D. Scorticati, A. Illiberi, G. R. B. Römer, T. Bor, W. Ogieglo, M. Klein Gunnewiek, A. Lenkferink, C. Otto, J. Z. Skolski, F. Grob, D. de Lange, A. Huis in 't Veld
Ultra-short pulsed laser sources, with pulse durations in the ps and fs regime, are commonly exploited for cold ablation. However, operating ultra-short pulsed laser sources at fluence levels well below the ablation threshold allows for fast and selective thermal processing. The latter is especially advantageous for the processing of thin films. A precise control of the heat affected zone, as small as tens of nanometers, depending on the material and laser conditions, can be achieved. It enables the treatment of the upper section of thin films with negligible effects on the bulk of the film and no thermal damage of sensitive substrates below. By applying picosecond laser pulses, the optical and electrical properties of 900 nm thick SnO2 films, grown by an industrial CVD process on borofloat®-glass, were modified. The treated films showed a higher transmittance of light in the visible and near infra-red range, as well as a slightly increased electrical sheet resistance. Changes in optical properties are attributed to thermal annealing, as well as to the occurrence of Laser- Induced Periodic Surface Structures (LIPSSs) superimposed on the surface of the SnO2 film. The small increase of electrical resistance is attributed to the generation of laser induced defects introduced during the fast heating-quenching cycle of the film. These results can be used to further improve the performance of SnO2-based electrodes for solar cells and/or electronic devices.
Hyperspectral coherent anti-Stokes Raman scattering (CARS) microscopy is quickly becoming a prominent imaging
modality because of its many advantages over the traditional paradigm of multispectral CARS. In particular, recording a
significant portion of the vibrational spectrum at each spatial pixel allows image-wide spectral analysis at much higher
rates than can be achieved with spontaneous Raman. We recently developed a hyperspectral CARS method, the driving
principle behind which is the fast acquisition and display of a hyperspectral datacube as a set of intuitive images wherein each material in a sample appears with a unique trio of colors. Here we use this system to image and analyze two types of polymorphic samples: the pseudopolymorphic hydration of theophylline, and the packing polymorphs of the sugar alcohol mannitol. In addition to these solid-state form modifications we have observed spectral variations of crystalline mannitol and diprophylline as functions of their orientations relative to the optical fields. We use that information to visualize the distributions of these compounds in a pharmaceutical solid oral dosage form.
Traditionally, the composition of bone and cartilage is determined by standard histological methods. We used Raman microscopy, which provides a molecular "fingerprint" of the investigated sample, to detect differences between the zones in human fetal femur cartilage without the need for additional staining or labeling. Raman area scans were made from the (pre)articular cartilage, resting, proliferative, and hypertrophic zones of growth plate and endochondral bone within human fetal femora. Multivariate data analysis was performed on Raman spectral datasets to construct cluster images with corresponding cluster averages. Cluster analysis resulted in detection of individual chondrocyte spectra that could be separated from cartilage extracellular matrix (ECM) spectra and was verified by comparing cluster images with intensity-based Raman images for the deoxyribonucleic acid/ribonucleic acid (DNA/RNA) band. Specific dendrograms were created using Ward's clustering method, and principal component analysis (PCA) was performed with the separated and averaged Raman spectra of cells and ECM of all measured zones. Overall (dis)similarities between measured zones were effectively visualized on the dendrograms and main spectral differences were revealed by PCA allowing for label-free detection of individual cartilaginous zones and for label-free evaluation of proper cartilaginous matrix formation for future tissue engineering and clinical purposes.
We demonstrate a method for performing nonlinear microspectroscopy that provides an intuitive and unified
description of the various signal contributions, and allows the direct extraction of the vibrational response. Three
optical fields create a pair of Stokes Raman pathways that interfere in the same vibrational state. Frequency
modulating one of the fields leads to amplitude modulations on all of the fields. This vibrational molecular
interferometry (VMI) technique allows imaging at high speed free of non-resonant background, and is able to
distinguish between electronic and vibrational contributions to the total signal.
