Proceedings Volume 7376

Laser Applications in Life Sciences

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Proceedings Volume 7376

Laser Applications in Life Sciences

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Volume Details

Date Published: 24 November 2010
Contents: 12 Sessions, 51 Papers, 0 Presentations
Conference: Laser Applications in Life Sciences 2010 2010
Volume Number: 7376

Table of Contents

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Table of Contents

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  • Front Matter
  • Nano-Biophotonics
  • Laser-Tissue Interactions
  • Laser Biomedical Diagnostics, Sensing and Therapy
  • Single Cells and Molecules; Optical Trapping and Manipulation
  • THz Waves in Biophotonics
  • Vibrational Spectroscopy, Structure and Dynamics of Biological Systems
  • Molecular and Bio-Imaging
  • Laser Microscopies
  • Novel Optical Devices for Biomedicine
  • Printing Techniques and their Applications in Biotechnology
  • Symposium on Water in Bioenvironment: Spectroscopy and Simulation
Front Matter
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Front Matter: Volume 7376
This PDF file contains the front matter associated with SPIE Proceedings Volume 7376, including the Title Page, Copyright information, Table of Contents, Introduction (if any), and the Conference Committee listing
Nano-Biophotonics
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Ripple-patterned substrates for light enhancement applications
We report on surface structuring of sapphire, silicon carbide, and silicon by femtosecond laser pulses in multipulse irradiation mode. The formed ripples on the flat surface or on the vertical walls with hierarchical structures whose feature sizes are ranging from the irradiation wavelength down to ~ 50 nm are prospective templates for surface enhanced Raman scattering after coating with plasmonic metals. We study complex patterns of fine ripples with periods Λr, as small as λ/Rp, where Rp (see manuscript) 3 - 5. The mechanisms suggested for such Rp values are discussed: temperature and density of breakdown plasma, angle of incidence, mechanism of second harmonic generation (SHG) and absorption. Predictions of the surface and bulk models of ripple formation are compared with experimental values of Rp-factor. We propose a model of ripple formation on the surface, which is based on the known in-bulk sphere-to-plane formation of breakdown plasma in the surface proximity. In semiconductor 4H:SiC normal ripples with periods 190 and 230 nm were recorded with 800 nm and 1030 nm fs-laser pulses respectively. We show that the period of ripples is defined by the dielectric properties of crystalline (solid) phase rather than the molten phase in the case of silicon. Generation of SHG on the surface of sample and plasma nano-bubbles are discussed: surface-SHG is found not important in ripples' formation as revealed by comparative study of periods on Al2O3 and TiO2 at 800 nm wavelength of irradiation. We propose that ripple periodicity is pinned to the smallest possible standing wave cavity (λ/n)/2 inside material of refractive index n.
CdTe quantum dots stabilization by protein in aqueous solution
Marija Matulionyte, Deividas Motekaitis, Vilius Poderys, et al.
Semiconductor nanoparticles are promising fluorescent markers. However it is very little known about interaction of quantum dots with biological molecules. In this study interaction of CdTe quantum dots coated with thioglycolic acid (TGA) with bovine serum albumin was investigated. Steady state and time resolved spectroscopy and atomic force microscopy methods were used. It was explored how bovine serum albumin affects stability and spectral properties of quantum dots in aqueous media. CdTe-TGA quantum dots in aqueous solution appeared to be not stable and precipitated. Interaction with bovine serum albumin significantly enhanced stability and photoluminescence quantum yield of quantum dots and prevented from aggregating.
The development of SPR device for biomarker detection
Pirkko Tervo, Janne Katainen, Adama Sesay, et al.
We have built an imaging SPR device with incident angle calibration system allowing selection of the most sensitive measurement area. Applying the device, an assay for salivary immunoglobulin A (IgA) was developed. Surface plasmon resonance (SPR) provides a highly sensitive, fast and label-free detection method for biomarkers. The specific binding of measured analyte onto the active bio-sensitive layer of the SPR sensor surface causes a change in the refractive index. Imaging SPR allows the analysis of the several different immobilization areas at the same time. Our imaging SPR device utilises a 750nm superluminescent light emitting diode (SLED) with manual incident angle calibration system, which enables us to choose the most sensitive measurement area. This makes the measurement faster and more sensitive for samples of varying refractive index. We demonstrated the applicability of the instrument by developing an immunoassay detection method for immunoglobulin A. IgA is a dominant immunoglobulin in secretions in the mucosal immune system and it has a role in upper respiratory tract infection (URTI). It is also a marker of physical stress.
Development of diagnostic SPR based biosensor for the detection of pharmaceutical compounds in saliva
Susanna Sonny, Adama M. Sesay, Vesa Virtanen
The aim of the study is to develop diagnostic tests for the detection of pharmaceutical compounds in saliva. Oral fluid is increasingly being considered as an ideal sample matrix. It can be collected non-invasively and causes less stress to the person being tested. The detection of pharmaceutical compounds and drugs in saliva can give valuable information on individual bases on dose response, usage, characterization and clinical diagnostics. Surface plasmon resonance (SPR) is a highly sensitive, fast and label free analytical technique for the detection of molecular interactions. The specific binding of measured analyte onto the active gold sensing surface of the SPR device induces a refractive index change that can be monitored. To monitor these pharmaceutical compounds in saliva the immunoassays were developed using a SPR instrument. The instrument is equipped with a 670nm laser diode and has two sensing channels. Monoclonal antibodies against the pharmaceutical compounds were used to specifically recognise and capture the compounds which intern will have an effect of the refractive index monitored. Preliminary results show that the immunoassays for cocaine and MDMA (3,4-methylenedioxymethamphetamine) are very sensitive and have linear ranges of 0.01 pg/ml - 1 ng/ml and 0.1 pg/ml - 100 ng/ml, respectively.
