cell and subcellular components of the intact heart and correlate these with electrophysiology.

Photoacoustic (PA) imaging is an emerging technology that could map the functional contrasts in deep biological tissues in high resolution by “listening” to the laser induced thermoelastic waves. Almost all of the current studies in PA imaging are focused on the intensity of the PA signals as an indication of the optical absorbance of the biological tissues. Our group has for the first time demonstrated that the frequency domain power distribution of the broadband PA signals encode the texture information within the regions-of-interest (ROI). Following the similar method of ultrasound spectral analysis (USSA), photoacoustic spectrum analysis (PASA) could evaluate the relative concentrations and, more importantly, the dimensions of microstructures of the optically absorbing materials in biological tissues, including lipid, collagen, water and hemoglobin. By providing valuable insights into tissue pathology, PASA should benefit basic research and clinical management of many diseases, and may help achieve eventual “noninvasive biopsy”. In this work, taking advantage of the optical absorption contrasts contributed by lipid and hemoglobin at 1200-nm and 532-nm wavelengths respectively, we investigated the capability of PASA in identifying histological changes corresponding to fat accumulation livers through the study on ex vivo and in situ mouse models. The PA signals from the mouse livers were acquired using our PA and US dual-modality imaging system, and analyzed in the frequency domain. After quantifying the power spectrum by fitting it to a first order model, three spectral parameters, including the intercept, the midband fit and the slope, were extracted and used to differentiate fatty livers from normal livers. The comparison between the PASA parameters from the normal and the fatty livers supports our hypotheses that PASA can quantitatively identify the microstructure changes in liver tissues for differentiating normal and fatty livers.

Several recent studies have described the use of infrared (IR) nanoimaging for non-invasive chemical discrimination of subcellular features and intracellular exogenous agents. In this work we outline a number of improvements in both quantitative IR nanoimage analysis and optical system improvements which enable recovery of nanoscale subcellular chemical localization with improved chemical precision. Additionally, we demonstrate how a combination of IR absorption nanoimaging and topographic data can produce subcellular chemical density and complexity maps, which can illustrate several cellular features of interest, including the label free localization of nuclei for both healthy and cancerous cell lines with sub 40nm accuracy. As many cell processes related to disease are governed by the position and dynamics of subcellular features, we present the ability to map biochemical inhomogeneity of cancer cells at nanoscale resolution as a means to explore the subcellular biomechanics underlying carcinogenesis.

We investigated more than 500 clinical cases to receive the spectral properties of basal cell (136 patients) and squamous cell carcinoma (28), malignant melanoma (41) and different cutaneous dysplastic and benign cutaneous lesions. Excitation at 365, 385 and 405 nm using LEDs sources is applied to obtain autofluorescence spectra, and broad-band illumination in the region of 400-900 nm is used to detect diffuse reflectance spectra of all pathologies investigated. USB4000 microspectrometer (Ocean Optics Inc, USA) is applied as a detector and fiber-optic probe is used for delivery of the light. In the case of in vivo tumor measurements spectral shape and intensity changes are observed that are specific for a given type of lesion. Autofluorescence origins of the signals coming from skin tissues are mainly due to proteins, such as collagen, elastin, keratin, their cross-links, co-enzimes – NADH and flavins and endogenous porphyrins. Spectral features significant into diffuse spectroscopy diagnosis are related to the effects of re-absorption of hemoglobin and its forms, as well as melanin and its concentration in different pathologies. We developed significant database and revealed specific features for a large class of cutaneous neoplasia, using about 30 different spectral peculiarities to differentiate cutaneous tumors. Sensitivity and specificity obtained exceed 90%, which make optical biopsy very useful tool for clinical practice. These results are obtained in the frames of clinical investigations for development of significant “spectral features” database for the most common cutaneous malignant, dysplastic and benign lesions. In the forthcoming plans, our group tries to optimize the existing experimental system for optical biopsy of skin, and to introduce it and the diagnostic algorithms developed into clinical practice, based on the high diagnostic accuracy achieved.

Localization and determination of blood region parameters in skin tissue can serve as a valuable supplement to standard non invasive techniques, especially in accessing the degree of depth of burns on skin and for the classification of vascular malformations. Quantitative optical examination of skin local blood region requires the use of depth sensitive techniques and preferential probing for assessment of data from specific layers of skin tissue. This work incorporates the depth sensitivity of diffuse reflectance spectroscopy and optimal source to detector fiber separation for maximum reflectance collection efficiency from local blood region in skin. Monte Carlo simulation for diffuse reflectance was performed on a multi layered skin tissue model consisting of epidermis, perfused dermis and localized blood region. It was found that the slope of the spatially resolved reflectance curve plotted with respect to the source to detector separation distance in semi log scale varies with the depth of the local blood region at specific wavelengths corresponding to the absorption wavelengths of hemoglobin. From the depth information obtained from the spatially resolved reflectance data, the optimum source to detector separation (SDS) is determined for maximum collection efficiency from the chromophore layer. The results obtained from simulation suggest the design of a linearly variable source to detector separation probe for preferential analysis of the depth of a specific tissue layer and subsequent determination of optimal source to detector separation for extracting the layer information.

Determining the optical properties of biological tissues in vivo from spectral intensity measurements performed at their surface is still a challenge. Based on spectroscopic data acquired, the aim is to solve an inverse problem, where the optical parameter values of a forward model are to be estimated through optimization procedure of some cost function. In many cases it is an ill-posed problem because of small numbers of measures, errors on experimental data, nature of a forward model output data, which may be affected by statistical noise in the case of Monte Carlo (MC) simulation or approximated values for short inter-fibre distances (for Diffusion Equation Approximation (DEA)). In case of optical biopsy, spatially resolved diffuse reflectance spectroscopy is one simple technique that uses various excitation-toemission fibre distances to probe tissue in depths. The aim of the present contribution is to study the characteristics of some classically used cost function, optimization methods (Levenberg-Marquardt algorithm) and how it is reaching global minimum when using MC and/or DEA approaches. Several methods of smoothing filters and fitting were tested on the reflectance curves, I(r), gathered from MC simulations. It was obtained that smoothing the initial data with local regression weighted second degree polynomial and then fitting the data with double exponential decay function decreases the probability of the inverse algorithm to converge to local minima close to the initial point of first guess.

Our sight is a major contributor to our quality of life. The treatment of diseases like macular degeneration and glaucoma, however, presents a challenge as the delivery of medication to ocular tissue is not well understood. The instrument described here will help quantify targeted delivery by non-invasively and simultaneously measuring light reflected from and fluorescence excited in the eye, used as position marker and to track compounds respectively. The measurement concept has been proven by monitoring the diffusion of fluorescein and a pharmaceutical compound for treating open angle glaucoma in vitro in a cuvette and in ex vivo porcine eyes. To obtain a baseline of natural fluorescence we measured the change in corneal and crystalline lens autofluorescence in volunteers over a week. We furthermore present data on 3D ocular autofluorescence. Our results demonstrate the capability to measure the location and concentration of the compound of interest with high axial and temporal resolution of 178 μm and 0.6 s respectively. The current detection limit is 2 nM for fluorescein, and compounds with a quantum yield as low as 0.01 were measured to concentrations below 1 μM. The instrument has many applications in assessing the diffusion of fluorescent compounds through the eye and skin in vitro and in vivo, measuring autofluorescence of ocular tissues and reducing the number of animals needed for research. The instrument has the capability of being used both in the clinical and home care environment opening up the possibility of measuring controlled drug release in a patient friendly manner.

Diffuse reflectance spectroscopy has been applied as a non-invasive method to measure tissue optical properties, which are associate with anatomical information. The algorithm widely used to extract, optical parameters from reflectance spectra is the regression method, which is time-consuming and frequently converge to local maxima. In this study, the effects of parameters changes on spectra are analyzed in different fiber geometries, source-detector separations and wavelengths. In the end of this paper, a new fitting algorithm is proposed base on parameters features found. The new algorithm is expected to enhance the accuracy of parameters extracted and save 75% of the process time.

Optical tweezers constitute an increasingly used tool for the study of biomechanical properties of cells. Here we report experiments for the projection induction of NIH3T3 fibroblast cells, using a single-trap optical tweezers. The system is based on a 1064-nm, 50mW infrared gaussian laser beam, a 100x microscope objective with 1.25 numerical aperture and a temperature-controlled warming plate to maintain cell viability. Eighteen cells were exposed to the focussed laser beam in different cell zones and another 18 cells were observed without laser stimulation as a control population. The results show that the probability of lamelipodia growth increases on exposed cells by a factor 1.5.

