Imperfect refractive index matching in scanning laser optical tomography and a method for digital correction.

Significance: Scanning laser optical tomography (SLOT) is a volumetric multi-modal imaging technique that is comparable to optical projection tomography and computer tomography. Image quality is crucially dependent on matching the refractive indexes (RIs) of the sample and surrounding medium, but RI matching often requires some effort and is never perfect. Aim: Reducing the burden of RI matching between the immersion medium and sample in biomedical imaging is a challenging and interesting task. We aim at implementing a post processing strategy for correcting SLOT measurements that have errors caused by RI mismatch. Approach: To better understand the problems with poorly matched Ris, simulated SLOT measurements with imperfect RI matching of the sample and medium are performed and presented here. A method to correct distorted measurements was developed and is presented and evaluated. This method is then applied to a sample containing fluorescent polystyrene beads and a sample made of olydimethylsiloxane with embedded fluorescent nanoparticles. Results: From the simulations, it is evident that measurements with an RI mismatch larger than 0.02 and no correction yield considerably worse results compared to perfectly matched measurements. RI mismatches larger than 0.05 make it almost impossible to resolve finer details and structures. By contrast, the simulations imply that a measurement with an RI mismatch of up to 0.1 can still yield reasonable results if the presented correction method is applied. The experiments validate the simulated results for an RI mismatch of about 0.09. Conclusions: The method significantly improves the SLOT image quality for samples with imperfectly matched Ris. Although the absolutely best imaging quality will be achieved with perfect RI matching, these results pave the way for imaging in SLOT with RI mismatches while maintaining high image quality.

Mueller Matrix Analysis of Collagen and Gelatin Containing Samples Towards More Objective Skin Tissue Diagnostics.

Electrospun polycaprolactone:gelatin (PCL:GT) fibre scaffolds are widely employed in the field of tissue implants. Here, the orientation of fibres plays an important role in regard to implantation due to the impact on the mechanical properties. Likewise, the orientation of collagen fibres in skin tissue is relevant for dermatology. State-of-the-art fibre orientation measurement methods like electron microscopy are time consuming and destructive. In this work, we demonstrate polarimetry as a non-invasive approach and evaluate its potential by measuring the Mueller matrix (MM) of gelatin and collagen containing samples as simple skin tissue phantoms. We demonstrate that it is possible to determine the orientation of PCL:GT fibre scaffolds within one MM measurement. Furthermore, we determine the structural orientation in collagen film samples. Currently, the diagnosis of skin diseases is often performed by image analysis or histopathology respectively, which are either subjective or invasive. The method presented, here, provides an interesting alternative approach for such investigations. Our findings indicate that the orientation of collagen fibres within skin lesions might be detectable by MM measurements in the future, which is of interest for skin diagnostics, and will be further investigated during the next step.

Mueller Matrix Measurement of Electrospun Fiber Scaffolds for Tissue Engineering.

Electrospun fiber scaffolds are gaining in importance in the area of tissue engineering. They can be used, for example, to fabricate graded implants to mimic the tendon bone junction. For the grading of the tensile strength of the fiber scaffolds, the orientation of the fibers plays a major role. This is currently measured by hand in scanning electron microscope (SEM) images. In this work, a correlation between polarimetric information generated by measuring the Mueller matrix (MM) and the orientation of the fibers of electrospun fiber scaffolds is reported. For this, the MM of fiber scaffolds, which were manufactured with different production parameters, was measured and analyzed. These data were correlated with fiber orientation and mechanical properties, which were evaluated in an established manner. We found that by measurement of the MM the production parameters as well as the relative orientation of the fibers in space can be determined. Thus, the MM measurement is suitable as an alternative tool for non-contact, non-destructive determination of the production parameters and, thus, the degree of alignment of electrospun fiber scaffolds.

Monte Carlo simulation of the influence of internal optical absorption on the external Raman signal for biological samples.

Proper understanding of Raman spectroscopic signals from biological samples requires the quantification of internal signal absorption and its effect on the Raman spectra detected outside the samples under study. In this paper, we describe an efficient Monte Carlo method to simulate Raman scattering in biological tissues and solutions and compare the findings with experimental results obtained in samples with different absorber concentrations and optical properties. As an illustrative example, we focus on solutions of beta-carotene (bCar) in ethanol with different concentrations of absorber (ink) added. We find good agreement between simulation and experiment, thus indicating a way to quantify the influence of internal signal absorption in Raman measurements.