Laser-induced microbubbles in gold and oxide nanoparticle suspensions: photoacoustic detection
A tunable pulsed laser with nano-second pulse duration is used to generate microbubbles in highly diluted nanoparticle (Au, TiO2 and ZnO) suspensions. The microbubble explosion may produce shock wave which is in-phase detected by a low-frequency piezoelectric transducer. The effects of particle size and category on the threshold laser fluence of shock wave generation and the wave intensity are investigated. The interaction between laser and nanoparticles has significant application in biomedicine such as photothermal diagnostic and therapy, as well as cosmetic or drug delivery in skin.
Complex of water-soluble CdSe/ZnS quantum dots and chlorin e6: interaction and FRET
J. Valanciunaite, A. Skripka, G. Streckyte, et al.
In this work we present the spectral study on complex formation between CdSe/ZnS-amino (620 nm) quantum dots (QDs) and second-generation photosensitizer chlorin e6 (Ce6). In the presence of QDs, significant changes in the absorption and fluorescence properties of Ce6 were observed. The fluorescence spectrum of bound Ce6 molecules displayed the new intensive fluorescence band at 670 nm, similar to that observed in organic solvent chloroform. With an increase in QDs:Ce6 molar ratio up to 1:20, the spectrum of QDs-Ce6 showed the decrease in intensity of QDs fluorescence band at 620 nm, while the intensity of fluorescence band at 670 nm was increasing simultaneously. The fluorescence excitation spectrum of bound Ce6 molecules exposed a contribution of QDs spectrum. The fluorescence decay measurements of QDs-Ce6 solution displayed the shortening of QDs fluorescence lifetime at increasing Ce6concentration. We conclude, that Ce6 molecules interact with the hydrophobic groups of QDs coating, which results in the changes of spectral properties of Ce6. Upon binding, Ce6 molecules are located close enough to the shell/core of QDs for Förster resonance energy transfer (FRET) from QDs to Ce6 to occur.
Laser-Tissue Interactions
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3D Monte Carlo model of optical transport in laser-irradiated cutaneous vascular malformations
Boris Majaron, Matija Milanič, Wangcun Jia, et al.
We have developed a three-dimensional Monte Carlo (MC) model of optical transport in skin and applied it to analysis of port wine stain treatment with sequential laser irradiation and intermittent cryogen spray cooling. Our MC model extends the approaches of the popular multi-layer model by Wang et al.1 to three dimensions, thus allowing treatment of skin inclusions with more complex geometries and arbitrary irradiation patterns. To overcome the obvious drawbacks of either "escape" or "mirror" boundary conditions at the lateral boundaries of the finely discretized volume of interest (VOI), photons exiting the VOI are propagated in laterally infinite tissue layers with appropriate optical properties, until they loose all their energy, escape into the air, or return to the VOI, but the energy deposition outside of the VOI is not computed and recorded. After discussing the selection of tissue parameters, we apply the model to analysis of blood photocoagulation and collateral thermal damage in treatment of port wine stain (PWS) lesions with sequential laser irradiation and intermittent cryogen spray cooling.
Accelerated 3D Monte Carlo light dosimetry using a graphics processing unit (GPU) cluster
William Chun Yip Lo, Lothar Lilge
This paper presents a basic computational framework for real-time, 3-D light dosimetry on graphics processing unit (GPU) clusters. The GPU-based approach offers a direct solution to overcome the long computation time preventing Monte Carlo simulations from being used in complex optimization problems such as treatment planning, particularly if simulated annealing is employed as the optimization algorithm. The current multi- GPU implementation is validated using a commercial light modelling software (ASAP from Breault Research Organization). It also supports the latest Fermi GPU architecture and features an interactive 3-D visualization interface. The software is available for download at http://code.google.com/p/gpu3d.
The photodynamic therapy effect of aluminum and zinc tetrasulfophthalocyanines on melanoma cancer cells
K. Maduray, A. Karsten, B. Odhav, et al.
Photodynamic therapy (PDT) represents a novel treatment that uses a photosensitizer (PS), light source (laser) of an appropriate wavelength and oxygen to induce cell death in cancer cells. The aim of this study was to investigate the photodynamic effects of aluminum tetrasulfophthalocyanines (AlTSPc) and zinc (ZnTSPc) tetrasulfophthalocyanines activated with a 672nm wavelength laser on melanoma cancer, dermal fibroblast and epidermal keratinocyte cells. Each cell line was photosensitized with either AlTSPc or ZnTSPc for 2 h before using a diode laser with a wavelength of 672nm to deliver a light dose of 4.5 J/cm2 to the cells. The cell viability of melanoma cells were decreased to approximately 50% with concentrations of 40 μg/ml for AlTSPc and 50 μg/ml for ZnTSPc. These PS concentrations caused a slight decrease in the cell viability of fibroblast and keratinocyte cells. Both photosensitizers in the presence of high concentrations (60 μg/ml-100 μg/ml) showed cytotoxicity effects on melanoma cells in its inactive state. This was not observed in fibroblast and keratinocyte cells. Cell death in PDT treated melanoma cells was induced by apoptosis. Therefore, AlTSPc and ZnTSPc exhibit the potential to be used as a PS in PDT for the treatment of melanoma cancer.