Guiding, controlling and studying cellular functions are challenging themes in the biomedical field, as they are fundamental prerequisites for new therapeutic strategies from tissue regeneration to controlled drug delivery. In recent years, multidisciplinary studies in nanotechnology offer new tools to investigate important biophysical phenomena in response to the local physical characteristics of the extracellular environment, some examples are the mechanisms of cell adhesion, migration, communication and differentiation. Indeed for reproducing the features of the extracellular matrix in vitro, it is essential to develop active devices that evoke as much as possible the natural cellular environment. Our investigation is in the framework of studying and clarifying the biophysical mechanisms of the interaction between cells and the microenvironment in which they exist. We implement an optical tweezers setup to investigate cell material interaction and we use Digital Holography as non-invasive imaging technique in microscopy. We exploit Holographic Optical Tweezers arrangement in order to trap and manage functionalized micrometric latex beads to induce mechanical deformation in suspended cells. A lot of papers in literature examine the dynamics of the cytoskeleton when cells adhere on substrates and nowadays well established cell models are based on such research activities. Actually, the natural cell environment is made of a complex extracellular matrix and the single cell behavior is due to intricate interactions with the environment and are strongly correlated to the cell-cell interactions. Our investigation is devoted to understand the inner cell mechanism when it is mechanically stressed by point-like stimulus without the substrate influence.

For the monitoring of biological samples, physical parameters such as size, shape and refractive index are of crucial importance. However, up to now the morphological in-vitro analysis of in-vitro cells has been limited to 2D analysis by classical optical microscopy such as phase-contrast or DIC. Here we show an approach that exploits the capability of optical tweezers to trap and put in self-rotation bovine spermatozoa flowing into a microfluidic channel. At same time, digital holographic microscopy allows to image the cell in phase-contrast modality for each different angular position, during the rotation. From the collected information about the cell’s phase-contrast signature, we demonstrate that it is possible to reconstruct the 3D shape of the cell and estimate its volume. The method can open new pathways for rapid measurement of in-vitro cells volume in microfluidic lab-on-a-chip platform, thus having access to 3D shape of the object avoiding tomography microscopy, that is an overwhelmed and very complex approach for measuring 3D shape and biovolume estimation.

The lac operon is a well known example of gene expression regulation, based on the specific interaction of Lac repressor protein (LacI) with its target DNA sequence (operator). LacI and other DNA-binding proteins bind their specific target sequences with rates higher than allowed by 3D diffusion alone. Generally accepted models predict a combination of free 3D diffusion and 1D sliding along non-specific DNA. We recently developed an ultrafast force-clamp laser trap technique capable of probing molecular interactions with sub-ms temporal resolution, under controlled pN-range forces. With this technique, we tested the interaction of LacI with two different DNA constructs: a construct with two copies of the O1 operator separated by 300 bp and a construct containing the native E.coli operator sequences. Our measurements show at least two classes of LacI-DNA interactions: long (in the tens of s range) and short (tens of ms). Based on position along the DNA sequence, the observed interactions can be interpreted as specific binding to operator sequences (long events) and transient interactions with nonspecific sequences (short events). Moreover, we observe continuous sliding of the protein along DNA, passively driven by the force applied with the optical tweezers.

Raman tweezers represents a unique method for identification of different microorganisms on the basis of Raman scattering. Raman tweezers allows us to fix and sterile manipulate with the trapped object and in the same time check the growth, viability, response to the external environment etc. by Raman signal evaluating. The investigations presented here include distinction of bacteria in general (staphylococcal cells), identification of bacteria strains (biofilm-positive and biofilm-negative) by using principal component analysis (PCA) and monitoring the influence of antibiotics.

Singlet oxygen (¹O₂) is commonly recognized to be a major phototoxic component for inducing the biological damage during photodynamic therapy (PDT). In this study, a novel configuration of a thermoelectrically-cooled near-infrared sensitive InGaAs camera was developed for imaging of photodynamically-generated ¹O₂ luminescence. The validation of ¹O₂ luminescence images for solution samples was performed with the model photosensitizer Rose Bengal (RB). Images of ¹O₂ luminescence generated in blood vessels in vivo in a well-controlled dorsal skinfold window chamber model were also recorded during PDT. This study demonstrated the capacity of the newly-developed imaging system for imaging of ¹O₂ luminescence, and the first reported images of ¹O₂ luminescence in blood vessels in vivo. This system has potential for elucidating the mechanisms of vascular targeted PDT.

Well-being applications demand unobtrusive treatment methods in order to reach user acceptance. In the field of light therapy this needs to be carefully addressed because, in most cases, light treatment system size has to be significant with respect to human body scale. At the same time we observe the push to make wearable devices that deliver the treatment on the go. Once scaled up, standard flexible electronics (FPC) fail to conform to body curvatures leading to decrease in comfort. A solution to this problem demands new or modified methods for fabrication of the electronic circuits that fulfill the conformability demand (flexing, but also stretching). Application of Stretchable Molded Interconnect (SMI) technology, that attempts to address these demands, will be discussed. The unique property of SMI is that its manufacturing draws mainly from standard PCB and FCB technologies to inherit the reliability and conductivity. At the same time, however, it allows soft, flexible and stretchable circuits with biomimetic haptics and high optical efficiency. In this work a demonstrator device for blue light therapy of RSI is presented that illustrates the strengths as well as challenges ahead of conformable light circuits. We report system electro-optical efficiency, possible irradiance levels within skin thermal comfort and efficiency under cyclic, tensile stretching deformation.

In 1980-2000 besides the laser surgery an intensive evolution of Low Level Laser Therapy (LLLT) had started in medicine, especially in Russia as well as in several other East-European countries. At the same time the biophysical mechanisms of LLLT are still the subject of disputes. One of the most popular clinical effects at Low Level Laser Irradiation (LLLI) being mentioned in medical publications for justification of the LLLT healing outcome is a stimulation of blood microcirculation in irradiated area. It was declared a priori at a dawn of LLLT and is now a basis of medical interpretation of healing mechanisms of LLLT at least in Russia. But in past 20 years a lot of investigation was carried out on optical registration of microhaemodynamic parameters in vivo as well as a number of noninvasive diagnostic tools was created for that. So, today it is possible to experimentally check the blood microcirculation stimulation hypothesis. Our study was aimed on that during the past 10 years. The most precision and accurate experiments we have carried out recently using simultaneously three different noninvasive diagnostic techniques: Laser Doppler Flowmetry, Tissue Reflectance Oximetry and Infrared Thermography. All these methods didn’t confirm the effect on the blood microcirculation stimulation in skin or mucosa at irradiation with the power density below 50 mW/cm² and irradiation time up to 5-6 minutes. Above this threshold the heating on 0,8…1 °C of tissue in the field of irradiation and the corresponding synchronous increase of all parameters of microhemodynamics were observed.

The emergence of antibiotic resistant bacteria causes significant increase in deaths due to wound infections around the world. Nowadays, it could be impossible to find appropriate antibiotics to treat some bacterial strains, especially multidrug resistant types. The aim of this study is to use photodynamic therapy that destroys these kinds of bacteria with the interaction of Indocyanine green (ICG) and 808-nm diode laser. In this study, antibacterial Photodynamic Therapy technique that we call ICG-IR Laser PDT was applied on antibiotic-resistant strains of Staphylococcus aureus that infected two different types of wound model (excisional and abrasion wound model) in vivo. Wistar albino rats were used to create animal wound models. Excisional or abrasion wounds were formed on the dorsal skin of the rats. They were infected with Staphylococcus aureus. 300 mW and 500 mW of 808-nm diode laser were applied on the wounds for 30 minutes and 15 minutes of exposure duration, respectively. ICG concentrations applied topically were 500, 1000, 1500 and 2000 μg/ml. Then the tissue was dissected properly and homogenized in buffer solution. From this solution, bacterial cell count was determined by serial dilution method. 1-2 log reduction in viable cell count was observed after these applications. The temperature increase in the tissue was between 6-8°C during these applications. From these findings, it was understood that this method with 808-nm and ICG is promising but it must be improved by further dosimetry studies.