The use of nanoparticles in biomedical applications is emerging rapidly. Recent developments have led to numerous
studies of noble metal nanoparticles, down to the level of single molecule detection in living cells. The application of
noble metal nanoparticles in diagnostics and treatment of early stage carcinomas is the subject of many present studies.
Gold nanoparticles are particularly interesting for optical biomedical applications due to their biocompatibility and
moreover, their enhanced absorption cross-sections. The latter is a result of surface plasmon resonance, which can be
tuned by altering the shape of the nanoparticles enabling usage of the near infrared tissue transparent optical window.
This paper presents a brief overview of the variety of shapes, size and surface chemistries of the gold nanoparticles used
for cancer detection and treatment, as well as their effects in different tumour models that have recently been
investigated, both in vitro and in vivo.
In biological samples the resonant CARS signal of less abundant constituents can be overwhelmed by the nonresonant
background, preventing detection of those molecules. We demonstrate a method to obtain the phase of
the oscillators in the focal volume that allows discrimination of those hidden molecules. The phase is measured
with respect to the local excitation fields using a cascaded
phase-preserving chain. It is measured point-bypoint
and takes into account refractive index changes in the sample, phase curvature over the field-of-view and
interferometric instabilities. The detection of the phase of the vibrational motion can be regarded as a vibrational
extension of the linear (refractive index) phase contrast microscopy introduced by Zernike around 1933.
In this article we show that heterodyne CARS, based on a controlled and stable phase-preserving chain, can be
used to measure amplitude and phase information of molecular vibration modes. The technique is validated by
a comparison of the imaginary part of the heterodyne CARS spectrum to the spontaneous Raman spectrum of
polyethylene. The detection of the phase allows for rejection of the non-resonant background from the data. The
resulting improvement of the signal to noise ratio is shown by measurements on a sample containing lipid.
We demonstrate heterodyne detection of CARS signals using a cascaded phase-preserving chain to generate the CARS input wavelengths and a coherent local oscillator. The heterodyne amplification by the local oscillator reveals a window for shot noise limited detection before the signal-to-noise is limited by amplitude fluctuations. We demonstrate an improvement in sensitivity by more than 3 orders of magnitude for detection using a photodiode. This will enable CARS microscopy to reveal concentrations below the current mMolar range.
The phagocyte NADPH oxidase is a crucial enzyme in the innate immune response of leukocytes against invading microorganisms. The superoxide (O2-) that is generated by this enzyme upon infection is directly and indirectly used in bacterial killing. The catalytic subunit of NADPH oxidase, the membrane-bound protein heterodimer flavocytochrome b558, contains two heme moieties. Here, we first briefly discuss our recent confocal resonant Raman (RR) spectroscopy and microscopy experiments on flavocytochrome b558 in both resting and phagocytosing neutrophilic granulocytes. Such experiments allow the determination of the redox state of flavocytochrome b558 inside the cell, which directly reflects the electron transporting activity of NADPH oxidase. Subsequently, we report that incubation of murine RAW 264.7 macrophages with PolyActive microspheres for 1 week in culture medium leads to morphological and biochemical changes in the macrophages that are characteristic for the generation of macrophage-derived foam cells. Lipid-laden foam cells are the hallmark of early atherosclerotic lesions. Using nonresonant Raman spectroscopy and microscopy, we demonstrate that the numerous intracellular droplets in macrophages exposed to microspheres are rich in cholesteryl esters. The finding that phagocytic processes may trigger foam cell formation reinforces the current belief that (chronic) infection and inflammation are linked to the initiation and progression of atherosclerotic lesions. The study of such a connection may reveal new therapeutic targets for atherosclerosis treatment or prevention.
