This PDF file contains the front matter associated with SPIE Proceedings Volume 8798, including the Title Page, Copyright Information, Table of Contents, and the Conference Committee listing.

We describe here the conjugation of polymethylmethacrylate nanoparticles to particular oligonucleotide switches, termed molecular beacons (MBs), as potential intracellular nanosensors. Survivin mRNA targeting MBs have been used with Atto647N and Blackberry 650 as fluorophore/quencher pair. The nanosensors have been characterized in vitro by investigating the analytical performances of the chosen molecular beacon and its functionalities after conjugation to the nanoparticles.

Pupil phase masks for enhanced depth of field microscopy were investigated by using a spatial light modulator. The phase masks were evaluated with simulations in terms of the mean square error between in-focus and out-of-focus point spread functions. The resulting best-performing phase masks were tested for fluorosphere samples using a microscope add-on containing the SLM. First, z-stacks of fixed fluorospheres in an agarose medium were recorded in order to measure the extended depth of field. The same measurements were also performed on fluorospheres subjected to Brownian motion in an aqueous solution. The results show that with deconvolution and appropriate filtering it is possible to obtain sharp fluorosphere images with an extended depth of field of at least 10 μm.

The uptake and intracellular distribution of the cytostatic drug doxorubicin is visualized in 2D and 3D systems of human breast cancer cells and fibroblasts by fluorescence microscopy and spectroscopy. Fluorescence lifetime imaging (FLIM) and scattering experiments with high angular resolution are suggested to probe apoptotic reactions. A light scattering microscope as well as a light sheet module for 3D fluorescence microscopy have been developed and are used for this purpose.

Complex biological systems often require measurements of multiple parameters with high temporal and spatial resolution. Multimodal approaches and the combination of methods are therefore a powerful tool to address such scientific questions. Laser speckle imaging (LSI) is an optical method that monitors dynamic changes in cortical blood flow (CBF) with high temporal resolution. Positron emission tomography (PET) allows for quantitative imaging of physiological processes and is a gold standard method to determine absolute cerebral blood flow. We developed a setup that allows simultaneous measurement with both modalities. Here, we simultaneously measured CBF with PET and LSI in rats and analyzed how the correlation of PET and LSI is modified when (1) different methods are used for the calculation of speckle inverse correlation time (ICT), (2) speckle data is acquired through thinned or craniectomized skull, (3) influence of surface vessels is removed from the speckle data. For the latter, a method for automated vessel segmentation from LSI data was developed. We obtained the best correlation (R² = 0.890, p<0.001) when correcting for surface vessel structures taking into account the contribution of static scatterers while keeping the coherence factor constant. However, using the originally published relation, which allows a 900 times faster computation of blood flow maps, still provided a good correlation (R² = 0.879, p<0.001). Given the good correlation between LSI and PET we used our data to calibrate the speckle ICT. Thus, LSI provides CBF in absolute units at high temporal resolution.

We present a single-photon camera for fluorescence imaging capable of providing both intensity and lifetime images, with an accuracy better than 100ps; the camera was fabricated in standard CMOS technology. As a first step towards the study of biologically relevant samples, it was used to characterize in-vitro cultured melanoma cells labeled with indocyanine green (ICG) and ICG conjugated with cyclic pentapeptide (RGDfK). The application field would be fluorescence-guided surgical oncology.

Action potentials in cardiac myocytes have durations in the order of magnitude of 100 milliseconds. In biomedical investigations the documentation of the occurrence of action potentials is often not sufficient, but a recording of the shape of an action potential allows a functional estimation of several molecular players. Therefore a temporal resolution of around 500 images per second is compulsory. In the past such measurements have been performed with photometric approaches limiting the measurement to one cell at a time. In contrast, imaging allows reading out several cells at a time with additional spatial information. Recent developments in camera technologies allow the acquisition with the required speed and sensitivity. We performed action potential imaging on isolated adult cardiomyocytes of guinea pigs utilizing the fluorescent membrane potential sensor di-8-ANEPPS and latest electron-multiplication CCD as well as scientific CMOS cameras of several manufacturers. Furthermore, we characterized the signal to noise ratio of action potential signals of varying sets of cameras, dye concentrations and objective lenses. We ensured that di-8-ANEPPS itself did not alter action potentials by avoiding concentrations above 5 μM. Based on these results we can conclude that imaging is a reliable method to read out action potentials. Compared to conventional current-clamp experiments, this optical approach allows a much higher throughput and due to its contact free concept leaving the cell to a much higher degree undisturbed. Action potential imaging based on isolated adult cardiomyocytes can be utilized in pharmacological cardiac safety screens bearing numerous advantages over approaches based on heterologous expression of hERG channels in cell lines.