In this paper, simulation and mathematical analysis for the determination of pulse energy from a Q-switched Yb³⁺-doped fibre laser is required in Port Wine Stain (PWS) treatment. The pulse energy depends on average power, gain, volume, repetition rate and pulse duration. In some treatments such as Selective Photothermolysis (SP), the peak power at the end of the optical fibre and pulse duration can be obtained and modified via a cavity design. For that purpose, a 585nm optical fibre laser full design which considers all of the above besides the average losses through the optical devices proposed for the design and the Ytterbium optical fibre overall gain will be presented.

Tissue oxygen saturation (StO₂) is known quite useful parameter for medical applications. A spectroscopic method has been developed to diagnose pathologic tissues due to lack of normal blood circulation by measuring tissue oxygen saturation. In the study, human blood samples with different level of oxygen saturations have been prepared and spectra were taken using an optical fiber probe to investigate correlation between the oxygen saturations and the spectra. The experimental set up for the spectroscopic measurements was consists of a miniature NIR light spectrometer, an optical fiber probe, a halogen-tungsten light source and a laptop. A linear correlation between the oxygen saturation of the blood samples and the ratio of the light of wavelengths 660 nm to 790 nm has been found from the spectra. Then, oxygen saturations of the blood samples were estimated from the spectroscopic measurements within an error of 2.9%. Furthermore, it has been shown that the linear dependence between the ratio and the oxygen saturation of the blood samples was valid for the blood samples with different hematocrits. Tissue oxygen saturation has been estimated from the spectroscopic measurements were taken from the fingers of healthy volunteers using the correlation between the spectra and blood oxygen saturation. The tissue StO₂ measured was 80% as expected. The technique developed to measure tissue oxygen saturation has potential to diagnose premalignant tissues, follow up prognosis of cancerous tissues, and evaluation of ischemia reperfusion tissues.

Diabetes is a serious health condition considered to be one of the major healthcare epidemics of modern era. An effective treatment of this disease can be only achieved by reliable continuous information on blood glucose levels. In this work we present a minimally invasive, chip-based near infrared (NIR) sensor, combined with microdialysis, for continuous glucose monitoring (CGM). The sensor principle is based on difference absorption spectroscopy in the 1^st overtone band of the near infrared spectrum. The device features a multi-emitter LED and InGaAs-Photodiodes, which are located on a single electronic board (non-disposable part), connected to a personal computer via Bluetooth. The disposable part consists of a chip containing the fluidic connections for microdialysis, two fluidic channels acting as optical transmission cells and total internally reflecting mirrors for in- and out-coupling of the LED light to the chip and to the detectors. The sensor is combined with an intraveneous microdialysis to separate the glucose from the cells and proteins in the blood and operates without any chemical consumption. In vitro measurements showed a linear relationship between glucose concentration and the integrated difference signal with a coefficient of determination of 99 % in the relevant physiological concentration range from 0 to 400 mg/dl. In vivo measurements on 10 patients showed that the NIR-CGM sensor data reflects the blood reference values adequately, if a proper calibration and signal drift compensation is applied. The MARE (mean absolute relative error) value taken over all patient data is 13.8 %. The best achieved MARE value is at 4.8 %, whereas the worst is 25.8 %, with a standard deviation of 5.5 %.

Near infrared spectroscopy offers a promising technological platform for continuous glucose monitoring in the human body. NIR measurements can be performed in vivo with an implantable single-chip based optical NIR sensor. However, the application of NIR spectroscopy for accurate estimation of the analyte concentration in highly scattering biological systems still remains a challenge. For instance, a thin tissue layer may grow in the optical path of the sensor. As most biological tissues allow only a small fraction of the collimated light to pass, this might result in a large reduction of the light throughput. To quantify the effect of presence of a thin tissue layer in the optical path, the bulk optical properties of tissue samples grown on sensor dummies which had been implanted for several months in goats were characterized using Double Integrating Spheres and unscattered transmittance measurements. The measured values of diffuse reflectance, diffuse transmittance and collimated transmittance were used as input to Inverse Adding-Doubling algorithm to estimate the bulk optical properties of the samples. The estimates of absorption and scattering coefficients were then used to calculate the light attenuation through a thin tissue layer. Based on the lower reduction in unscattered transmittance and higher absorptivity of glucose molecules, the measurement in the combination band was found to be the better option for the implantable sensor. As the tissues were found to be highly forward scattering with very low unscattered transmittance, the diffuse transmittance measurement based sensor configuration was recommended for the implantable glucose sensor.

We propose and study an integrated refractive index sensor which is based on a plasmonic slot cavity integrated on silicon-on-insulator wafers. In this device, the guided mode is vertically coupled to the cavity which is a metal-dielectricmetal waveguide and is separated from a photonic wire waveguide by a silicon dioxide spacer. We perform an in-depth study that links the geometrical parameters to the coupling and sensitivity. The strong coupling from the dielectric waveguide to the plasmonic slot waveguide cavity allows a local change in refractive index to be detectable with a high sensitivity of around 600 nm/RIU in a femto-liter volume. These results are obtained with three-dimensional time domain simulations made with the CST Microwave Studio. The sensing performance of the devices are presented and compared to the practical needs to achieve localized biomolecular sensing.

Advanced multiplexed assays have recently become an indispensable tool for clinical diagnostics. These techniques provide simultaneous quantitative determination of multiple biomolecules in a single sample quickly and accurately. The development of multiplex suspension arrays is currently of particular interest for clinical applications. Optical encoding of microparticles is the most available and easy-to-use technique. This technology uses fluorophores incorporated into microbeads to obtain individual optical codes. Fluorophore-encoded beads can be rapidly analyzed using classical flow cytometry or microfluidic techniques. We have developed a new generation of highly sensitive and specific diagnostic systems for detection of cancer antigens in human serum samples based on microbeads encoded with fluorescent quantum dots (QDs). The designed suspension microarray system was validated for quantitative detection of (1) free and total prostate specific antigen (PSA) in the serum of patients with prostate cancer and (2) carcinoembryonic antigen (CEA) and cancer antigen 15-3 (CA 15-3) in the serum of patients with breast cancer. The serum samples from healthy donors were used as a control. The antigen detection is based on the formation of an immune complex of a specific capture antibody (Ab), a target antigen (Ag), and a detector Ab on the surface of the encoded particles. The capture Ab is bound to the polymer shell of microbeads via an adapter molecule, for example, protein A. Protein A binds a monoclonal Ab in a highly oriented manner due to specific interaction with the Fc-region of the Ab molecule. Each antigen can be recognized and detected due to a specific microbead population carrying the unique fluorescent code. 100 and 231 serum samples from patients with different stages of prostate cancer and breast cancer, respectively, and those from healthy donors were examined using the designed suspension system. The data were validated by comparing with the results of the “gold standard” enzyme-linked immunosorbent assay (ELISA). They have shown that our approach is a good alternative to the diagnostics of cancer markers using conventional assays, especially in early diagnostic applications.

We have studied different strategies of use of luminescence thermometry with upconverting nanoparticles in the biological range of temperatures, among them, the thermal sensing ability of fluoresncent lifetime of Er,Yb:NaY₂F₅ nanoparticles. Er,Yb:NaY₂F₅O nanocrystals show great potentiality as thermal sensors at the nanoscale for biomedical applications due to the incorporation of additional non-radiative relaxation mechanisms that shorten the emission lifetime generated by the oxygen present in the structure. Here we report ex-vivo temperature determination by laser induced heating in chicken breast using lifetime-based thermometry in these up-conversion nanoparticles.

