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.
We report the utilization of Third-Harmonic Generation microscopy for label-free live cell imaging of lipid droplets in the hypha of filamentous fungus Phycomyces blakesleeanus. THG microscopy images ...showed bright spherical features dispersed throughout the hypha cytoplasm in control conditions and a transient increase in the number of bright features after complete nitrogen starvation. Colocalization analysis of THG and lipid-counterstained images disclosed that the cytoplasmic particles were lipid droplets. Particle Size Analysis and Image Correlation Spectroscopy were used to quantify the number density and size of lipid droplets. The two analysis methods both revealed an increase from 16 × 10
to 23 × 10
lipid droplets/µm
after nitrogen starvation and a decrease in the average size of the droplets (range: 0.5-0.8 µm diameter). In conclusion, THG imaging, followed by PSA and ICS, can be reliably used for filamentous fungi for the in vivo quantification of lipid droplets without the need for labeling and/or fixation. In addition, it has been demonstrated that ICS is suitable for THG microscopy.
Changes in the structural properties of the skin due to collagen alterations are an important factor in diabetic skin complications. Using a combination of photonic methods as an optic diagnostic ...tool, we investigated the structural alteration in rat dermal collagen I in diabetes, and after short-term l-arginine treatment. The multiplex approach shows that in the early phase of diabetes, collagen fibers are partially damaged, resulting in the heterogeneity of fibers, e.g., "patchy patterns" of highly ordered/disordered fibers, while l-arginine treatment counteracts to some extent the conformational changes in collagen-induced by diabetes and mitigates the damage. Raman spectroscopy shows intense collagen conformational changes via amides I and II in diabetes, suggesting that diabetes-induced structural changes in collagen originate predominantly from individual collagen molecules rather than supramolecular structures. There is a clear increase in the amounts of newly synthesized proline and hydroxyproline after treatment with l-arginine, reflecting the changed collagen content. This suggests that it might be useful for treating and stopping collagen damage early on in diabetic skin. Our results demonstrate that l-arginine attenuates the early collagen I alteration caused by diabetes and that it could be used to treat and prevent collagen damage in diabetic skin at a very early stage.
Remodelling of collagen fibers has been described during every phase of cancer genesis and progression. Changes in morphology and organization of collagen fibers contribute to the formation of ...microenvironment that favors cancer progression and development of metastasis. However, there are only few data about remodelling of collagen fibers in healthy looking mucosa distant from the cancer. Using SHG imaging, electron microscopy and specialized softwares (CT-FIRE, CurveAlign and FiberFit), we objectively visualized and quantified changes in morphology and organization of collagen fibers and investigated possible causes of collagen remodelling (change in syntheses, degradation and collagen cross-linking) in the colon mucosa 10 cm and 20 cm away from the cancer in comparison with healthy mucosa. We showed that in the lamina propria this far from the colon cancer, there were changes in collagen architecture (width, straightness, alignment of collagen fibers and collagen molecules inside fibers), increased representation of myofibroblasts and increase expression of collagen-remodelling enzymes (LOX and MMP2). Thus, the changes in organization of collagen fibers, which were already described in the cancer microenvironment, also exist in the mucosa far from the cancer, but smaller in magnitude.
The structure of teeth can be altered by diet, age or diseases such as caries and sclerosis. It is very important to characterize their mechanical properties to predict and understand tooth decay, ...design restorative dental procedures, and investigate their tribological behavior. However, existing imaging techniques are not well suited to investigating the micromechanics of teeth, in particular at tissue interfaces. Here, we describe a microscope based on Brillouin light scattering (BLS) developed to probe the spectrum of the light scattered from tooth tissues, from which the mechanical properties (sound velocity, viscosity) can be inferred with a priori knowledge of the refractive index. BLS is an inelastic process that uses the scattering of light by acoustic waves in the GHz range. Our microscope thus reveals the mechanical properties at the micrometer scale without contact with the sample. BLS signals show significant differences between sound tissues and pathological lesions, and can be used to precisely delineate carious dentin. We also show maps of the sagittal and transversal planes of sound tubular dentin that reveal its anisotropic microstructure at 1 µm resolution. Our observations indicate that the collagen-based matrix of dentine is the main load-bearing structure, which can be considered as a fiber-reinforced composite. In the vicinity of polymeric tooth-filling materials, we observed the infiltration of the adhesive complex into the opened tubules of sound dentine. The ability to probe the quality of this interfacial layer could lead to innovative designs of biomaterials used for dental restorations in contemporary adhesive dentistry, with possible direct repercussions on decision-making during clinical work.