Single Transparent Piezoelectric Detector for Optoacoustic Sensing-Design and Signal Processing.

In this article, we present a simple and intuitive approach to create a handheld optoacoustic setup for near field measurements. A single piezoelectric transducer glued in between two sheets of polymethyl methacrylate (PMMA) facilitates nearfield depth profiling of layered media. The detector electrodes are made of indium tin oxide (ITO) which is both electrically conducting as well as optically transparent, enabling an on-axis illumination through the detector. By mapping the active detector area, we show that it matches the design form precisely. We also present a straightforward approach to determine the instrument response function, which allows to obtain the original pressure profile arriving at the detector. To demonstrate the validity of this approach, the measurement on a simple test sample is deconvolved with the instrument response function and compared to simulation results. Except for the sputter instrumentation, all required materials and instruments as well as the tools needed to create such a setup are available to standard scientific laboratories.

Raman Sensing and Its Multimodal Combination with Optoacoustics and OCT for Applications in the Life Sciences.

Currently, many optical modalities are being investigated, applied, and further developed for non-invasive analysis and sensing in the life sciences. To befit the complexity of the study objects and questions in this field, the combination of two or more modalities is attempted. We review our work on multimodal sensing concepts for applications ranging from non-invasive quantification of biomolecules in the living organism to supporting medical diagnosis showing the combined capabilities of Raman spectroscopy, optical coherence tomography, and optoacoustics.

Multivariate discrimination of heat shock proteins using a fiber optic Raman setup for in situ analysis of human perilymph.

Raman spectroscopy has proven to be an effective tool for molecular analysis in different applications. In clinical diagnostics, its application has enabled nondestructive investigation of biological tissues and liquids. The human perilymph, for example, is an inner ear liquid, essential for the hearing sensation. The composition of this liquid is correlated with pathophysiological parameters and was analyzed by extraction and mass spectrometry so far. In this work, we present a fiber optic probe setup for the Raman spectroscopic sampling of inner ear proteins in solution. Multivariate data analysis is applied for the discrimination of individual proteins (heat shock proteins) linked to a specific type of hearing impairment. This proof-of-principle is a first step toward a system for sensitive and continuous in vivo perilymph investigation in the future.

Polymer Based Whispering Gallery Mode Humidity Sensor.

Whispering gallery mode (WGM) resonators are versatile high sensitivity sensors, but applications regularly suffer from elaborate and expensive manufacturing and read-out. We have realized a simple and inexpensive concept for an all-polymer WGM sensor. Here, we evaluate its performance for relative humidity measurements demonstrating a sensitivity of 47 pm/% RH. Our results show the sensor concepts' promising potential for use in real-life applications and environments.

Trimodal system for in vivo skin cancer screening with combined optical coherence tomography-Raman and colocalized optoacoustic measurements.

A new multimodal system for rapid, noninvasive in vivo skin cancer screening is presented, combining optical coherence tomography (OCT) and optoacoustic (OA) modalities to provide precise tumor depth determination with a Raman spectroscopic modality capable of detecting the lesion type and, thus, providing diagnostic capability. Both OA and Raman setups use wide field skin illumination to ensure the compliance with maximum permissible exposure (MPE) requirements. The Raman signal is collected via the OCT scanning lens to maximize the signal-to-noise ratio of the measured signal while keeping radiation levels below MPE limits. OCT is used to optically determine the tumor thickness and for volumetric imaging whereas OA utilizes acoustic signals generated by optical absorption contrast for thickness determination at potentially higher penetration depths compared to OCT. Preliminary results of first clinical trials using our setup are presented. The measured lesion depth is in good agreement with histology results, while Raman measurements show distinctive differences between normal skin and melanocytic lesions, and, moreover, between different skin areas. In future, we will validate the setup presented for reliable detection of pathophysiological parameters, morphology and thickness of suspicious skin lesions.

Simulation of Raman scattering including detector parameters and sampling volume.

Raman spectroscopy can be employed to measure the chemical composition of a sample, which can in turn be used to extract biological information. The aim of this paper is to introduce an efficient simulation technique for Raman spectroscopy in turbid (scattering) media taking into account relevant detector parameters and the sampling volume. We simulate the process of photon motion in turbid media by means of the Monte Carlo (MC) method. The numerical simulation of Raman scattering consists of two stages: calculation of the photon fluence at each point of the medium and subsequent generation of the corresponding amount of Raman photons at each point. The developed model allows simulation of both confocal and optical fiber probe Raman setups. In more detail, the model efficiently simulates Raman signals for different single and multi-layer phantoms and geometries, including focused and collimated (i.e., the fiber-based case) excitation laser beams as well as different values for the numerical aperture and the excitation beam radius. In the future, our results offer the potential to improve the design of Raman systems for in vivo applications in biomedical research.