Used polarized light for fluorescence excitation one could obtain response related to the anisotropy features of extracellular matrix. The fluorophore anisotropy is attenuated during lesions’ growth and level of such decrease could be correlated with the stage of tumor development. Our preliminary investigations are based on in vivo point-by-point measurements of excitation-emission matrices (EEM) from healthy volunteers skin on different ages and from different anatomical places using linear polarizer and analyzer for excitation and emission light detected. Measurements were made using spectrofluorimeter FluoroLog 3 (HORIBA Jobin Yvon, France) with fiber-optic probe in steady-state regime using excitation in the region of 280-440 nm. Three different situations were evaluated and corresponding excitation-emission matrices were developed – with parallel and perpendicular positions for linear polarizer and analyzer, and without polarization of excitation and fluorescence light detected from a forearm skin surface. The fluorescence spectra obtained reveal differences in spectral intensity, related to general attenuation, due to filtering effects of used polarizer/analyzer couple. Significant spectral shape changes were observed for the complex autofluorescence signal detected, which correlated with collagen and protein cross-links fluorescence, that could be addressed to the tissue extracellular matrix and general condition of the skin investigated, due to morphological destruction during lesions’ growth. A correlation between volunteers’ age and the fluorescence spectra detected was observed during our measurements. Our next step is to increase developed initial database and to evaluate all sources of intrinsic fluorescent polarization effects and found if they are significantly altered from normal skin to cancerous state of the tissue, this way to develop a non-invasive diagnostic tool for dermatological practice.

Many researchers during past 20 years have used the laser fluorescence spectroscopy (LFS) for in vivo tissue diagnosis. But in the up-to-date medical in vivo LFS there is a problem of quantification of the fluorophores concentrations in optically-turbid biotissues basing on measurements of the laser induced fluorescence on a surface of the tissues. The purpose of our work is both experimental and theoretical study of the character of dependences of measured fluorescence intensities on tissues’ optical properties and on fluorophores concentrations in tissues. In the experimental part of our study the measurements of the superficial fluorescence on phantoms at different known concentration of fluorophores in them were carried out. As a result experimental dependences of the registered intensities of the laser induced fluorescence emission on fluorophores concentration were plotted. In the theoretical part of our study the analytical solution for a fluorescence emission by Kokhanovsky’s method based on the well-known two-flux Kubelka-Munk approach (KMA) was used. Besides, in the study the Kokhanovsky’s method was modified by its association with our improved KMA, allowing us to receive exact solutions for boundary intensities collected by an optical probe. As a result a set of theoretical curves describing the influence of fluorophore concentration in tissues on the registered intensities was obtained as well. Both experimental and theoretical results show a good qualitative agreement between each other. Also these results show that the dependence of the fluorescence intensity from tissues’ optical properties and from the concentration of fluorophores can be both nonlinear and non-monotonic.

The aim of the study was to develop a prototype system of the smart garment for real time telemetric monitoring of human cardiovascular activity. Two types of photoplethysmography (PPG) sensors for low noise and artefact free signal recording from various sites of the human body that were suitable for integration into smart textile were investigated. The reflectance sensors with single and multiple photodiodes based on "pulse-duration-based signal conversion" signal acquisition principle were designed and evaluated. The technical parameters of the system were measured both on bench and in vivo. Overall, both types of PPG sensors showed acceptable signal quality SNR 86.56±3.00 dB, dynamic range 89.84 dB. However, in-vivo condition tests revealed lower noise and higher accuracy achieved by applying the multiple photodiodes sensor. We concluded that the proposed PPG device prototype is simple and reliable, and therefore, can be utilized in low-cost smart garments.

Clostridium chauvoei is the causative agent of blackleg, which is an endogenous bacterial infection. Mainly cattle and other ruminants are affected. The symptoms of blackleg are very similar to those of malignant edema, an infection caused by Clostridium septicum. [1, 2] Therefore a reliable differentiation of Clostridium chauvoei from other Clostridium species is required. Traditional microbiological detection methods are time consuming and laborious. Additionally, the unique identification is hindered by the overgrowing tendency of swarming Clostridium septicum colonies when both species are present. [1, 3, 4] Thus, there is a crucial need to improve and simplify the specific detection of Clostridium chauvoei and Clostridium septicum. Here we present an easy and fast Clostridium species discrimination method combining magnetic beads and fluorescence spectroscopy. Functionalized magnetic particles exhibit plentiful advantages, like their simple manipulation in combination with a large binding capacity of biomolecules. A specific region of the pathogenic DNA is amplified and labelled with biotin by polymerase chain reaction (PCR). These PCR products were then immobilized on magnetic beads exploiting the strong biotin-streptavidin interaction. The specific detection of different Clostridium species is achieved by using fluorescence dye labeled probe DNA for the hybridization with the immobilized PCR products. Finally, the samples were investigated by fluorescence spectroscopy. [5]

Laser bonding is a promising minimally invasive approach, emerging as a valid alternative to conventional suturing techniques. It shows widely demonstrated advantages in wound treatment: immediate closuring effect, minimal inflammatory response and scar formation, reduced healing time. This laser based technique can overcome the difficulties in working through narrow surgical corridors (e.g. the modern “key-hole” surgery as well as the endoscopy setting) or in thin tissues that are impossible to treat with staples and/or stitches. We recently proposed the use of chitosan matrices, stained with conventional chromophores, to be used in laser bonding of vascular tissue. In this work we propose the same procedure to perform laser bonding of vocal folds and dura mater repair. Laser bonding of vocal folds is proposed to avoid the development of adhesions (synechiae), after conventional or CO2 laser surgery. Laser bonding application in neurosurgery is proposed for the treatment of dural defects being the Cerebro Spinal Fluid leaks still a major issue.
Vocal folds and dura mater were harvested from 9-months old porks and used in the experimental sessions within 4 hours after sacrifice.
In vocal folds treatment, an IdocyanineGreen-infused chitosan patch was applied onto the anterior commissure, while the dura mater was previously incised and then bonded. A diode laser emitting at 810 nm, equipped with a 600 μm diameter optical fiber was used to weld the patch onto the tissue, by delivering single laser spots to induce local patch/tissue adhesion. The result is an immediate adhesion of the patch to the tissue. Standard histology was performed, in order to study the induced photothermal effect at the bonding sites. This preliminary experimental activity shows the advantages of the proposed technique in respect to standard surgery: simplification of the procedure; decreased foreign-body reaction; reduced inflammatory response; reduced operating times and better handling in depth.

In this study, gold nanoplasmonic biosensors using localized surface plasmon resonance (LSPR) were fabricated for the diagnosis of cancer. We optimized the structures of the gold nanodot array (GNA) via the experiments for the optical characteristics. In addition, the nanoimprint lithography was employed for realizing nanoplasmonic structures, which is a more efficient technique for mass production than nanolithography such as electron beam lithography (EBL) or focused ion beam (FIB) lithography that is a quite intricate, time-consuming and expensive process.
After the UV nanoimprinting process using a film stamp and metal films were deposited using an electron-beam evaporator, followed by the lift-off step. Consequently, the nanoplasmonic MNA was realized on 5-inch glass wafer and the pitch, diameter and height of MNA were 300nm, 150 nm and 20 nm, respectively. The wavelength of nanoplasmonic resonance peak represented from the MNA sensors was about 740nm under aqueous ambient.
The capture antibodies of the lung and the pancreas cancer marker, respectively, were immobilized on the surfaces of MNA sensor. Using a compact fiber-optic spectrometer and a reflection optical probe, we were able to confirm the binding of cancer markers with their antibodies due to the immunoreactions between each cancer marker and its corresponding antibody on the sensor surfaces. The amount of the cancer markers in serum were analyzed through the observation of nanoplasmonic resonance wavelength-shift on the reflection spectra. To amplify a sensitivity of detection demonstrated by the nanoplasmonic resonance peak shift, we applied enzyme-precipitation reaction on the surface of MNA biosensor. The enzyme-catalyzed precipitation method in the GNA biosensor could be extended to detect other clinical biomarkers at extremely low concentrations in actual clinical samples.

Alveolar macrophages (AM) are cells from immune defense inside the lung. They engulf particles in vacuoles from the outer membrane. Volume and surface are important parameters to characterize the particle uptake. AM change their shape within a few seconds, therefore it is hard to obtain by confocal laser scanning microscopy, which is commonly used to generate 3D-images. So we used an intensified dark field microscopy (DFM) as an alternative method to generate contrast rich AM gray tone image slices used for 3D-reconstructions of AM cells by VTK software applications. From these 3D-reconstructions approximate volume and surface data of the AM were obtained and compared to values found in the literature. Finally, simple geometrical 3D-models of the AM were created and compared to real data. Averaged volume and surface data from the DFM images are close to values found in the literature. Furthermore, calculation of volume and surface data from DFM images could be done faster if simplified geometrical 3D-models of the cells were used.