IR spectroscopy vs. Raman scattering by measurement of glucose concentration
O. Abdallah, J. Hansmann, A. Bolz, et al.
By developing a non-invasive device for glucose concentration measurement, two promising methods were compared for that aim. The Raman scattering using Laser at the wavelength 785 nm and the light scattering in R- and IR-range are demonstrated. An easy accessible and low-cost method for glucose concentration monitoring and management to avoid its complications will be a great help for diabetic patients. Raman Scattering is a promising method for noninvasively measuring of glucose and for the diagnostic of pathological tissue variations. Despite the power and the time of measurement can be reduced using enhanced Raman scattering, it will be difficult to develop a compatible device with low power Laser and low price for a non-invasive method for home monitoring. As using IR-spectroscopy at wavelengths slightly below 10000 nm, the absorption of glucose can be well discriminated from that of water, LED`s or LD´s at these wavelengths are very expensive for this purpose. At wavelengths about 6250 and 7700 glucose has a less light absorption than water. Also slightly above 3000 nm glucose has a high absorption. There are also possibilities for the measurement in the NIR at wavelengths between 1400 nm and 1670 nm. Scattering measurements at wavelengths below 900 nm and our measurements with the wavelength about 640 nm give reproducible glucose dependence on the reflected light from a glucose solution at a constant temperature. A multi-sensor with different wavelengths and temperature sensor will be a good choice for in-vivo glucose monitoring.
Composite fillings microleakage after TEM00 Er: YAG laser texturing of human tooth enamel surface
A. V. Belikov, K. V. Shatilova, A. V. Skrypnik, et al.
The results of comparative investigation of methylene-blue microleakage between tooth enamel surface and light-cure composites various fluidity are presented. An enamel surface was treated by traditional methods or laser method (laser texturing). The role of adhesive systems is investigated at enamel texturing by the TEM00 Er: YAG radiation. It is shown, that microleakage was not observed when enamel was textured by the TEM00 Er: YAG laser radiation and covered with flowable composite "Revolution" (Kerr) without adhesive system. It is established, that for laser textured surfaces methylene-blue microleakage depends on distance between microcraters.
Hard tooth tissue removal efficiency by single-mode low energy Er:YAG laser
A. V. Belikov, A. V. Skrypnik, K. V. Shatilova
The ablation of dental hard tissue by the single-mode (TEM00) Er: YAG laser radiation was investigated. For the first time dependence of removal efficiency of enamel and dentin of human teeth on the single pulse energy TEM00 Er: YAG laser is represented. Attention is drawn to the correlation of laser energy and micro-craters form. The fact that the efficiency of human tooth enamel and dentin removal by TEM00 Er: YAG laser radiation are close to each other is deduced from experiments. Removal efficiency for enamel reaches the maximum value at pulse energy near 1 mJ and is equal 250±20 mm3/kJ. Removal efficiency for dentine also reaches the maximum at pulse energy near 1 mJ and is equal 265±20 mm3/kJ.
Laser-induced skin temperature oscillations
S. Podtaev, A. Dumler, N. Muravyov, et al.
Adaptive wavelet analysis algorithms are used to study skin temperature oscillations caused by periodic changes in the blood flow resulting from oscillations in the vasomotor smooth muscle tone. Reduction in the amplitude of temperature fluctuations with frequency arises because the skin, owing to its low thermal diffusivity, has the properties of a lowfrequency filter. In view of their small amplitude, oscillations in the spectral range, reflecting the influence of heartbeat and respiration, cannot be distinguished from the external thermal noise. To analyze changes in oscillations of skin temperature during the laser stimulation (10 mW/cm2, 630 nm) we extract three frequency bands, corresponding to myogenic, neurogenic and endothelial vascular tone regulation mechanisms. Red laser irradiation causes temperature fluctuations changes within spectral ranges corresponding endothelial functioning and neurogenic activity.
Skin phantoms with realistic vessel structure for OCT measurements
We present here a novel phantom for optical coherence tomography (OCT) made of polyvinyl chloride-plastisol (PVCP). The optical properties of PVCP were estimated by the Mie theory and deduced from OCT measurements. Titanium dioxide (TiO2) powder and black plastic colour (light-absorbing plastic ink) were used to introduce scattering to the phantom and create capillary structure, respectively.
Laser Biomedical Diagnostics, Sensing and Therapy
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Interferometry for topographical diagnostics of RBCs in optical tweezers
Ranjeet Kumar, Sarita Saraswati, Chandra Shakher, et al.
Simultaneous non-invasive trapping and topography of erythrocytes by interferometric fringe projection profilometry has been presented. Sinusoidal fringes were generated with the help of compact Michelson interferometer (CMI) developed by coating a thin reflective layer (~100μm) of Al2O3 on one face of a cubic beam splitter. An external mirror was mounted on XYZ translational stage to control the fringe frequency and orientation. Red He-Ne laser was used to generate the sinusoidal probe -interferogram using CMI to project onto the green-laser (cw) trapped healthy and deceased RBCs separately. Information coded reflected interferograms exhibits characteristic fringe-deviation with respect to probe-pattern for both the healthy and defective RBCs. Fourier transform analysis was adopted to retrieve the phase-map which can be exploited for topography, size determination and refractive-index of RBC. Refractive-index change is directly related with hemoglobin concentration of RBCs at any specific physiological state and hence information about health status and disease progression can be anticipated.