Integrated optics micoresonators (μ-resonators) are microstructures with dimensions typically in the order of tens of
microns down to a few microns, whose response depends critically on optical wavelength and material properties. Recent experimental studies have shown that they are suitable as refractive index sensors, absorption sensors, and microresonator-assisted single and two-photon fluorescence. The absorption and fluorescence spectra are material-specific properties, that the devices can readily detect by using different excitation wavelengths. Therefore, the devices
are suitable for non-specific agent detection. Due to their inherent small size and the ease of cascading several microresonators, they are suitable building blocks for a sensing array allowing sensing/detection of multiple quantities/agents on a single chip, by e.g., using different chemo-optical transduction layers on top of the
microresonators. Such devices have a chip-area of only a few 100 μm2, making them suitable for sensing ultra-small analyte volumes (which is advantageous for bio-chemical sensing). In this contribution, sensing arrays based on integrated optics microresonators and their prospects for Homeland Security applications are discussed. Several device-concepts based on integrated optics microresonators will be treated. Their performance is analyzed using realistic parameters and experimental results of microresonator devices realized in silicon oxynitride (SiON) technology. The potential integration of theses devices with microelectronics, micro-mechanics and micro total analysis systems is
discussed.
Singular value decomposition was applied to the set of the
non-resonant Raman spectra, recorded during Raman imaging of the
single apoptotic cell. The basis vectors and the corresponding
singular values were assessed in terms of their statistical
significance. The noise-containing basis vectors were rejected
while keeping the meaningful ones. In this way the Raman images of
the apoptotic cell were successfully reconstructed from the
spectrally-filtered data matrix. The results are demonstrated on
the spatial distribution of the DNA, protein and phospholipids,
present in the fragments of the apoptotic cell.
The potential of integrated optical micro cavities (MC) for use in enhanced optical spectroscopy has been studied. The MC devices can sustain high morphological enhancement of optical field due to excitation of high-Q whispering gallery modes. The evanescent near field of the MC can be used to excite spectroscopic signal of molecules pout on top of the MC. Estimation shows that both local excitation field and emitted field can be increased y 2-3 orders of magnitude in the MC on resonance. In total, a gain of 4-8 orders of magnitude in the Raman/fluorescent signal of a molecule near the MC can be expected. In addition, the MC delivers a tunable and measurable enhancement, which is a real benefit in terms of enhanced optical microspectroscopy on-chip. High-Finesse integrated optics cylindrical micro cavities capable of significant field enhancement have been fabricated. Use of various waveguide/MC coupling schemes and design parameters allowed optimization of the devices for the largest intra-cavity power. The result for different micro cavities show prominent enhancement of intra-cavity field correlating with its mode spectrum. The characterization of MC and measurements performed demonstrate feasibility of the MC-based device for optical spectroscopy.
In this research project a confocal Raman micro spectrometer (CRM) will be designed and incorporated in a scanning electron microscope (SEM). The aim is to develop a new analytical instrumentation to investigate samples on their morphology, atomic composition and molecular composition. Application of the CRM-SEM is in the field of bio-material research where bio-compatibility with an d bio-degeneration by cells and tissues play an important role. The purposes of CRM is for using in medical, biological, chemical and other field science and technology. The energy of these vibration states depends on the molecular structure and environmental condition like PH, temperature. Therefore, the Raman spectrum contains information about the chemical composition of a substance and the structure of molecules. With CRM we can obtain a 3D image of molecular distribution in living cells or composite materials. In this way it provides directly and non-invasively, unique information about the spatial distribution of molecules in inhomogeneous systems. It is important information for such molecules as DNA, protein and other, where a lot of properties their molecules depends on configuration in space. Combining the capacities of the SEM and the CRM will add a powerful device for material investigation.
Scanning Confocal Fluorescence Microscopy is used for single molecule studies on DNA-protein complexes that occur in Nucleotide Excision Repair (NER). During DNA-damage elimination by the NER-pathway, complex protein structures assemble over DNA. It is our aim to resolve the architecture of these DNA-protein complexes and to study dynamic changes that occur in these structures. For this purpose NER- complexes are partly reconstituted onto DNA-substrates using NER-proteins fused to different Green Fluorescent Protein mutants. Here we describe the recombinant production of NER- GFP fusion proteins. Characterization of GFP fluorescence is shown together with results of GFP single molecule imaging. First results with NER-GFP fusion proteins are presented as well.