Optical trapping for isolation and sorting of cells and particles inside microfluidic channels is an efficient non-destructive manipulation technique in the field of biophotonics. In recent years, vertical-cavity surface-emitting lasers (VCSELs) have been proven to be excellent light sources for particle manipulation inside microfluidic channels. The small dimension and low power consumption of these devices enable direct integration with the channels. With such integration, however, the simultaneous manipulation or trapping of multiple particles require the usage of densely packed VCSEL arrays with very small device pitch, which makes the fabrication process more expensive and more complicated. We present an innovative technique for simultaneous optical multi-particle manipulation using one rectangular-shaped top-emitting AlGaAsGaAs VCSEL resonator having an active aperture area of around 100 × 14 μm². The VCSEL emission wavelength is about 850 nm, which is suitable for usage in biophotonics, as biological materials present very little absorption in the near-infrared spectral range. Furthermore, this oblong VCSEL can potentially be integrated with polydimethylsiloxane (PDMS) microfluidic channels to form miniaturized optofluidic chips for ultra-compact particle handling and manipulation. We show efficient single as well as multiple polystyrene particle trapping and sorting inside PDMS microfluidic channels.

In this paper we report on the use of digital holographic microscopy for 3D real time imaging of cultured neurons and neural networks, in vitro. Digital holographic microscopy is employed as an assessment tool to study the biophysical origin of neurodegenerative diseases. Our study consists in the morphological characterization of the axon, dendrites and cell bodies. The average size and thickness of the soma were 21 and 13 μm, respectively. Furthermore, the average size and diameter of some randomly selected neurites were 4.8 and 0.89 μm, respectively. In addition, the spatiotemporal growth process of cellular bodies and extensions was fitted to by a non-linear behavior of the nerve system. Remarkably, this non-linear process represents the relationship between the growth process of cellular body with respect to the axon and dendrites of the neurons.

Here we report a preliminary study based on the application of Raman spectroscopy and surface enhanced Raman spectroscopy (SERS) to investigate the compositional differences between exosomes derived from ovarian carcinoma cells (cell line A2780) grown in normoxia (normal O₂ conditions) and hypoxia (1% O₂ conditions). Exosomes are integral to cell signalling, and are of interest in the study of how cells communicate within their environment. We are particularly interested in identifying whether hypoxia induced senescent cells can communi- cate via exosomes with neighbouring tumour cells, thereby causing them to become senescent and therefore radio and chemo resistant. With this goal in mind, we performed a preliminary study on the application of Raman spectroscopy and SERS to analyse the biomolecular fingerprint of both groups of exosomes and to investigate whether there exists a different biomolecular composition associated with exosomes derived from hypoxic cells in comparison to those from normoxic cells. We also applied multivariate statistical techniques for the classification of both groups of exosomes.

Spectral profiles of tissues from patients with breast carcinoma, malignant carcinoid and non-small cell lung carcinoma were acquired using native fluorescence spectroscopy. A novel spectroscopic ratiometer device (S³-LED) with selective excitation wavelengths at 280 nm and 335 nm was used to produce the emission spectra of the key biomolecules, tryptophan and NADH, in the tissue samples. In each of the samples, analysis of emission intensity peaks from biomolecules showed increased 340 nm/440 nm and 340 nm/460 nm ratios in the malignant samples compared to their paired normal samples. This most likely represented increased tryptophan to NADH ratios in the malignant tissue samples compared to their paired normal samples. Among the non-small cell lung carcinoma and breast carcinomas, it appeared that tumors of very large size or poor differentiation had an even greater increase in the 340 nm/440 nm and 340 nm/460 nm ratios. In the samples of malignant carcinoid, which is known to be a highly metabolically active tumor, a marked increase in these ratios was also seen.

Biostimulation is still a controversial subject in wound healing studies. The effect of laser depends of not only laser parameters applied but also the physiological state of the target tissue. The aim of this project is to investigate the biostimulation effects of 635nm laser irradiation on the healing processes of cutaneous wounds by means of morphological and histological examinations.

3-4 months old male Wistar Albino rats weighing 330 to 350 gr were used throughout this study. Low-level laser therapy was applied through local irradiation of red light on open skin excision wounds of 5mm in diameter prepared via punch biopsy. Each animal had three identical wounds on their right dorsal part, at which two of them were irradiated with continuous diode laser of 635nm in wavelength, 30mW of power output and two different energy densities of 1 J/cm² and 3 J/cm². The third wound was kept as control group and had no irradiation. In order to find out the biostimulation consequences during each step of wound healing, which are inflammation, proliferation and remodeling, wound tissues removed at days 3, 7, 10 and 14 following the laser irradiation are morphologically examined and than prepared for histological examination. Fragments of skin including the margin and neighboring healthy tissue were embedded in paraffin and 6 to 9 um thick sections cut are stained with hematoxylin and eosin.

Histological examinations show that 635nm laser irradiation accelerated the healing process of cutaneous wounds while considering the changes of tissue morphology, inflammatory reaction, proliferation of newly formed fibroblasts and formation and deposition of collagen fibers. The data obtained gives rise to examine the effects of two distinct power densities of low-level laser irradiation and compare both with the non-treatment groups at different stages of healing process.

Sleep problems are getting worse and worse in modern world. They have a severe impact on psychological and physical health, as well as social performances. From our previous study, the brainwave α rhythm, θ wave and β wave were affected by radiating the palm of the subjects with low-level laser array. In addition, from other study, the LED array stimulator (LEDAS) also has the similar effects. In the present study, LED light was used to radiate the left palm of the subjects too, and the effects were assessed with the multiple sleep latency test (MSLT) and heart-rate variability (HRV) analysis. The results revealed that it doesn’t have significant meaning between these two groups. However, the tendency of the sleep latency (SL) in the LED group was shorter than that in the control group. In addition, the autonomic nervous system (ANS) analysis showed that the sympathetic nervous system was getting larger in the LED group than that in the control group, and total ANS activity were mainly getting larger in the LED group. We infer that this LED stimulation could reduce SL and balance ANS activity of the night-shift people. In the future, the further study will be conducted on normal subjects.

The widespread introduction of laser noninvasive diagnostic techniques in medicine gave rise interest to theoretical description of light propagation in turbid media. One of the purposes for that is a preliminary simulation of incoming radiation for diagnostic spectrophotometry equipment. For complex diagnostic devices combining the Laser Doppler Flowmetry (LDF) and the tissue reflectance oximetry (TRO) it is necessary to know a ratio of signals in each diagnostic channel for a proper choice of the radiation power of laser sources, sensitivity of photodetectors, etc. In LDF the lightbeating backscattered signal mixed from moving red blood cells and static inhomogeneities inside the tissue is the useful signal, while in TRO both signals from static and moving scatterers are registered in the sum. The aim of our study was an estimation of the ratio between flux with the Doppler shifted signal and the total backscattered flux. For this purpose the simple analytical model describing the backscattered radiation for a two-layered tissue with different levels of blood volume in the second layer was under consideration. The physical model was based on the improved Kubelka-Munk approach. This approach involves an additional parameter of the density of scatterers, so it is useful for the Doppler signal intensity calculation as well. To assess the intensity of the Doppler component the single-scattering approximation inside the tissue’s second layer was used. It was found that the fraction of the Doppler component in the total backscattered flux can vary in the range of 1-10% for the blood volume of 1-20%.

In this paper we consider the original method of solving noise reduction problem for visualization’s quality improvement of SD-OCT skin and tumors biomedical images. The principal advantages of OCT are high resolution and possibility of in vivo analysis. We propose a two-stage algorithm: 1) process of raw one-dimensional A-scans of SD-OCT and 2) remove a noise from the resulting B(C)-scans. The general mathematical methods of SD-OCT are unstable: if the noise of the CCD is 1.6% of the dynamic range then result distortions are already 25-40% of the dynamic range. We use at the first stage a resampling of A-scans and simple linear filters to reduce the amount of data and remove the noise of the CCD camera. The efficiency, improving productivity and conservation of the axial resolution when using this approach are showed. At the second stage we use an effective algorithms based on Hilbert-Huang Transform for more accurately noise peaks removal. The effectiveness of the proposed approach for visualization of malignant and benign skin tumors (melanoma, BCC etc.) and a significant improvement of SNR level for different methods of noise reduction are showed. Also in this study we consider a modification of this method depending of a specific hardware and software features of used OCT setup. The basic version does not require any hardware modifications of existing equipment. The effectiveness of proposed method for 3D visualization of tissues can simplify medical diagnosis in oncology.