Polarization structure and spectral properties of aminoacid's crystal layers
S. B. Yermolenko, P. V. Ivashko
The qualities of the polarization-correlation structure of the images of thin film of aminoacid are researched. The samples of 20 aminoacid were used in the experiment on the modified micropolarimeter. Research methods are Mueller matrices polarimetry and spectropolarimetry in 200-2000 nm, as well as spectrometry in 2500-25000 nm. The two-dimensional distribution of Stocks image and Mueller matrix elements of aminoacid are obtained, the correlated analysis of the necessary images was carried out. The specific for every amino acids uniaxial character of the crystals was corroborated.
Spectropolarimetry features of biotissue's malignant changes
I. Gruia, S. B. Yermolenko, C. Gavrila, et al.
This paper follows to combine optical and biochemical techniques for identification the cell membrane transformation in the dynamic of growth and development of experimental solid tumour. It is researched that in all the cases the linear dichroism appears in biotissues (the human esophagus, the muscle tissue of rats, prostate tissue) with the cancer disease the magnitude of which depends on the type of the tissue and on the time of the cancer process development. As the linear dichroism is lacking for healthy tissues, then the obtained results can have diagnostic values with the purpose of detection and estimation of the stage of the cancer disease development.
Multispectral imaging of pigmented and vascular cutaneous malformations: the influence of laser treatment
The paper investigates influence and efficacy of laser therapy on pigmented and vascular cutaneous malformations by multispectral imaging technique. Parameter mapping of skin pigmented and vascular lesions and monitoring of the laser therapy efficacy are performed by multispectral imaging in wavelength range 450-700nm by scanning step - 10nm. Parameter maps of the oxyhemoglobin deoxyhemoglobin and melanin derived from the images are presented. Possibility of laser therapy efficacy monitoring by comparison of the parameter maps before and after laser treatment has been demonstrated. As both cutaneous pigmented and vascular malformations are commonly found lesions, the parameter mapping would be a valuable method to use routinely.
Rapid flow cytometry analysis of antimicrobial properties of nettle powder and cranberry powder
Maarit Hattuniemi, Johanna Korhonen, Mari Jaakkola, et al.
Both nettle (Urtica dioica) and cranberry (Vaccinium oxycoccus) are widely known to have good influence on health. The aim of this study was to investigate antimicrobial properties of nettle powder and cranberry powder against Escherichia coli (E. coli) and monitor the growth of the bacteria by a rapid flow cytometry (FCM) method. For FCM measurements samples were stained with fluorescent dyes. The inhibitory effects of plant material on growth of E. coli were estimated by comparing the results of control sample (E. coli) to E. coli samples with plant material. FCM offers both a brilliant tool to investigate the kinetics of the growth of bacterium, since subsamples can be taken from the same liquid medium during the growing period and with fluorescent dyes a rapid method to investigate viability of the bacterium.
Arterial stiffness estimation based photoplethysmographic pulse wave analysis
Matti Huotari, Kari Maatta, Juha Kostamovaara
Arterial stiffness is one of the indices of vascular healthiness. It is based on pulse wave analysis. In the case we decompose the pulse waveform for the estimation and determination of arterial elasticity. Firstly, optically measured with photoplethysmograph and then investigating means by four lognormal pulse waveforms for which we can find very good fit between the original and summed decomposed pulse wave. Several studies have demonstrated that these kinds of measures predict cardiovascular events. While dynamic factors, e.g., arterial stiffness, depend on fixed structural features of the vascular wall. Arterial stiffness is estimated based on pulse wave decomposition analysis in the radial and tibial arteries. Elucidation of the precise relationship between endothelial function and vascular stiffness awaits still further study.
Single Cells and Molecules; Optical Trapping and Manipulation
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Microfluidic system for single cell sorting with optical tweezers
Thomas Bruns, Laszlo Becsi, Marc Talkenberg, et al.
A microfluidic system was developed and combined with optical tweezers for single cell sorting. This system consists of a glass chip of 300 μm thickness with an etched crosswise channel structure, a silicon layer for sealing and a PMMA substrate for tubular coupling. Selected cells are trapped and moved in perpendicular direction to the main flow for recovery in special reservoirs and further evaluation (e.g. by polymerase chain reaction, PCR). In addition, maximum light doses and exposure times for maintaining cell viability were determined.
THz Waves in Biophotonics
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Electric oscillations generated by collective vibration modes of microtubule
Michal Cifra, Daniel Havelka, Ondrej Kucera
Microtubules are important organizing structures of eukaroytic cells. They are electrically polar and have collective vibration modes from kHz to low THz region. In approximation of microtubule subunits (tubulin molecules) as rigid particles, we calculate electric field generated by optical branch of axial longitudinal vibration modes of microtubule. This oscillatory electric field, due to its complex spatial distribution, may play an important role in cellular temporal and spatial organization.
Protein association investigated by THz spectroscopy and molecular modeling
Maria Mernea, Octavian Calborean, Livia Petrescu, et al.
Macromolecular crowding is a common intracellular phenomenon that causes conformational changes of proteins and protein association. We investigated these macromolecular crowding effects on a highly concentrated BSA solution using THz spectroscopy and molecular modeling. We modeled several BSA 50% w/w solutions comprising two BSA molecules in a water box and selected a single model based on the agreement with THz experiments. We further modeled BSA association at concentrations higher than 50% w/w and selected a possible dimer model based on the strength of the interaction between the two proteins. The flexibility of the BSA dimer was compared with the flexibility of BSA from the solution. Monomeric BSA from the solution model presents mobile regions scattered through all the structure, with differences of disposition and extent between the two molecules. Dimerization changes BSA flexibility, as the two molecules from the dimer present compact regions of both high flexibility and low flexibility. The low flexibility regions include their interaction sites.