Optical properties of the human round window membrane.

Optical techniques are effective tools for diagnostic applications in medicine and are particularly attractive for the noninvasive analysis of biological tissues and fluids in vivo. Noninvasive examinations of substances via a fiber optic probe need to consider the optical properties of biological tissues obstructing the optical path. This applies to the analysis of the human perilymph, which is located behind the round window membrane. The composition of this inner ear liquid is directly correlated to inner ear hearing loss. In this work, experimental methods for studying the optical properties of the human round window membrane ex vivo are presented. For the first time, a comprehensive investigation of this tissue is performed, including optical transmission, forward scattering, and Raman scattering. The results obtained suggest the application of visible wavelengths (>400 nm) for investigating the perilymph behind the round window membrane in future.

Efficient procedure for the measurement of preresonant excitation profiles in UV Raman spectroscopy.

Resonance Raman spectroscopy (RRS) is a promising technique for investigating samples with low concentrations of single constituents or many different constituents. The wavelength dependent resonance enhancement (resonance profile) of the respective molecule yields information about the targeted species and reveals the optimal wavelength for high resolution RRS. A significant increase of the Raman scattered intensity can already be achieved in the vicinity of the molecules' absorption band (preresonance). Measuring such preresonance and resonance profiles requires precise control of excitation conditions and careful assessment of the spectral accuracy of the setup. We present a comprehensive procedure for the acquisition of preresonance profiles in Raman spectroscopy. An experimental setup for recording the single spectra is combined with an efficient algorithm for data postprocessing. The procedure is demonstrated on amino acids measured in the UV and can be applied to any molecule and wavelength range.

Comparative study of presurgical skin infiltration depth measurements of melanocytic lesions with OCT and high frequency ultrasound.

A reliable, fast, and non-invasive determination of melanoma thickness in vivo is highly desirable for clinical dermatology as it may facilitate the identification of surgical melanoma margins, determine if a sentinel node biopsy should be performed or not, and reduce the number of surgical interventions for patients. In this work, optical coherence tomography (OCT) and high frequency ultrasound (HFUS) are evaluated for quantitative in vivo preoperative assessment of the skin infiltration depth of melanocytic tissue. Both methods allow non-invasive imaging of skin at similar axial resolution. Comparison with the Breslow lesion thickness obtained from histopathology revealed that OCT is slightly more precise in terms of thickness determination while HFUS has better contrast. The latter does not require image post-processing, as necessary for the OCT images. The findings of our pilot study suggest that non-invasive OCT and HFUS are able to determine the infiltration depth of lesions like melanocytic nevi or melanomas preoperatively and in vivo with a precision comparable to invasive histopathology measurements on skin biopsies. In future, to further strengthen our findings a statistically significant study comprising a larger amount of data is required which will be conducted in an extended clinical study in the next step. Comparison of optical coherence tomography and high frequency ultrasound B-Scans and a H&E stained histology of a melanocytic nevus.

Confocal Raman microscopy and fluorescent in situ hybridization - A complementary approach for biofilm analysis.

We combine confocal Raman microscopy (CRM) of wet samples with subsequent Fluorescent in situ hybridization (FISH) without significant limitations to either technique for analyzing the same sample of a microbial community on a cell-to-cell basis. This combination of techniques allows a much deeper, more complete understanding of complex environmental samples than provided by either technique alone. The minimalistic approach is based on laboratory glassware with micro-engravings for reproducible localization of the sample at cell scale combined with a fixation and de- and rehydration protocol for the respective techniques. As proof of concept, we analyzed a floc of nitrifying activated sludge, demonstrating that the sample can be tracked with cell-scale precision over different measurements and instruments. The collected information includes the microbial content, spatial shape, variant chemical compositions of the floc matrix and the mineral microparticles embedded within. In addition, the direct comparison of CRM and FISH revealed a difference in reported cell size due to the different cell components targeted by the respective technique. To the best of our knowledge, this is the first report of a direct cell-to-cell comparison of confocal Raman microscopy and Fluorescent in situ hybridization analysis performed on the same sample. An adaptation of the method to include native samples as a starting point is planned for the near future. The micro-engraving approach itself also opens up the possibility of combining other, functionally incompatible techniques as required for further in-depth investigations of low-volume samples.