Osteoarthritis is one of the most prevalent joint diseases in the world. Although the cause of osteoarthritis is not exactly clear, the disease results in a degradation of the quality of the articular cartilage including collagen and other extracellular matrix components. We have investigated alterations in the structure of collagen fibers in the cartilage tissue of the human knee using mulitphoton microscopy. Due to inherent high nonlinear susceptibility, ordered collagen fibers present in the cartilage tissue matrix produces strong second harmonic generation (SHG) signals. Significant morphological differences are found in different Osteoarthritic grades of cartilage by SHG microscopy. Based on the polarization analysis of the SHG signal, we find that a few locations of hyaline cartilage (mainly type II collagen) is being replaced by fibrocartilage (mainly type I cartilage), in agreement with earlier literature. To locate the different types and quantify the alteration in the structure of collagen fiber, we employ polarization-SHG microscopic analysis, also referred to as _-tensor imaging. The image analysis of p-SHG image obtained by excitation polarization measurements would represent different tissue constituents with different numerical values at pixel level resolution.

In this paper, plasmon-resonant nanostructures, such as gold nanostars and their silica-coated composites, were used for enhancement of OCT image contrast of water flows in glass capillaries. The contrasting properties of the synthesized nanostars and nanocomposites with silica shell thickness of about 5 nm and 50 nm were compared in the framework of capillary stasis model. The most intensive signal was detected from the nanocomposites with the thickest silica shell. The nanocomposites were characterized by optical spectroscopy and electron microscopy. Nontoxicity of nanostars and nanocomposites up to ~ 3 mg/mL concentration was showed by MTT assay suggesting practical applications of the nanostructures for bioimaging.

Digital (droplet-based) microfluidic systems apply electromagnetic characteristics as the fundamental fluid actuation mechanism. These systems are often implemented in two-dimensional architectures, overcoming one-dimensional continuous flow channel practical issues. The fundamental operation for digital microfluidics requires the creation of an electric field distribution to achieve desired fluid actuation. The electric field distribution is typically non-uniform, enabling creation of net dielectrophoresis (DEP) force. The DEP force magnitude is proportional to the difference between microdroplet and surrounding medium complex dielectric constants, and the gradient of the electric field magnitude squared. Force sign/direction can be manipulated to achieve a force towards higher (positive DEP) or lower (negative DEP) electrostatic energy by tailoring the relative difference between microdroplet and surrounding medium complex dielectric constants through careful selection of the devices fabrication materials. The DEP force magnitudes and directions are applied here for well-controlled and high-speed microdroplet actuation. Control and speed characteristics arise from significant differences in the microdroplet/medium conductivity and the use of a micropin architecture with strong electric field gradients. The implementation, referred to here as a DEP microjet, establishes especially strong axial propulsion forces. Single- and double-micropin topologies achieve strong axial propulsion force, but only the double-micropin topology creates transverse converging forces for stable and controlled microdroplet actuation. Electric field distributions for each topology are investigated and linked to axial and transverse forces. Experimental results are presented for both topologies. The double-micropin topology is tested with biological fluids. Microdroplet actuation speeds up to 25 cm/s are achieved—comparable to the fastest speeds to-date.

Two different optical fiber probes for combined Raman and fluorescence spectroscopic measurements were designed, developed and used for tissue diagnostics. Two visible laser diodes were used for fluorescence spectroscopy, whereas a laser diode emitting in the NIR was used for Raman spectroscopy. The two probes were based on fiber bundles with a central multimode optical fiber, used for delivering light to the tissue, and 24 surrounding optical fibers for signal collection. Both fluorescence and Raman spectra were acquired using the same detection unit, based on a cooled CCD camera, connected to a spectrograph. The two probes were successfully employed for diagnosing melanocytic lesions in a good agreement with common routine histology. The obtained results demonstrated that the multimodal approach is crucial for improving diagnostic capabilities. Further investigations were performed on colon and brain tissue samples in order to have a benchmark for diagnosing a broader range of tissue lesions and malignancies. The system presented here can improve diagnostic capabilities on a broad range of tissues and has the potential of being used for endoscopic inspections in the near future.

The developed portable multi-spectral photoplethysmograph (MS-PPG) optical biosensor device, intended for analysis of peripheral blood volume pulsations at different vascular depths, has been clinically verified. Multi-spectral monitoring was performed by means of a four – wavelengths (454 nm, 519 nm, 632 nm and 888 nm) light emitted diodes and photodiode with multi-channel signal output processing. Two such sensors can be operated in parallel and imposed on the patient’s skin. The clinical measurements confirmed ability to detect PPG signals at four wavelengths simultaneously and to record temporal differences in the signal shapes (corresponding to different penetration depths) in normal and pathological skin. This study analyzed wavelengths relations between systole and diastole peak difference at various tissue depths in normal and pathological skin. The difference between parameters of healthy and pathological skin at various skin depths could be explain by oxy- and deoxyhemoglobin dominance at different wavelengths operated in sensor. The proposed methodology and potential clinical applications in dermatology for skin assessment are discussed.

Nowadays, biophotonics is widely used in neuroscience. The effectiveness of biophotonic techniques, such as fluorescence imaging and optogenetics, is affected by the optical properties of the examined tissue. Therefore, knowledge of these properties is essential to carefully plan experiments. Mice and rats are widely used in neuroscience studies. However, reports about optical properties of their brains are very rare. We measured optical absorption μ_a and reduced scattering μ’_s coefficients of native rat brain in the visible and near-infrared wavelength region, using contact spatially resolved spectroscopy (SRS). In this study, we estimate μ_a and μ’_s for the rat cortex and discuss their stability in time. Additionally, variations in optical properties within and between samples were characterized. The results extend the range of known optical properties for the rat cortex, especially in the visible range, relevant to optogenetics. μ_a and μ’_s are stable within a time span of four hours, and show low variation in and between brain samples. This indicates that a suitable protocol was used to estimate optical properties of rodent brain tissue. Since contact SRS is a non-destructive method, this technique could be used also to measure μ_a and μ’_s in living animals. Moreover, the probe has small dimensions, allowing the characterization of optical properties in different structures of the brain.

We present a single-photon camera for fluorescence imaging, with a time resolution better than 100ps, capable of providing both intensity and lifetime images. the camera was fabricated in standard CMOS technology. With this FluoCam we show the possibility to study sub-nanosecond fluorescence mechanisms. The FluoCam was used to characterize a near-infrared probe, indocyanine green, conjugated with multimeric cyclic pentapeptide (cRGD). The fluorescent probe-conjugated was used to target and mark tumors with better specificity, in particular aiming at targeting the integrins α_vβ₃ and α_vβ₅. As a first step towards clinical studies, preliminary results obtained in-vivo are presented. The first envisioned clinical application would be image-guided surgical oncology to help the surgeon to remove tumor tissue by a better discrimination from normal tissues and also to improve the detection of metastatic lymph nodes. A further application could be the in-vivo determination of the αvβ₃ and α_vβ₅ targets to select patients for therapy with RGD chemotherapy conjugates.

Spectral imaging techniques are widely used in medical and biological research for example in skin cancer diagnostics. In this paper methods and equipment for spectral imaging of micro objects such as cells, their structure and histological samples are discussed.

A tilted fiber Bragg grating (TFBG) was integrated as the dispersive element in a high performance biomedical imaging system. The spectrum emitted by the 23 mm long active region of the fiber is projected through custom designed optics consisting of a cylindrical lens for vertical beam collimation and successively by an achromatic doublet onto a linear detector array. High resolution tomograms of biomedical samples were successfully acquired by the frequency domain OCT-system. Tomograms of ophthalmic and dermal samples obtained by the frequency domain OCT-system were obtained achieving 2.84 μm axial and 10.2 μm lateral resolution. The miniaturization reduces costs and has the potential to further extend the field of application for OCT-systems in biology, medicine and technology.

The diffuse reflectance multispectral imaging technique has been used for distant mapping of in vivo skin chromophores (hemoglobin and melanin). The fluorescence multispectral imaging is not so common for skin applications due to complicity of data acquisition and processing, but could provide additional information about skin fluorophores. Both techniques are compatible, and could be combined into a multimodal solution.