Vibrational spectra of corticosteroid hormones in the terahertz range
O. P. Cherkasova, M. M. Nazarov, D. A. Sapozhnikov, et al.
The terahertz time-domain and Raman spectra of corticosteroid hormones in the region of low-frequency infrared vibrations have been measured. On the ground of quantum chemical calculations of the frequencies and normal modes the assignments of vibrational bands in the THz-spectra are performed.
Vibrational Spectroscopy, Structure and Dynamics of Biological Systems
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Sum frequency generation spectroscopy of amyloid fibrils and oligomers at air/water interface
Gediminas Niaura, Rima Budvytyte, Zenonas Kuprionis, et al.
Structure of beta-amyloid oligomers and fibrils at air/water interface was studied by sum frequency generation (SFG) spectroscopy. It was found that both small (1-3 nm) and large (4-10 nm) oligomers as well as fibrils adsorb in ordered fashion at air/water interface. Intense resonances from methyl group stretching vibrations at 2875 and 2937 cm-1 and "ice-like" structured water bands were characteristic for adsorbed fibrils. Similar spectral pattern was observed for large oligomers. However, small oligomers exhibited intense SFG resonance near 2912 cm-1 due to methylene or methine C-H stretching vibration. No "ice-like" water band was observed in this case. The possibility of discrimination between the small (toxic) oligomers, large (non-toxic) oligomers and fibrils was demonstrated.
Electron detachment and recombination in aqueous solutions studied with 2- and 3-pulse femtosecond spectroscopy
Hristo Iglev, Alfred Laubereau
The electron photodetachment of the aqueous halides and hydroxide is studied after excitation in the lowest CTTS state. The initially excited state is followed by an intermediate assigned to a donor-electron pair that displays a competition of recombination and separation. The geminate dynamics measured in the various CTTS systems reveal a strong influence of the parent radical. The electron survival probability decreases significantly from 0.77 to 0.29 going from F- to OH-. Results for I- show that excitation of a higher-lying CTTS state opens a new relaxation channel, which directly leads to fully hydrated electron, while the relaxation channel discussed above is not significantly affected. Using pump-repump-probe spectroscopy the pair species is verified via a secondary excitation with separation of the pairs so that the yield of released electrons is increased. Three pulse spectroscopy on aqueous hydroxide give evidence for an additional ultrafast (~700 fs) geminate recombination channel in this system. Comparison of these data with similar experiments on neat water after two-photon excitation with total energy of 9.2 eV demonstrates the important role of (OH-)* for the indirect photoionization of water.
Improved signal extraction method for single-pulse heterodyne CARS spectroscopy
Yu Nagashima, Takayuki Suzuki, Sumio Terada, et al.
Single-pulse heterodyne CARS (coherent anti-Stokes Raman scattering) detection scheme using shaped femtosecond pulses is one of the most sophisticated approach for managing the problem of non-resonant background disturbance in CARS measurement. However, with the signal processing method conducted in the original report,1-3 we found that background suppression and resonant peak extraction were sometimes difficult and incomplete. We discuss the reason of this unsuccessful signal processing and propose an improved method for signal extraction realizing the better quality of extracted spectra.
Cancer tissue screening using surface enhanced Raman scattering
S. Cinta Pinzaru, C. A. Dehelean, A. Falamas, et al.
The present work summarizes our recent results in combining the FT-Raman & micro-Raman techniques with the high sensitivity of the surface enhanced Raman scattering (SERS) to characterize and monitor cancer disease in the tissue biopsy. The Raman imaging technique used for investigating the samples identified SERS signal in the Ag colloidal incubated tissues which provided a great deal of molecular information about the studied samples, indicating at the same time the chemisorption of the Ag nanoparticles inside the biological tissues. A comparison between two different set-ups, a dispersive instrument and an FT-Raman one employed for the investigation of the same samples is also presented and discussed.
Surface plasmon dispersive spectroscopy of thin films at terahertz frequencies
A. K. Nikitin, O. V. Khitrov, A. P. Kyrianov, et al.
A number of surface plasmon (SP) techniques and devices for terahertz (THz) dispersive spectroscopy of thin films have been developed and reviewed. The techniques are based on the strong dependence of SP complex refractive index κ on the transition layer optical constants. Three of the mentioned techniques employ interference in parallel or quasi parallel beams of bulk and (or) surface waves. These three are remarkable for their accuracy and enable investigators to determine both (real and imaginary) parts of κ in one measuring procedure. Some devices implementing the techniques are static and can have measuring time equal to one pulse duration. Besides, two noninterferometric techniques intended for determining only the real part of κ and assuming tunable monochromatic THz sources are described.
Biophysical studies on the structure and function of molecules from the vertebrate myelin sheath
Chaozhan Wang, Matti Myllykoski, Petri Kursula
The myelin sheath is a crucial structure for the proper functioning of the vertebrate nervous system. We employ diverse methods to study the structure, function, and dynamics of the molecules specifically present in myelin. Eventually, we hope to better understand the details of the tightly packed myelin structure and the etiology of myelin-related diseases. The paper will provide background into the molecular structure of myelin, and recent results from our laboratory, dealing with the structure and function of selected myelin proteins, will be highlighted.
Molecular and Bio-Imaging
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New staining methods for yeast like fungi under special consideration of human pathogenic fungi
Astrid Paulitsch-Fuchs, Fritz Treiber, Erik Grasser, et al.
