Mechanical and optical properties are the main criteria for assessing the health of dental tissue in contemporary dentistry. Dentinal pathological changes can be detected by visuo-tactile and radiographic methods to guide clinicians in establishing a relevant diagnosis and an adapted therapy. However, such approaches cannot give information on the dentinal microstructure. Recently, laser ultrasonic techniques have been deployed to evaluate the mechanical properties of enamel [1,2] However, such techniques lack the resolution to reveal the transitions differences between tissue layers.
In this work, we used Brillouin light scattering spectroscopy as a non-contact alternative to probe mechanical changes in dentin and dentin-resin interface at GHz hypersonic frequencies. We obtained maps of the Brillouin frequency shift and linewidth that can be interpreted as maps of sound velocity and viscosity. In addition, we observed the specimens by a homemade nonlinear microscopy setup [3]. A 730 nm wavelength Titanium-sapphire laser was used as an excitation source for two-photon excitation fluorescence microscopy (TPEF), while 1040 nm wavelengthYb:KGW laser was used for second harmonic generation (SHG).
Our results show significant changes between healthy tissues and pathological lesions. Such results can help to precisely delineate destructed dentin during clinical procedures, paving the way to minimally invasive strategies. In addition, our simultaneous analysis of Brillouin maps and nonlinear images brings valuable information on structure-related mechanical properties of dentin and dentin-resin adhesive interface.
1. Wang et al, Experimental and numerical studies for nondestructive evaluation of human enamel using laser ultrasonic technique. Appl Opt 52, 6896-6905 (2013).
2. Wang et al., Laser ultrasonic evaluation of human dental enamel during remineralisation treatment. Biomed Opt Express 2, 345-355 (2011).
3. Rabasović M et al. Nonlinear microscopy of chitin and chitinous structures: a case study of two cave-dwelling insects. J Biomed Opt 20, 016010 (2015)
The present study describes utilization of two photon excitation fluorescence (2PE) microscopy for visualization of the hemoglobin in human and porcine erythrocytes and their empty membranes (i.e., ghosts). High-quality, label- and fixation-free visualization of hemoglobin was achieved at excitation wavelength 730 nm by detecting visible autofluorescence. Localization in the suspension and spatial distribution (i.e., mapping) of residual hemoglobin in erythrocyte ghosts has been resolved by 2PE. Prior to the 2PE mapping, the presence of residual hemoglobin in the bulk suspension of erythrocyte ghosts was confirmed by cyanmethemoglobin assay. 2PE analysis revealed that the distribution of hemoglobin in intact erythrocytes follows the cells’ shape. Two types of erythrocytes, human and porcine, characterized with discocyte and echinocyte morphology, respectively, showed significant differences in hemoglobin distribution. The 2PE images have revealed that despite an extensive washing out procedure after gradual hypotonic hemolysis, a certain amount of hemoglobin localized on the intracellular side always remains bound to the membrane and cannot be eliminated. The obtained results open the possibility to use 2PE microscopy to examine hemoglobin distribution in erythrocytes and estimate the purity level of erythrocyte ghosts in biotechnological processes.
Aleksandar Krmpot, Stanko Nikolić, Marco Vitali, Dimitrios Papadopoulos, Sho Oasa, Per Thyberg, Simone Tisa, Masataka Kinjo, Lennart Nilsson, Walter Gehring, Lars Terenius, Rudolf Rigler, Vladana Vukojevic
Quantitative confocal fluorescence microscopy imaging without scanning is developed for the study of fast dynamical processes. The method relies on the use of massively parallel Fluorescence Correlation Spectroscopy (mpFCS). Simultaneous excitation of fluorescent molecules across the specimen is achieved by passing a single laser beam through a Diffractive Optical Element (DOE) to generate a quadratic illumination matrix of 32×32 light sources. Fluorescence from 1024 illuminated spots is detected in a confocal arrangement by a matching matrix detector consisting of the same number of single-photon avalanche photodiodes (SPADs). Software was developed for data acquisition and fast autoand cross-correlation analysis by parallel signal processing using a Graphic Processing Unit (GPU). Instrumental performance was assessed using a conventional single-beam FCS instrument as a reference. Versatility of the approach for application in biomedical research was evaluated using ex vivo salivary glands from Drosophila third instar larvae expressing a fluorescently-tagged transcription factor Sex Combs Reduced (Scr) and live PC12 cells stably expressing the fluorescently tagged mu-opioid receptor (MOPeGFP). We show that quantitative mapping of local concentration and mobility of transcription factor molecules across the specimen can be achieved using this approach, which paves the way for future quantitative characterization of dynamical reaction-diffusion landscapes across live cells/tissue with a submillisecond temporal resolution (presently 21 μs/frame) and single-molecule sensitivity.