The multispectral imaging system Nuance based on liquid crystal tunable filters was adapted for diffuse reflectance and fluorescence spectral imaging of in vivo skin. Uniform illumination was achieved by LED ring light. Combination of four LEDs (warm white, 770 nm, 830 nm and 890 nm) was used to support diffuse reflectance mode in spectral range 450-950 nm. 405 nm LEDs were used for excitation of skin autofluorescence. Multispectral imaging system was adapted for spectral working range of 450-950 nm with scanning step of 10 nm and spectral resolution of 15 nm. An average field of view was 50x35 mm in size with spatial resolution 0,05 mm (the pixel size). Due to spectrally different illumination intensity and system sensitivity, various exposure times (from 7…500 ms) were used for each image acquisition.

The proposed approach was tested for different skin lesions: benign nevus, hemangioma, basalioma and halo nevus. Spectral image cubes of different skin lesions were acquired and analyzed to test its diagnostic potential.

Laser speckle contrast analysis (LASCA) offers a non-contact, full-field, and real-time mapping of capillary blood flow and can be considered as an alternative method to Laser Doppler perfusion imaging. LASCA technique has been implemented in several commercial instruments. However, these systems are still too expensive and bulky to be widely available. Several optical techniques have found new implementations as connection kits for mobile phones thus offering low cost screening devices.

In this work we demonstrate simple implementation of LASCA imaging technique as connection kit for mobile phone for primary low-cost assessment of skin blood flow. Stabilized 650 nm and 532 nm laser diode modules were used for LASCA illumination. Dual wavelength illumination could provide additional information about skin hemoglobin and oxygenation level.

The proposed approach was tested for arterial occlusion and heat test. Besides, blood flow maps of injured and provoked skin were demonstrated.

Structured illumination microscopy (SIM) is a recent microscopy technique that enables one to go beyond the diffraction limit using patterned illumination. The high frequency information is encoded through aliasing into the observed image. By acquiring multiple images with different illumination patterns aliased components can be separated and a highresolution image reconstructed. Here we investigate image processing methods that perform the task of high-resolution image reconstruction, namely square-law detection, scaled subtraction, super-resolution SIM (SR-SIM), and Bayesian estimation. The optical sectioning and lateral resolution improvement abilities of these algorithms were tested under various noise level conditions on simulated data and on fluorescence microscopy images of a pollen grain test sample and of a cultured cell stained for the actin cytoskeleton. In order to compare the performance of the algorithms, the following objective criteria were evaluated: Signal to Noise Ratio (SNR), Signal to Background Ratio (SBR), circular average of the power spectral density and the S₃ sharpness index. The results show that SR-SIM and Bayesian estimation combine illumination patterned images more effectively and provide better lateral resolution in exchange for more complex image processing. SR-SIM requires one to precisely shift the separated spectral components to their proper positions in reciprocal space. High noise levels in the raw data can cause inaccuracies in the shifts of the spectral components which degrade the super-resolved image. Bayesian estimation has proven to be more robust to changes in noise level and illumination pattern frequency.

There are many techniques and instruments that are currently available to give better results for measuring the quality of human skin. In this study, two non-invasive spectroscopy instruments have been used namely NIRQuest spectrometer and ASD FieldSpec® 3 Spectroradiometer. Both of these spectroscopy instruments were used to find the correlation technique with the commercial instruments (DermaLab® USB Sebum Module). Initially an experiment was conducted to find intensities peak of the absorption of oleic acid as a part of sebum composition. From the spectra peak of the absorbance, the wavelength will be determined. Next step was to measure the reflectance of human skin sebum by using two spectroscopic instruments. The analysis will carry on at the wavelength that have been chosen from the previous study and also from the wavelength of the fatty acid to find the best wavelength that contribute in sebum composition. From several analyses, the wavelengths that contribute in sebum were 1208, 1414, 1726, and 1758 nm that obtained the value of R² 0.8444 for NIRQuest Spectrometer and 0.8532 for ASD FieldSpec® 3 Spectroradiometer. For future research this non- invasive techniques can be used in dermatology field for the use of various skin analysis. Besides that, the less wavelength used is an advantage to develop instruments with less amount of wavelength sensor. It can reduce the cost of development.

In the recent years, there has been a considerable surge in the application of spectroscopy for disease diagnosis. Raman and fluorescence spectra provide characteristic spectral profile related to biochemical and morphological changes when tissues progress from normal state towards malignancy. Spectroscopic techniques offer the advantage of being minimally invasive compared to traditional histopathology, real time and quantitative. In biomedical optical diagnostics, freshly excised specimens are preferred for making ex-vivo spectroscopic measurements. With regard to fresh tissues, if the lab is located far away from the clinic it could pose a problem as spectral measurements have to be performed immediately after dissection. Tissue samples are usually placed in a fixative agent such as 4% formaldehyde to preserve the samples before processing them for routine histopathological studies. Fixation prevents the tissues from decomposition by arresting autolysis. In the present study, we intend to investigate the possibility of using formalin fixed samples for discrimination of brain tumours from dysplastic tissue using Raman and fluorescence spectroscopy. Formalin fixed samples were washed with phosphate buffered saline for about 5 minutes in order to remove the effects of formalin during spectroscopic measurements. In case of fluorescence spectroscopy, changes in spectral profile have been observed in the region between 550−670 nm between dysplastic and tumor samples. For Raman measurements, we found significant differences in the spectral profiles between dysplasia and tumor. In conclusion, formalin fixed samples can be potentially used for the spectroscopic discrimination of tumor against dysplastic tissue in brain samples.

Microfluidics technologies have received great attention and appear in many bioanalyses applications. A recent microfluidics subset has appeared as droplet-based digital microfluidics (DMF). Here, microdroplets are manipulated in a two-dimensional on-chip plane using electric fields, contrasting the one-dimensional pressure-based channel flow of continuous flow microfluidics. These DMF systems fundamentally offer reconfigurability, whereby one device performs many bioanalysis tasks. A subset of DMF systems called optoelectrowetting is also of recent interest due to its ability for intricate microdroplet routing processes in the on-chip plane. For an optoelectrowetting chip, the DMF structure is modified with optically triggered electrodes with arrayed photoconductive switches. The arrayed photoconductive switches are optically-activated so microdroplets in the vicinity are routed to the illuminated switch. Unfortunately, such systems still require intricate electrode arrays, limiting microdroplet actuation resolution by the electrode size. This work proposes an on-chip optofluidic device with a continuous and planar semiconductor layer as the photoconductive mechanism. An illuminated section of the semiconductor layer acts as a localized electrode, with the photogenerated charge-carriers attracting nearby microdroplets. Given this planar topology, the illuminating beam is used to move the microdroplets continuously over the on-chip plane with precise optical control. The resolution for such a process is ultimately limited by charge-carrier diffusion, so an alternative material, a nanocomposite, is introduced to the on-chip device design. The nanocomposite consists of 20 nm semiconductor nanoparticles embedded in an insulative polymer host. This gives restricted diffusion length, being on the nanometer-scale of the nanoparticle diameter. Experimental device operation is demonstrated.

OCT is a promising tool for performing fast and cheap noninvasive biopsies. High speed imaging helps to reduce motion artifacts that cause decreased sensitivity and resolution. Using a point scanning configuration one is ultimately limited in sensitivity. Therefore parallel configurations are a potentially attractive solution to further enhance the speed capabilities of future OCT systems. Even more, if full field configurations are employed one can exploit the intrinsic phase correlation over the field of view for digital wavefront correction techniques. Full field OCT has nevertheless limitations concerning the missing confocal gating. The sensitivity is decreased in the presence of specular reflexes from optical interfaces, furthermore light scattering cross talk between pixel causes additional signal degradation. A good compromise between parallel detection capabilities and confocal gating seems therefore line field OCT. We built a bench top line field system employing a frequency swept source enabling 2D/3D imaging at up to 200 kA-scans/s with an axial resolution of 8μm and a depth range of 3.53mm in air. To prevent specular reflexes reaching the line scan camera, an off axis configuration of the optical path together with spatial filters placed in conjugate planes of the system was used. Geometrical optics based digital refocusing through the full depth range was shown on a sample target containing FeO particles, on a biological sample and in vivo. Furthermore, we assessed the regime where line field has an advantage over point scanning OCT in terms of sensitivity.