A new method for in-cellular staining of yeast like fungi with Oregon Green and SYTOX Green is presented enabling their detection as well as the observation of cellular details via confocal laser scanning microscopy. Fluorochromes play an important role in many scientific disciplines including medicine, cell biology and botany. For the visualisation of fungal cell walls Calcofluor White is the flourochrome of choice. The necessity of an UV laser for its excitation makes it unpracticable for daily use. Safranin O, DAPI, 2NBDG, Ethidium Bromide and Acridin-orange are commonly used stains for nuclei in fugal microscopy. The attention was given to the possibility of using the differences in staining patterns to distinguish certain pathogenic yeast species e.g. Candida albicans and Candida krusei. Our results show that high quality microscopy of yeast like organisms can readily be achieved by the use of two suitable fluorochromes.
Infrared absorbing dyes tailored for detection and therapy of solid tumors
Fábio A. Schaberle, Luis G. Arnaut, Carlos Serpa, et al.
We explore the use as contrast agents for photoacoustic tomography (PAT) of photosensitizers originally developed for the photodynamic therapy of cancer. We show that halogenated bacteriochlorins can enhance the sensitivity of PAT and increase the depth of the field probed by this technique.
Development of a novel stigmatic imaging mass spectrometer using laser ionization and a multi-turn time-of-flight mass spectrometer
Kunio Awazu, Hisanao Hazama, Hidetoshi Yoshimura, et al.
A stigmatic and microscopic imaging mass spectrometer has been developed using matrix-assisted laser desorption/ionization and a multi-turn time-of-flight mass spectrometer, MULTUM-IMG. Ion images of rhodamine 6G masked by fine grids with pitches of 63.5, 25.4, and 12.7 μm were clearly observed in the linear mode. Separation of stigmatic ion images according to the time-of-flight, i.e., the mass-to-charge ratio of ions was also successfully demonstrated with a micro-dot pattern made with crystal violet and methylene blue. Stigmatic ion images of a micro-dot pattern made with crystal violet was also observed and the ion image of the pattern was maintained after 10 cycles in MULTUM-IMG. A section of a mouse brain stained with crystal violet and methylene blue was observed in the linear mode, and the stigmatic total ion image of crystal violet and methylene blue was in good agreement with the optical microphotograph of the hippocampus in the section.
Widefield TSCSPC-systems with large-area-detectors: application in simultaneous multi-channel-FLIM
Sergei Stepanov, Sergei Bakhlanov, Evgeny Drobchenko, et al.
Novel proximity-type Time- and Space-Correlated Single Photon Counting (TSCSPC) crossed-delay-line (DL)- and multi-anode (MA)-systems of outstanding performance and homogeneity were developed, using large-area detector heads of 25 and 40 mm diameter. Instrument response functions IRF(space) = (60 ± 5) μm FWHM and IRF(time) = (28 ± 3) ps FWHM were achieved over the full 12 cm2 area of the detector. Deadtime at throughput of 105 cps is 10% for "high-resolution" system and 5% in the "video"-system at 106 cps, at slightly reduced time- and space resolution. A fluorescence lifetime of (3.5 ± 1) ps can be recovered from multi-exponential dynamics of a single living cyanobacterium (Acaryochloris marina). The present large-area detectors are particularly useful in simultaneous multichannel applications, such as 2-colour anisotropy or 4-colour lifetime imaging, utilizing dual- or quad-view image splitters. The long-term stability, low- excitation-intensity (< 100 mW/cm2) widefield systems enable minimal-invasive observation, without significant bleaching or photodynamic reactions, thus allowing long-period observation of up to several hours in living cells.
A wide-field multi-parameter FLIM and FRAP setup to investigate the fluorescence emission of individual living cyanobacteria
Marco Vitali, Matthias Reis, Thomas Friedrich, et al.
Multi-parameter fluorescence lifetime imaging microscopy is a powerful tool to investigate the spatial dependence of the fluorescence decay signal of chromophores in living cells. A multi-channel detection system based on a position sensitive Quadrant Anode photomultiplier and time-correlated single photon counting (TCSPC) was applied to monitor the fluorescence decay in individual living cells of the cyanobacterium Thermosynechococcus elongatus. The fluorescence lifetime imaging system was used to detect at a frame-rate of 1 Hertz the recovery after photobleaching of Phycobilisomes (PBS), a photosynthetic pigment-protein complex with light harvesting functions. Simultaneous monitoring of the fluorescence decays of the PBS- and the Chlorophyll a (Chl a)-containing antenna systems unveils a heterogeneity of the cyanobacterial population with respect to the fluorescence lifetime. Furthermore the FLIM images clearly show that the decay of the fluorescence signal in the targeted area becomes longer after the bleaching at 633 nm for the whole measuring time. The results suggest that the diffusing PBSs, which replace the bleached ones in the targeted area, can only partially reestablish excitation energy transfer to Chl a.
Laser Microscopies
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FRET-sensor for imaging with lifetime resolution
Alexander L. Rusanov, Tatiana V. Ivashina, Leonid M. Vinokurov, et al.
FRET-sensor with nonfluorescent protein as an acceptor was synthesized to observe caspase-3 activity in lifetime mode. We inserted caspase-3 cleavable linker between red highly fluorescent protein TagRFP and chromoprotein KFP. Dynamic light scattering was used to determine size of the fusion protein. Incubation with caspase-3 lead to increase both fluorescence intensity and lifetime of the construction. Cleavage of the linker between proteins was confirmed by electrophoresis and immunoblotting. FLIM-microscopy showed the differences between fluorescence decays of A549 cell line expressed TagRFP and TagRFP-23-KFP.