KEYWORDS: Glucose, Raman spectroscopy, Signal to noise ratio, Luminescence, Spectroscopy, In vivo imaging, Raman scattering, Blood, Light scattering, Error analysis
We have investigated the possibilities of applying Raman spectroscopy for the in-vivo determination of blood glucose levels. To this end we measured Raman spectra of glucose dissolved in pure water and in the presence of other analytes such as glycogen and proteins. Secondly, we determined the fluorescence of blood serum for different excitation wavelengths. Since all measurements were done in an absolute way, we were able to predict if the Raman signal level of glucose was high enough to permit the in-vivo determination of the physiological glucose levels in blood.
The results are presented of the study of intracellular localization of Co-phthalocyanines. The methods were used of Raman and fluorescence microspectroscopy.
We report polarization sensitive coherent Stokes Raman scattering (CSRS) measurements of oxy- and deoxyhemoglobin in aqueous solutions that were carried out under electronic resonance with the Q absorption bands. All independent susceptibility (Chi) (3) components as well as anisotropic and anti-symmetric scattering contributions were resolved within frequency nondegenerate CSRS scheme. Eight bands of oxy- and five of deoxyhemoglobin were observed in the range 1500 - 1680 cm-1. Each set of dispersion profiles was simultaneously fitted with a set of parameters including band positions, widths, amplitudes, phases and, importantly, all the CSRS depolarization ratios. On this basis the major bands were assigned to non-totally symmetric (nu) 10, (nu) 11, and (nu) 19 modes of the porphyrin macrocycle. These displayed a clear correlation of vibrational phase with the mode symmetry. Of eight bands resolved in oxyHb spectra three were attributed to features of intermediate deoxyHb, caused by a partial photolysis of oxyhemes. On a nanosecond time scale they were found essentially similar to those found for stable deoxyHb. A detectable isotropy was observed for all non-totally symmetric modes of both oxy- and deoxyhemes. The (nu) 10 and (nu) 11 modes were found to exhibit anti-symmetry as well. The decrease in depolarization ratio (rho) 1212R of anomalously polarized (nu) 19 mode from 7.7 (oxyheme) to 4.3 (deoxyheme) was detected. The latter evidenced heme deformation related with a further doming that occurs upon release of oxygen.
One of the most interesting problems of modern biophysics is the problem of enzyme conformational changes during the catalytic act. There are some bands in the vibrational spectra of proteins that may be sensitive to the conformational state of the macromolecule. The aim of the present study is to reveal the changes in protein vibrational spectra associated with the interaction with the substrate by means of highly sensitive PSCARS spectroscopy and to refer these spectroscopic changes to the possible conformations of the protein molecule. The peculiarities of the experimental method applied are associated mainly with a very high nonresonant background to resonant signal ratio. This makes necessary the polarization suppression of the background, the sufficient quality of which may only be achieved with very high polarization quality of the pumping beams and rather thin samples (about 2 mm). The PSCARS spectra were measured of protein chymotrypsin and its complex with antranilic acid (model of substrate in this system) solutions in water and heavy water. The spectra were obtained within three frequency ranges: 800 - 900 cm-1, 1180 - 1300 cm-1, 1580 - 1700 cm-1. The vibrational bands analyzed were amide I, amide III, and several bands belonging to tyrosine and tryptophan. It was demonstrated that all the bands studied were sensitive to the ligand binding. The fitting procedure was applied to all the PSCARS spectra and the vibrational bands' parameters were determined (positions, bandwidths, amplitudes). Hereafter we present some details on the results obtained for amide I band.
Due to the coherent nature of the third order CARS process the spectra obtained are often seriously corrupted by the noise brought by the laser pulse-to-pulse fluctuations. A broadband (multiplex) CARS technique is known to be an efficient mean to diminish the noise contributed by the laser power fluctuations as well as the imperfections in the spatial and temporal coherence of laser beams. In this work, we coupled the broadband CARS scheme with the `scanning-multichannel' detection technique. By combining several multichannel Raman spectra detected by a CCD camera at different positions of a monochromator, the authors have achieved (1) enhanced dynamic range, (2) large spectral length, (3) improved accuracy of peak position and bandwidth determination due to an increased density of measurement points and (4) elimination of the variation in the sensitivity and noise characteristics of each detector element.