Atherosclerosis is a primary cause of critical ischemic disease and disease attributed atherosclerosis is major mortality in the world today. The risk of critical event is involved the content of lipid in plaque. Near-infrared multispectral imaging (MSI) is suitable for the evaluation of plaque because it can provide spectroscopic information and spatial image quickly with simple measurement system. In this paper, the optimal wavelengths to detect plaque were investigated in the near-infrared wavelength range with atherosclerotic phantom. Supercontinuum light was illuminated on a grating spectrometer for the selection of a specific wavelength, and the wavelength-limited light was irradiated to the phantom. Two phantoms were observed by near-infrared camera in the wavelength range from 1150 to 1790 nm. Plaque phantom can be detected with three wavelengths containing an absorption peak of lipid at 1210 nm or 1730 nm. Especially, the absorption peak at 1730 nm had advantage over 1210 nm even considering the difference of penetration depth. The multispectral images were blurred with decreasing the number of wavelengths. These result showed the possibility of MSI using three wavelengths including 1210 nm and 1730 nm for enhancing diagnosis of atherosclerotic plaque.

Dysplastic progression is known to be associated with changes in morphology and internal structure of cells. A detailed assessment of the influence of these changes on cellular scattering response is needed to develop and optimize optical diagnostic techniques. In this study, we first analyzed a set of quantitative histopathologic images from cervical biopsies and we obtained detailed information on morphometric and photometric features of segmented epithelial cell nuclei. Morphometric parameters included average size and eccentricity of the best-fit ellipse. Photometric parameters included optical density measures that can be related to dielectric properties and texture characteristics of the nuclei. These features enabled us to construct realistic three-dimensional computational models of basal, parabasal, intermediate, and superficial cell nuclei that were representative of four diagnostic categories, namely normal (or negative for dysplasia), mild dysplasia, moderate dysplasia, and severe dysplasia or carcinoma in situ. We then employed the finite-difference time-domain method, a popular numerical tool in electromagnetics, to compute the angle-resolved light scattering properties of these representative models. Results indicated that a high degree of variability can characterize a given diagnostic category, but scattering from moderately and severely dysplastic or cancerous nuclei was generally observed to be stronger compared to scattering from normal and mildly dysplastic nuclei. Simulation results also pointed to significant intensity level variations among different epithelial depths. This suggests that intensity changes associated with dysplastic progression need to be analyzed in a depth-dependent manner.

Simultaneous LIF, LIBS and PA spectroscopic techniques are applied to investigate abnormal lymph tissues due to hodgkin disease. The distinct differences in the spectra are taken into account for early and rapid identification of malignant tissues.

Living colon carcinoma cells were investigated by confocal Raman microspectroscopy. An in vitro model of tumor progression was established. Evaluation of data sets by cluster analysis reveals that lipid bodies might be a valuable diagnostic parameter for early carcinogenesis.

We designed and developed two different optical fibre probes for combined Raman and fluorescence spectroscopic measurements on human tissues. The experimental setup combines fluorescence spectroscopy and Raman spectroscopy in a multimodal approach. Two laser diodes, respectively emitting in the UV (378 nm) and in the visible (445 nm), were used for fluorescence spectroscopy. An additional laser diode emitting in the NIR (785 nm) was used for Raman spectroscopy. Laser light was delivered to the tissue under examination through a multimode optical fibre located in the centre of the fibre bundle probe. The surrounding 24 optical fibres were used for collection of the signal of interest and for delivering light to a common detection unit. Both fluorescence and Raman spectra were acquired on a cooled CCD camera, connected to a spectrograph. The device was successfully used for diagnosing melanocytic lesions in a good agreement with common routine histology. Additional measurements were performed on other human tissue samples, such as colon tissue and brain tissue in order to test the capability of the device for diagnosing a broader range of tissue lesions and malignancies. The system has the potential to improve diagnostic capabilities on a broad range of tissues and to be used for endoscopic inspections in the near future.

To perform a contactless plethysmographic imaging, we investigated a method to estimate the concentrations of oxygenated and deoxygenated blood in human skin tissue from RGB images, based on the Monte Carlo simulation.

Time-lapsed, three-dimensional multiphoton microscopy showed that application of air-drying and glycerol to animal tissue induced a well-expressed optical clearing. The effect was dynamic, reversible process, and can be used to enhance capabilities of nonlinear imaging.

We investigate different fibre probes for fluorescence measurements. Our design shows a threefold sensitivity improvement compared to a single fibre probe allowing bacteria concentration as low as 1 CFU/ml to be measured.