Mihailo Rabasović, Dejan Pantelić, Branislav Jelenković, Srećko Ćurčić, Maja Rabasović, Maja Vrbica, Vladimir Lazović, Božidar P. Ćurčić, Aleksandar Krmpot
We performed a study of the nonlinear optical properties of chemically purified chitin and insect cuticle using two-photon excited autofluorescence (TPEF) and second-harmonic generation (SHG) microscopy. Excitation spectrum, fluorescence time, polarization sensitivity, and bleaching speed were measured. We have found that the maximum autofluorescence signal requires an excitation wavelength below 850 nm. At longer wavelengths, we were able to penetrate more than 150-μm deep into the sample through the chitinous structures. The excitation power was kept below 10 mW (at the sample) in order to diminish bleaching. The SHG from the purified chitin was confirmed by spectral- and time-resolved measurements. Two cave-dwelling, depigmented, insect species were analyzed and three-dimensional images of the cuticular structures were obtained.
In this study we show the results for parametric non-degenerate four wave mixing (FWM) obtained using double lambda scheme at D1 line in hot potassium vapor. We have investigated the influence of one-photon detuning and two-photon detuning on the FWM gain. The laser frequency is locked at approximately 1GHz from the resonance 4S1/2 Fg=1 -< 4P1/2, using external reference cavity. The probe beam passes through acoustooptic modulator that enables controllable detuning around 460 MHz (ground state hyperfine splitting) in respect to the pump beam. The vacuum glass cell containing the potassium vapor was heated by hot air in order to achieve necessary concentration of atoms. The efficiency of FWM process is studied by measuring the gains of the conjugate beam the probe beam, simultaneously. The maximal gain was achieved for nonzero two photon detuning.
We present Hanle electromagnetically induced transparency (EIT) resonances obtained from the outer parts of the
Gaussian laser beam. The signal from the outer parts only was obtained by placing circular opaque masks of different
diameters in the center of the laser beam just in front of the detector. The Hanle EIT resonances obtained in that way are
narrower and for high laser intensities even more contrasted. Suggested explanation for the line narrowing is based on
lower power broadening in the wings of the Gaussian laser beam as well as on the traversing of the coherently prepared
atoms through the beam. The resonance contrast to linewidth ratio, when the central part of the beam is blocked, is
higher or equal to the ratio obtained when the whole laser beam is detected, for all laser intensities used in the
experiment. Due to high ratio of contrast and linewidth, resonances obtained in proposed way could be useful in
frequency metrology and magnetometry.
In this communication we present our results related to examination of Coherent Population Trapping resonance profile
on the D1 line of 87Rb when coupling two ground-state Zeeman sublevels belonging to different hyperfine ground-state
levels to a common excited-state one. For this, two coherent laser fields with frequency difference of about 6.8 GHz are
used. The resonance is observed as a narrow dip in the fluorescence in dependence on the modulation frequency (fm) of
the laser light, centered at 2fm = &Dgr;vhf , where &Dgr;vhf is the frequency difference between the ground-state hyperfine levels
of Rb. The resonance observation and its splitting in 7 components have been demonstrated under influence of nonshielded
laboratory magnetic field (MF). It has been shown that different components are with different sensitivity to the
MF gradients that is in agreement with the performed estimations.
We have measured non-linear Faraday effects for dark and bright long-lived Zeeman coherences in the D2 resonance line of 85Rb, for linearly polarized input laser light. Zeeman coherence in the bright state show enhanced nonlinear effects, larger values for the angle of polarization rotation and higher rates of the polarization rotation, similar to recent results for higher refractive index and dispersion in the bright state of Cs 1. Dark and bright states also show different dependence of both polarization rotation angle and of polarization rate on the laser intensity, while the resonance width for both states varies similarly with the laser intensity. We show small enhancement of such non-linear effects when the laser beam diameter increases from 1 mm to 5 mm. Ellipticity of transmitted laser beam vary strongly for amplitudes of the external magnetic field smaller then 50 mG, but the maximum value of the ellipticity remains below &pgr;/300.
Modification of Koester's interferometer is described in this paper, which is used for calibration of gauge blocks up to 100mm in length. Higher precision was obtained by using a camera (for measurement of fringe fractions) and lasers as the source of light. The process of digitalization and processing of the interference pattern images is also described, as well as rejection of noise caused by transmission of laser beams through the optical fiber.
We have demonstrated that the frequency of the laser radiation can be stabilized by controlling its coherence properties. Theoretical analysis of the laser radiation visibility function was performed and connection with the longitudinal mode structure was established. It was established that laser emission frequency could be stabilized by controlling the visibility of interference pattern. Calculations have shown that, in theory, stability around 4 kHz can be expected. As a proof-of-the-principle, a HeNe laser, stabilized by measurement of the interference pattern visibility in a modified Mechelson interferometer, was built. Stabilization was performed by temperature control of the laser tube in a digital feedback loop and total frequency variation of 3 MHz was obtained. Possible improvements of the experimental setup, together with some applications of multimode stabilized lasers are discussed.
Electromagnetically induced transparency and absorption is observed in a transmission of the coupling laser, locked to transition between hyperfine levels of the ground and excited states of 85Rb. The transmission was monitored in a V, Λ and N-type systems while the probe laser, with similar power as the coupling laser, scans across the hyperfine levels F' of the 2P3/2. The measured width of the EIT resonance increases with a square-root dependence of laser intensity. The EIA was observed only for a cycling transition and for F' = F+1, where F and F' are the angluar momentum of the hyper-fine levels of the 2S1/2 and 2P3/2, respectively. We demonstrated the effect of a three-photon EIA in a four-level scheme that involves the Raman transitions via virtual level.
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