Raman spectroscopic techniques are developed to study the presence, structure and dynamics of (bio-)organic molecules at surfaces. Coherent anti-Stokes Raman scattering (CARS) and spontaneous Raman scattering (spRS) are used in combination with the technology to fabricate optical waveguides. A mode-locked Nd-YLF laser system with approximately 8 ps. pulse lengths was used as well as a Q-switched nanosecond pulselength Nd-YAG laser. In principle the spRS technique is more simple, however the CARS technique allows background suppression by asymmetric mode selection. Improvement of the ratio of signal of the monolayer to that of the waveguide can hence be expected. A theory was developed that describes the radiative field of a dipole in a waveguide. This theory describes the angular distribution of the radiated power from a dipole positioned at a chosen position in the waveguide configuration. It is calculated that the power varies strongly as a function of direction of observation. In particular in the direction where coupling into substrate modes occurs this is predicted to be the case. The intensity of the Raman scattering in well defined directions can be an order of magnitude larger than in the average direction.
Isotropic and anisotropic Raman spectra of RNA and DNA solutions have been obtained with a scanning monochromator system. The registration of the spectra was optimized for the special purpose of extracting bandwidth information and the non-coincidence effect.
Elastic light scattering and Raman light scattering applied to the same eye-lens have been used, respectively, to extract information on the spatial variations in the intensity of scattered light and the protein content. A combination of the results allows one to obtain the distribution of the scattering coefficient, the size of the scattering particles and the molecular weight of the scattering particles. The design of the light scattering set up is such that the results can be compared with those obtained with a `Scheimpflug' camera. The increase in light scattering at the anterior and posterior cortex in young (< 20 - 30 yrs.) eye-lenses is in accordance with the theory for short-range crystalline order in eye-lenses.
Polarization sensitive coherent Raman spectroscopy (PCARS/PCSRS) was applied to study proteins in solutions in both two and three color configurations. In off-electron resonant PCARS the main problem is the nonresonant background, arising from the solvent. The advantage of PCARS is the resolving power as is demonstrated in the amide-I region of some proteins. Three color PCSRS was used to measure independently the (chi) (3) components of hemoglobin resonantly excited in the (alpha) and (beta) absorption bands. From a simultaneous fit on different PCSRS spectra vibrational parameters were determined including phases and depolarization ratios. Besides the polarization and phase characterization of hemoglobin bands, we decomposed the vibrational amplitudes and phases into electronic contributions. Together with amplitudes, the phases contain information on electronic enhancement, vibronic coupling and symmetry of the molecular vibrations.
A sensitive confocal Raman microspectrometer (CRM) has been developed in our
laboratory enabling the study of single living cells and chromosomes [1,2].
Characteristics of the CR14 are; efficient signal collection (by a high power
microscope objective), confocal detection achieved by positioning a small pinhole in
the image plane of the microscope objective, a high signal throughput of the
spectrograph (including a Chevron type dielectric band pass filter set for laser
1 ight suppression , with a 80-90% transmission of Raman signal [3] ) and virtually
photon-noise limited signal detection by a liquid nitrogen cooled CCD-camera
(quantum efficiency 40% at 700 nm). Laser light of 660 nm from a DCM-operated dye
laser is used for excitation. It prevents degradation of the samples in the focused
beam, a phenomenon observed when using the 514.5 nm line of an argon-ion laser. The
degradation in that case is ascribed to as yet unidentified photo-chemical processes
since the possibilities of excessive heating of the samples and multiphoton
absorption could be ruled out [51 . When using 660 nm laser light Raman spectra can
be obtained even of single fixed metaphase chromosomes in air without any signs of
damage [6] . Radial and axial spatial resolution of the CRM in the standard
configuration are 0.45 m and 1.3 un respectively [4]. This enables the recording of
Raman spectra of specific cellular or chromosomal regions in situ.
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