Time-resolved 3D confocal fluorescence microscopy on living cells
M. Schellenberg, E. Peev, M. Kloster, et al.
Time resolved 3D-microscopy using DMD-arrays utilizes the principles of confocal microscopy. Application fitted patterns optimize optical imaging of reflective, transparent, and fluorescent objects. High spatial resolution is achieved simultaneously with high temporal resolution due to fast DMD control. This enables to visualize and track processes in vivo within living biocells.
Diffraction tomography for biological cells imaging using digital holographic microscopy
I. Bergoënd, C. Arfire, N. Pavillon, et al.
Many biological objects are mainly transparent and weakly scattering, thus a promising (and already widely used) way of imaging them consists in considering optical refractive index variations. The method proposed here permits 3D imaging of the refractive index distribution with a tomographic approach. Usually, the classical Radon transform does not sufficiently take into account the physical interaction between light and biological cells, therefore diffraction has to be considered. Diffraction tomography is a method that permits 3D reconstruction of the refractive index, using many captures of the complex optical field, for example at various angles. Then, the 3D Fourier space can be filled with spatial frequencies coming from the different views. Our setup consists in rotating the object under fix illumination and detection. The complex scattered field needed for tomographic reconstruction is obtained from digital holographic microscopy, using one hologram per angle of view. The method is first validated with a spherical object. Mie scattering theory is used to simulate the measured field from which the tomographic reconstruction is performed. Experimental results on microbeads are also presented. The wide capability of 3D imaging using diffraction tomography in biology is shown.
In-line color digital holographic microscope for water quality measurements
Zoltán Göröcs, László Orzó, Márton Kiss, et al.
We introduce a color digital holographic microscope for measuring the biological content of water samples. Our approach uses single shot RGB exposure in an in-line holographic setup to obtain color images. With the application of appropriate numerical algorithms we can fulfill color crosstalk compensation, segmentation, and twin image removal tasks, and we obtain good quality color image reconstructions with 1μm resolution from a 1mm3 volume. We briefly compare the conventional color CCD/CMOS and the Foveon X3 sensor for color digital holographic applications. The in-line holographic setup and reconstruction algorithms are presented with demonstrative simulations, experimentally captured and numerically reconstructed images.
Using digital holographic microscopy for 4D tracking of colloid particles
Dmitry Ekimov, Ville Kaikkonen, Anssi Mäkynen
A setup and algorithm for 4D tracking of microscopic particles is proposed. The particles in certain volume are detected automatically and their coordinates as well as magnitude and phase distributions saved. The saved data can be used to analyze the number, spatial distribution, size, speed and track of the particles. Calculations show that it is possible to measure the 3D position of a particle having a speed as high as 10 m/s. Experiments from 15 to 2000 fps show that high quality video reconstruction of 1 and 6 μm particle flow is possible at least upto particle density of 200 particles/hologram.
Novel Optical Devices for Biomedicine
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Yellow-red semiconductor disk lasers for biophotonics applications
Tomi Leinonen, Antti Härkönen, Ville-Markus Korpijärvi, et al.
Dilute nitride semiconductor disk lasers offer a convenient way of producing 570-650 nm radiation required in medicine and life science. These lasers can produce multi-watt powers with narrow spectra and compact footprints similar to solid state lasers. The advantage of using semiconductor gain materials is their ability to reach wavelengths that are not attainable by traditional solid state lasers. Other advantages include a wide tuning range and the possibility for electrical modulation. Here we demonstrate a narrow band (<30 MHz) yellow (589 nm) disk laser with 2.7 W output power. The gain mirror of the laser is optically pumped with an 808 nm diode laser. The emission wavelength of the laser can be tuned over several nanometers by tilting the filter inside the laser cavity.
Evaluation of microfluidic channels with optical coherence tomography
J. Czajkowski, T. Prykäri, E. Alarousu, et al.
Application of time domain, ultra high resolution optical coherence tomography (UHR-OCT) in evaluation of microfluidic channels is demonstrated. Presented study was done using experimental UHR-OCT device based on a Kerr-lens mode locked Ti:sapphire femtosecond laser, a photonic crystal fibre and modified, free-space Michelson interferometer. To show potential of the technique, microfluidic chip fabricated by VTT Center for Printed Intelligence (Oulu, Finland) was measured. Ability for full volumetric reconstruction in non-contact manner enabled complete characterization of closed entity of a microfluidic channel without contamination and harm for the sample. Measurement, occurring problems, and methods of postprocessing for raw data are described. Results present completely resolved physical structure of the channel, its spatial dimensions, draft angles and evaluation of lamination quality.
RGB imaging of laser-excited skin autofluorescence bleaching rates
In-vivo skin photo-bleaching (intensity decrease during irradiation) has been investigated at 405 nm cw laser excitation. Digital RGB photo-camera was used for studies of the bleaching features by analysis of fluorescent images at separated R, G and B spectral bands. Diagnostic potential based on correlations between skin pigmentation and bleaching rates is discussed.
Measurement of light scattering from trapped particles
Mikael Collins, Antti Kauppila, Artashes Karmenyan, et al.