In the course of pulmonary research, understanding alveolar tissue dynamics plays a critical role in the treatment of patients suffering from acute lung diseases. As a gold standard technique for monitoring micro scale changes of lung tissue, real-time intra-vital microscopy (IVM) has been established to evaluate the behavior of the alveolar tissue. To allow profound qualitative and quantitative conclusions, characteristic features of the obtained images have to be thoroughly understood. These factors are strongly influenced by the imaging setup and physiological condition of the lung. To circumvent misinterpretations, a ray-tracing approach has been applied in this study using an idealized geometry of the mouse lung parenchyma deduced from optical coherence tomography (OCT) as a complementary imaging technique. Basic features of IVM images are double ring structures and disappearing of alveoli related to liquid infiltration. Ray propagation analysis reveals the formation of these features by two major reflection processes: partial reflection and total internal reflection. The results give rise to quantification errors of the alveolar area related to reflexes misinterpreted as alveolar borders and should further be used to yield a correction factor for future IVM lung tissue studies.

The structure of an artificial ligament was examined using Raman microscopy in combination with novel data analysis. Basis approximation and compressed principal component analysis are shown to provide efficient compression of confocal Raman microscopy images, alongside powerful methods for unsupervised analysis. This scheme allows the acceleration of data mining, such as principal component analysis, as they can be performed on the compressed data representation, providing a decrease in the factorisation time of a single image from five minutes to under a second. Using this workflow the interface region between a chemically engineered ligament construct and a bone-mimic anchor was examined. Natural ligament contains a striated interface between the bone and tissue that provides improved mechanical load tolerance, a similar interface was found in the ligament construct.

The one-shot-type spectroscopic-tomography is proposed to develop the medical-patient-condition monitoring systems. The optical-setup is configured with the relative-inclined phase-shifter for improving the time resolution and the phase-shift array for improving visibility. We obtained the line-spectroscopic imaging and could recognize the Hg bright-line-spectrum that is a component of the light-source. The realization of the optical stethoscope for early diagnosis of cancer can be expected by obtaining the 2-dimensional spectroscopic distribution with rotating interferometer.

Raman spectroscopy increasingly becomes a valuable analytical tool in biomedicine. A novel Raman microscope designed for biomedical applications was used to discriminate viability states and cell types of Hodgkin’s disease as well as different neural and invading glioblastoma cells within a human engineered neural tissue (ENT).

The interaction between gold nanoparticles and glucose and its effect on the fluorescence spectrum of nanoparticles were investigated experimentally. It was observed after this interaction the intensity of fluorescence peak becomes weaker and red shifted.

The inappropriate use of antibiotics leads to antibiotic resistance, which is a major health care problem. The current method for determination of bacterial susceptibility to antibiotics requires overnight cultures. However most of the infections cannot wait for the results to receive treatment, so physicians administer general spectrum antibiotics. This results in ineffective treatments and aggravates the rising problem of antibiotic resistance. In this work, a rapid method for diagnosis and antibiogram for a bacterial infection was developed using Surface Enhanced Raman Spectroscopy (SERS) with silver nanoparticles. The advantages of this novel method include its rapidness and efficiency which will potentially allow doctors to prescribe the most appropriate antibiotic for an infection. SERS spectra of three species of gram negative bacteria, Escherichia coli, Proteus spp., and Klebsiella spp. were obtained after 0 and 4 hour exposure to the seven different antibiotics. Bacterial strains were diluted in order to reach the concentration of (2x10⁵ cfu/ml), cells/ml which is equivalent to the minimum concentration found in urine samples from UTIs. Even though the concentration of bacteria was low, species classification was achieved with 94% accuracy using spectra obtained at 0 hours. Sensitivity or resistance to antibiotics was predicted with 81%-100% accuracy from spectra obtained after 4 hours of exposure to the different antibiotics. This technique can be applied directly to urine samples, and with the enhancement provided by SERS, this method has the potential to be developed into a rapid method for same day UTI diagnosis and antibiogram.

Bacterial identification is one of the applications for which classification using Raman spectra has proved to be successful. In this paper, we propose the use of Rank Order Kernels to classify Raman spectra in order to identify bacterial samples. Rank Order Kernels are two-dimensional image functions. The first step in the process transforms each Raman spectrum to a two-dimensional image. This is achieved by splitting the spectra into segments and calculating the ratio between the mean value of each and every other segment. The resulting two-dimensional matrix of ratios for each Raman spectrum is the image processed by the Rank Order Kernels. A similarity metric is used with a nearest neighbor algorithm for classification. The metric is based on rank order kernels. Our results show that the rank order kernel method is comparable in accuracy to other previously-used methods.

We developed a computerized optical probe for near real time bacterial detection in water. This microorganism detection technique, based on fluorescence enhanced by nucleic acids staining, shows promising results compared to conventional methods.