Closed-loop brain computer interfaces are rapidly progressing due to their applications in fundamental neuroscience and prosthetics. For optogenetic experiments, the integration of optical stimulation and electrophysiological recordings is emerging as an imperative engineering research topic. Optical stimulation does not only bring the advantage of cell-type selectivity, but also provides an alternative solution to the electrical stimulation-induced artifacts, a challenge in closedloop architectures. A closed-loop system must identify the neuronal signals in real-time such that a strategy is selected immediately (within a few milliseconds) for delivering stimulation patterns. Real-time spike sorting poses important challenges especially when a large number of recording channels are involved. Here we present a prototype allowing simultaneous optical stimulation and electro-physiological recordings in a closed-loop manner. The prototype was implemented with online spike detection and classification capabilities for selective cell stimulation. Real-time spike sorting was achieved by computations with a high speed, low cost graphic processing unit (GPU). We have successfully demonstrated the closed-loop operation, i.e. optical stimulation in vivo based on spike detection from 8 tetrodes (32 channels). The performance of GPU computation in spike sorting for different channel numbers and signal lengths was also investigated.

Two-step Raman spectroscopy phase method was proposed for differential diagnosis of malignant tumor in skin and lung tissue. It includes detection of malignant tumor in healthy tissue on first step with identification of concrete cancer type on the second step. Proposed phase method analyze spectral intensity alteration in 1300-1340 and 1640-1680 cm^-1 Raman bands in relation to the intensity of the 1450 cm^-1 band on first step, and relative differences between RS intensities for tumor area and healthy skin closely adjacent to the lesion on the second step. It was tested more than 40 ex vivo samples of lung tissue and more than 50 in vivo skin tumors. Linear Discriminant Analysis, Quadratic Discriminant Analysis and Support Vector Machine were used for tumors type classification on phase planes. It is shown that two-step phase method allows to reach 88.9% sensitivity and 87.8% specificity for malignant melanoma diagnosis (skin cancer); 100% sensitivity and 81.5% specificity for adenocarcinoma diagnosis (lung cancer); 90.9% sensitivity and 77.8% specificity for squamous cell carcinoma diagnosis (lung cancer).

Detection of fluorescent particles is an integral part of flow cytometry for analysis of selectively stained cells. Established flow cytometer designs achieve great sensitivity and throughput but require bulky and expensive components which prohibit mass production of small single-use point-of-care devices. The use of a combination of innovative technologies such as roll-to-roll printed microuidics with integrated optoelectronic components such as printed organic light emitting diodes and printed organic photodiodes enables tremendous opportunities in cost reduction, miniaturization and new application areas. In order to harvest these benefits, the optical setup requires a redesign to eliminate the need for lenses, dichroic mirrors and lasers. We investigate the influence of geometric parameters on the performance of a thin planar design which uses a high power LED as planar light source and a PIN-photodiode as planar detector. Due to the lack of focusing optics and inferior optical filters, the device sensitivity is not yet on par with commercial state of the art flow cytometer setups. From noise measurements, electronic and optical considerations we deduce possible pathways of improving the device performance. We identify that the sensitivity is either limited by dark noise for very short apertures or by noise from background light for long apertures. We calculate the corresponding crossover length. For the device design we conclude that a low device thickness, low particle velocity and short aperture length are necessary to obtain optimal sensitivity.

A faster healing process was observed in superficial skin wounds after irradiation with the EMOLED photocoagulator. The instrument consists of a compact handheld photocoagulation device, useful for inducing coagulation in superficial abrasions. The illumination is provided by a high power blue LED. Blue light is selectively absorbed by haemoglobin and converted into heat through a photothermal effect. In this study, 10 Sprague Dawley rats were mechanically abraded in four regions of their back: two regions were used as a control and the other two were treated with EMOLED. The photothermal effect was monitored by an infrared thermocamera in order to avoid accidental thermal damage. Visual observations, histopathological analysis and non-linear microscopic imaging performed after 8 days from the treatment showed no adverse reactions and no thermal damage in both treated areas and surrounding tissues. Moreover, a faster healing process and a better-recovered morphology was evidenced in the treated tissue with respect to the untreated tissue. Compared to the control regions, a reduced inflammatory response, a higher collagen content, and a skin morphology more similar to normal skin were observed in the treated regions. Collagen organization in the two regions was characterized using image pattern analysis algorithms on SHG images, demonstrating a fully recovered aspect of dermis as well as a faster neocollagenesis in the treated regions. This study demonstrates that the selective photothermal effect we used for inducing immediate coagulation in superficial wounds is associated to a minimal inflammatory response, which provides reduced recovery times and improved healing process.

We fabricated 1.4 nm nanogold and molecular dark quencher assembled quantum dot for estimating their performances in a target specific conformal changing molecular event. For the assembling, we immobilized each acceptor linked molecular beacons using interaction between biotin at molecular beacon and streptavidins on quantum dot. Through optical analysis of the purified hybrids of the acceptors and quantum dots, we could estimate numbers of the assembled acceptors per quantum dot and their efficiency of energy transfer depending on conformal changes of molecular beacons. We obtained maximum 95 % and 78% of energy transfer efficiency with 17 metallic nanocrystals and 41 black hole quencher 2, the molecular dark quencher per single quantum dote, respectively. Molecular beacons form linear helix from a hair-pin structure by hybridizing with complementary DNA. In the presence of target DNA, energy transfer efficiency of the organic quencher was 22 % while only 2 % decreased efficiency was obtained with the nanogold, indicating higher fluorescence recovery with the ordinary organic quencher. Considering the relatively low assembled number and the large size, a steric hindrance might be attributed to the low fluorescence recovery. Since the energy transfer efficiency obtained with the nanogold at a fixed distance is high enough, it would be still effective to apply nanogold a system, where nanogold is removed permanently from quantum dots.

Common in vitro toxicity tests of drugs, chemicals or nanomaterials involves the measurement of cellular endpoints like stress response, cell viability, proliferation or cell death. The assay systems determine enzyme activity or protein expression by optical read out of enzyme substrates or marker protein labeling. These standard procedures have several disadvantages. Cellular processes have to be stopped at a distinct time point for the read out, where usually only parts of the cells were affected by the treatment with substances. Typically, only one parameter is analyzed and detection of cellular processes requires several time consuming incubations and washing steps.

Here we have applied digital holographic microscopy (DHM) for a multimodal label-free analysis of drug toxicity. NIH 3T3 cells were incubated with 1 μM Taxol for 24 h. The recorded quantitative phase images were analyzed for cell thickness, cell volume, dry mass and cell migration. Taxol treated cells showed rapidly decreasing cell motility as measure of cell viability. A short increase in cell thickness and dry mass indicated cell division and growth in control cells, whereas Taxol treatment resulted in a continuous increase in cell height followed by a rapid decrease and a decrease of dry mass as indicators of cell death.

Multimodal DHM analysis of drug treatment by multiple parameters allows direct and label-free detection of several toxicity parameters in parallel. DHM can quantify cellular reactions minimally invasive over a long time period and analyze kinetics of delayed cellular responses. Our results demonstrate digital holographic microscopy as a valuable tool for multimodal toxicity testing.

Optical Coherence Tomography is a natural candidate for imaging biological structures just under tissue surface. Human thoracic aorta from aneurysms reveal elastin disorders and smooth muscle cell alterations when visualizing the media layer of the aortic wall, which is only some tens of microns in depth from surface. The resulting images require a suitable processing to enhance interesting disorder features and to use them as indicators for wall degradation, converting OCT into a hallmark for diagnosis of risk of aneurysm under intraoperative conditions. This work proposes gradient-based digital image processing approaches to conclude this risk. These techniques are believed to be useful in these applications as aortic wall disorders directly affect the refractive index of the tissue, having an effect on the gradient of the tissue reflectivity that conform the OCT image. Preliminary results show that the direction of the gradient contains information to estimate the tissue abnormality score. The detection of the edges of the OCT image is performed using the Canny algorithm. The edges delineate tissue disorders in the region of interest and isolate the abnormalities. These edges can be quantified to estimate a degradation score. Furthermore, the direction of the gradient seems to be a promising enhancement technique, as it detects areas of homogeneity in the region of interest. Automatic results from gradient-based strategies are finally compared to the histopathological global aortic score, which accounts for each risk factor presence and seriousness.