We have developed an optical tweezers setup combined with a laser light scattering measurement system to measure the elastic light scattering from trapped particles. The setup consists of a near infrared laser (λ=1064nm), a water immersion objective for trapping, a single or double structure sample cuvette, and a HeNe-laser for illuminating the trapped particles. The light is detected with an amplified photomultiplier and a lock-in amplifier. Light scattering images from the trapped particles are also shown with a CCD camera. An optical trap keeps the particle stable during the measurement. The measured scattering patterns from 23.25 μm diameter polystyrene spheres were shown to have good comparability with theoretical modelling. 6.0 μm particles were also measured. The light scattering from trapped red blood cells was much weaker than that from 23.25 μm and 6.0 μm polystyrene spheres, almost at the detection limit of our current detection system configuration. The stability of the polystyrene sphere was much better during the measurements than that of the red blood cell.
Printing Techniques and their Applications in Biotechnology
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Fabrication of polymer waveguide devices for sensor applications
The fabrication of polymer based waveguide devices by different methods is investigated in this work including lithographic, imprinting and focused-ion-beam processing. Also, the combination of luminescent substance with waveguide is evaluated to produce integrated optical micro system including both the light source and sensor structure on a single platform.
Symposium on Water in Bioenvironment: Spectroscopy and Simulation
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Hydrogen bonding in aqueous ethanol solutions studied by Raman spectroscopy
Tatiana Dolenko, Sergey Burikov, Masashi Hojo, et al.
The results of Raman spectroscopy research of aqueous ethanol solutions with various mixing ratios are presented. The analysis of the behavior of Raman spectra with changes in ethanol concentration from 0 to pure ethanol is given. The analysis of contour of stretching OH-band provided information about changes in hydrogen bonding along with increases in ethanol concentration. Obtained results showed that the maximum strength of H-bonding in aqueous ethanol solution corresponded to an ethanol concentration of 15...20 % w/w. The observed strengthening of H-bonding strongly supports the hypothesis of clathrate-like structures. These results were supported by application of MCR-ALS method.
Long-range attraction in aqueous colloidal suspensions
Qing Zhao, Jason Coult, Gerald H. Pollack
Long-range attractions in aqueous suspensions were observed between polymeric microspheres and also between microspheres and a gel bead. Attractive displacements were consistently seen even between like-charged entities, and they were observed over spans as large as 2 mm. Such behaviors are unexpected, and may reside in a long-range attraction mechanism.
Laser scattering in water and aqueous solutions of salts
Nikolai F Bunkin, Alexey V. Shkirin, Valeriy A. Kozlov, et al.
An automated laser polarimetric scatterometer, operating at a wavelength of 532nm, has been developed for measuring the total scattering matrix of liquid samples in a cylindrical geometry. The optical scheme of the polarimeter combines an electro-optic modulator with a rotary quarter-wave plate. The results of experiments involving distilled water and NaCl aqueous solutions are reported. The measured angular dependencies of the elements of the scattering matrix are compared with numerical computations based on the T-matrix method. The experimental scattering matrix data can be interpreted as a scattering on an ensemble of stochastic micron-scale clusters composed of polydisperse air bubbles having effective radii of 70-100 nm. The fractal dimension of such clusters was evaluated as 2.5-2.8. Their concentration increases with salt addition from 103 cm-3 in distilled water to 106 cm-3 in 0.8 M aqueous solution of NaCl.
Mass and charge transfer within a floating water bridge
Elmar C. Fuchs, Luewton L. F. Agostinho, Mathias Eisenhut, et al.
When high voltage is applied to pure water filled into two beakers close to each other, a connection forms spontaneously, giving the impression of a floating water bridge 1-8. This phenomenon is of special interest, since it comprises a number of phenomena currently tackled in modern water science. In this work, the charge and mass transfer through the water bridge are investigated with schlieren visualization and laser interferometry. It can be shown that the addition of a pH dye increases the H+ and OH- production with subsequent electrolysis, whereas schlieren and interferometric methods reveal another mechanism where charge and mass transfer appear to be coupled. Whereas this mechanism seems to be responsible for the electrolysis-less charge and mass transfer in the water bridge, it is increasingly superseded by the electrochemical mechanism with rising conductivity. Thus it can be shown that a pH dye does only indirectly visualize the charge transfer in the water bridge since it is dragged along with the water flow like any other dye, and additionally promotes conventional electrochemical conduction mechanisms, thereby enhancing electrolysis and reducing the masscoupled charge transport and thus destabilizing the bridge.
Spectroscopy on laser induced plasma in cavitation bubbles
Sandra Koch, Martin Reck, Walter Neu, et al.
Spectroscopic results with laser induced breakdown spectroscopy (LIBS) in liquids have shown a signal improvement using double pulse experiments compared to single pulse measurements. A setup of two Q-switched Nd:YAG lasers in an orthogonal arrangement have led to bright illuminated plasma inside cavitation bubbles. A Nd:YAG laser pulse focussed by a lens (6 ns@532 nm, 2.9*1019 W/m2) into the center of a water filled cuvette gives rise to a cavitation bubble. A second tightly focused Nd:YAG laser pulse (5 ns@1064 nm, 3.1*1019 W/m2 ) induces a plasma at approximately 70 μs delay within the cavitation bubble. These absolutely reliable processes allow to capture image sequences of plasma filled cavitation bubbles with an ultrafast camera. The optical emission is guided via a quartz fibre to a Czerny-Turner spectrograph and recorded by an intensified CCD camera. A delay time of more than 1.7 μs between plasma ignition and spectroscopic data taking is necessary to avoid inverse bremsstrahlung and residual lines arising from bright plasma emission. Characteristic spectral line intensities of manganese (Mn) and Palladium (Pd) are recorded and correlated with an internal calibration standard using strontium (Sr) and chromium (Cr) respectively. The LOD of manganese is 0.3 mg/L.