Mechanical properties of teeth can be altered by diet, age or diseases. Yet existing imaging modalities cannot reveal the micromechanics of the tooth. Here we developed a new type of microscope that uses the scattering of a laser light by naturally-occurring acoustic waves to probe mechanical changes in tooth tissues at a sub-micrometer scale without contact to the sample. We observe significant mechanical differences between healthy tissues and pathological lesions. The contrast in mechanical properties also reveals the microstructure of the polymer-dentin interfaces. We believe that this new development of laser spectroscopy is very important because it should lead to innovative designs of biomaterials used for dental restoration, and allow delineating precisely destructed dentin for minimally-invasive strategies.
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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.
Hemoglobin (Hb), a life-sustaining and highly abundant erythrocyte protein, is not readily fluorescent. A few studies have already reported Two-Photon Excited Fluorescence (TPEF) of Hb, however, the ...mechanisms through which Hb becomes fluorescent upon interaction with ultrashort laser pulses are not completely understood. Here, we characterized photophysically this interaction on Hb thin film and erythrocytes using fluorescence spectroscopy upon single-photon/two-photon absorption, and UV-VIS single-photon absorption spectroscopy. A gradual increase of the fluorescence intensity, ending up with saturation, is observed upon prolonged exposure of Hb thin layer and erythrocytes to ultrashort laser pulses at 730 nm. When compared to protoporphyrin IX (PpIX) and oxidized Hb by H2O2, TPEF spectra from a thin Hb film and erythrocytes showed good mutual agreement, broad peaking at 550 nm, supporting hemoglobin undergoes degradation and that same fluorescent specie(s) originating from the heme moiety are generated. The uniform square shaped patterns of the fluorescent photoproduct exhibited the same level of the fluorescence intensity even after 12 weeks from the formation, indicating high photoproduct stability. We finally demonstrated the full potential of the formed Hb photoproduct with TPEF scanning microscopy towards spatiotemporally controlled micropatterning in HTF and single human erythrocyte labelling and tracking in the whole blood.
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The aim of the current work was to assess obtain a single crystal of calcium
tungstate doped with neodymium - (CaWO4:Nd3+), and after that, the crystal
was characterized with various spectroscopic ...methods. The single crystal was
grown from the melt using the Czochralski method in air. By optimizing
growth conditions, -oriented CaWO4:Nd3+ crystal? up to 10 mm in
diameter were grown. Number of dislocations in obtained crystal was 102 per
cm2. Micro hardness was measured with the Vickers pyramid. Anisotropy in
direction was not observed. Selected CaWO4:Nd3+ single crystal was cut
into several tiles with the diamond saw. The plates were polished with a
diamond paste. The crystal structure is confirmed by X-ray diffraction. The
obtained crystal w?s studied by Raman and infrared spectroscopy. Seven Raman
and six IR optical active modes predicted by group theory are observed. FTIR
confirmed the occurrence of all the functional groups and bonds in this
material. From the FTIR spectrum, a strong peak of 862 cm-1 has been
obtained due to the stretching vibration of WO42- in scheelite structure,
and a weak but sharp band at 433 cm-1 has been noticed due to the
metal-oxygen (Ca-O) band. Estimated luminescence lifetime of 4F5/2 - the
4I9/2 transition is about 120 ?s; estimated luminescence lifetime of 4F3/2 -
the 4I9/2 transition is about 140 ?s. All performed investigations show that
the obtained CaWO4:Nd3+ single crystal has good optical quality, which was
the goal of this work.
We describe a streak camera system that is capable of both spatial and spectral measurements of laser-induced plasma. The system is based on a Hamamatsu C4334 streak camera and SpectraPro 2300i ...spectrograph. To improve the analysis of laser-induced plasma development, it is necessary to determine the timing of laser excitation in regard to the time scale on streak images. We present several methods to determine the laser signal timing on streak images—one uses the fast photodiode, and other techniques are based on the inclusion of the laser pulse directly on the streak image. A Nd:YAG laser (λ = 1064 nm, Quantel, Brilliant B) was employed as the excitation source. The problem of synchronization of the streak camera with the Q-switched Nd:YAG laser is also analyzed. A simple modification of the spectrograph enables easy switching between the spectral and spatial measurement modes.
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