All-polymer whispering gallery mode sensor system.

Sensors based on whispering gallery modes have been extensively investigated with respect to their possible application as physical or biological sensors. Instead of using a single resonator, we use an all polymer resonator array as sensing element. A tunable narrowband laser is coupled into a PMMA plate serving as an optical wave guide. PMMA spheres are placed in the evanescent field on the surface of the plate. Due to small size variations, some spheres are in resonance at a given wavelength while others are not. We show that this device is well suited for the determination of an unknown wavelength or for temperature measurements. Moreover, we discuss several general aspects of the sensor concept such as the number and size of sensing elements which are necessary for a correct measurement result, or the maximum acceptable linewidth of the laser.

Two efficient approaches for modeling of Raman scattering in homogeneous turbid media.

The quantitative analysis of Raman spectroscopic signals in biological tissue is generally difficult. Typical samples contain a multitude of molecular species and, in addition, measurements are altered by attenuation of the Raman signal. Realistic numerical modeling of the Raman process can help to facilitate the quantitative analysis of the Raman spectra, but approaches so far are scarce and often time-consuming. In this work, we report on two different and very efficient approaches for modeling of Raman scattering in turbid media irradiated by laser light. Both approaches utilize the Monte Carlo method to simulate the Raman scattering process. We compare the efficiency of both approaches and discuss possible future extensions and experimental validation.

Cladded self-written multimode step-index waveguides using a one-polymer approach.

Low-loss optical-coupling structures are highly relevant for applications in fields as diverse as information and communication technologies, integrated circuits, or flexible and highly-functional polymer sensor networks. For this suitable and reliable production methods are crucial. Self-written waveguides are an interesting solution. In this work, we present a simple and efficient one-polymer approach for self-written optical connections between light-guiding structures such as single-mode and multi-mode optical fibers or waveguides that relies on self focusing of the light inside a photopolymerizing mixture. The optical connections are produced in a two-step process by writing into monomer resin using cw laser light in the blue wavelength range and subsequent UV curing. Since only one photopolymerizing resin is required, we reduced the fabrication complexity compared to previous approaches to obtain a waveguide embedded in a rigid cladding material. We discuss the production method, the results obtained as function of relevant process parameters such as writing speed or curing time, and evaluate optical properties and coupling efficiencies.

Light source design for spectral tuning in biomedical imaging.

We propose an architecture with a remote phosphor-based modular and compact light-emitting diode (LED) light source in a noncontact dermoscope prototype for skin cancer screening. The spectrum and color temperature of the output light can easily and significantly be changed depending on spectral absorption characteristics of the tissues being imaged. The new system has several advantages compared to state-of-the-art phosphor converted ultrabright white LEDs, used in a wide range of medical imaging devices, which have a fixed spectrum and color temperature at a given operating point. In particular, the system can more easily be adapted to the requirements originating from different tissues in the human body, which have wavelength-dependent absorption and reflectivity. This leads to improved contrast for different kinds of imaged tissue components. The concept of such a lighting architecture can be vastly utilized in many other medical imaging devices including endoscopic systems.

Temperature-sensitive gating of hCx26: high-resolution Raman spectroscopy sheds light on conformational changes.

The temperature-sensitive gating of human Connexin 26 (hCx26) was analyzed with confocal Raman microscopy. High-resolution Raman spectra covering the spectral range between 400 and 1500 rel. cm(-1) with a spectral resolution of 1 cm(-1) were fully annotated, revealing notable differences between the spectrum recorded from solubilized hCx26 in Ca(2+)-buffered POPC at 10°C and any other set of protein conditions (temperature, Ca(2+) presence, POPC presence). Spectral components originating from specific amino acids show that the TM1/EL1 parahelix and probably the TM4 trans-membrane helix and the plug domain are involved in the gating process responsible for fully closing the hemichannel.

A computational model for previtamin D(3) production in skin.

Low levels of vitamin D have been implicated in a wide variety of health issues from calcemic diseases to cancer, diabetes and cardiovascular disease. For most humans, the majority of vitamin D(3) is derived from sunlight. How much vitamin D is produced under given exposure conditions is still widely discussed. We present a computational model for the production of (pre-)vitamin D within the skin. It accounts for spectral irradiance, optical properties of the skin and concentration profile of provitamin D. Results are computed for various sets of these parameters yielding the distribution of produced previtamin D in the skin.