Early detection and excision of melanoma skin cancer is crucial for a successful therapy. Dermoscopy in direct contact with the skin is routinely used for inspection, but screening is time consuming for high-risk patients with a large number of nevi. Features like symmetry, border, color and most importantly changes like growth or depigmentation of a nevus may indicate malignancy. We present a non-contact remote imaging system for human melanocytic nevi with homogenous illumination by an ultra-bright white LED. The advantage compared to established dermoscopy systems requiring direct skin contact is that deformation of raised nevi is avoided and full-body scans of the patients may time-efficiently be obtained while they are in a lying, comfortable position. This will ultimately allow for automated screening in the future. In addition, calibration of true color rendering, which is essential for distinguishing between benign and malignant lesions and to ensure reproducibility and comparison between individual check-ups in order to follow nevi evolution is implemented as well as suppression of specular highlights on the skin surface by integration of polarizing filters. Important features of the system which will be crucial for future integration into automated systems are the possibility to record images without artifacts in combination with short exposure times which both reduce image blurring caused by patient motion.

We have investigated multiple lens based non-contact illumination and detection configurations, including a conventional cone configuration and a novel cone shell configuration, for depth sensitive diffuse reflectance and fluorescence measurements numerically and experimentally.

Possibilities of measuring systems that uses ellipsoidal reflectors for determining the optical parameters of biological tissue are studied. The modified inverse Monte Carlo method was designed for biomedical photometric system “biological tissue - ellipsoidal mirror.”

We propose a silver coated microneedle to detect test molecules, including R6G and glucose, positioned at a depth of more than 700 μm below a skin phantom surface for mimicking intradermal surface-enhanced Raman scattering measurements.

The multi-spectral imaging technique to reveal skin malformations has been described in this work. Four spectral images taken at polarized monochromatic LED illumination (450nm, 545nm, 660nm and 940 nm) and polarized white LED light imaged by CMOS sensor via cross-oriented polarizing filter were analyzed to calculate chromophore maps. The algorithm based on skin color analysis and user-defined threshold selection allows highlighting of skin areas with predefined chromophore concentration semi-automatically. Preliminary results of clinical tests are presented.

Possibilities to map skin chromophores using a modified low-cost digital video-microscope is discussed. The device comprises CMOS digital imaging sensor, four-colour LED illumination system and image acquisition optics. The main goal is to obtain a set of spectral images of the skin area of interest for further conversion into maps of the main skin chromophores

We used infrared reflection microspectroscopy for chemical imaging of urinary calculi and showed that contribution of diffuse reflection, influencing the imaging results, can be suppressed by decreasing surface roughness and (or) increasing wavelength of infrared radiation applied for the imaging.

We have used infrared microspectroscopy for chemical analysis of urinary sediments. We showed that Mie scattering from urinary sediments as small as 10-100 μm is influencing the spectra and the influence can be suppressed and quality of the spectra can be improved by applying RMieS-EMSC procedure.

In the imaging of blood concentration change using near infrared bio-speckles, temporal averaging of speckle images is necessary for speckle reduction. To improve the temporal resolution in blood concentration imaging, use of spatial averaging is investigated to measured data in rat experiments. Results show that three frames in temporal averaging with (2×2) pixels in spatial averaging can be accepted to obtain the temporal resolution of ten concentration images per second.

Noninvasive multispectral imaging method was applied for different skin pathology such as nevus, basal cell carcinoma, and melanoma diagnostics. Developed melanoma diagnostic parameter, using three spectral bands (540 nm, 650 nm and 950 nm), was calculated for nevus, melanoma and basal cell carcinoma. Simple multispectral diagnostic device was established and applied for skin assessment. Development and application of multispectral diagnostics method described further in this article.

We present a method for the determination of absorption spectra in VIS and NIR spectra of turbid media without the need for calibration. Measurements of the absorption spectra of a phantom and butter are presented.

A method to identify vulnerable plaques that are likely to cause acute coronary events has been required. The object of this study is identifying vulnerable plaques by hyperspectral imaging in near-infrared range (NIR-HSI) for an angioscopic application. In this study, NIR-HSI of atherosclerotic tissue phantoms was demonstrated under simulated angioscopic conditions. NIR-HSI system was constructed by a NIR super continuum light and a mercury-cadmium-telluride camera. Spectral absorbance values were obtained in the wavelength range from 1150 to 2400 nm at 10 nm intervals. The hyperspectral images were constructed with spectral angle mapper algorithm. As a result, detections of the lipid area in the atherosclerotic tissue phantom under angioscopic observation conditions were achieved especially in the wavelength around 1200 nm, which corresponds to the second overtone of CH stretching vibration mode.

Two measurement setups with different optical adapters that enable multichannel rapid simultaneous measurement of calcium transients and length changes of adult cardiac cells are presented and compared on a sample experimental data taken from experiments.

We detected near-infrared luminescence from singlet oxygen in photosensitization reaction with talaporfin sodium rich condition excited by CW laser in vitro. The applicability of our singlet oxygen luminescence measurement system to in vivo is suggested.