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1.
Acta Biomater ; 2020 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-31988041

RESUMO

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. STATEMENT OF SIGNIFICANCE: 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.

2.
J Biophotonics ; 12(12): e201900218, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31452335

RESUMO

Modern document protection relies on the simultaneous combination of many optical features with micron and submicron structures, whose complexity is the main obstacle for unauthorized copying. In that sense, documents are best protected by the diffractive optical elements generated lithographically and mass-produced by embossing. The problem is that the resulting security elements are identical, facilitating mass-production of both original and counterfeited documents. Here, we prove that each butterfly wing-scale is structurally and optically unique and can be used as an inimitable optical memory tag and applied for document security. Wing-scales, exhibiting angular variability of their color, were laser-cut and bleached to imprint cryptographic information of an authorized issuer. The resulting optical memory tag is extremely durable, as verified by several century-old insect specimens still retaining their coloration. The described technique is simple, amenable to mass-production, low cost and easy to integrate within the existing security infrastructure.

3.
Anal Chem ; 91(17): 11129-11137, 2019 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-31364842

RESUMO

Functional fluorescence microscopy imaging (fFMI), a time-resolved (21 µs/frame) confocal fluorescence microscopy imaging technique without scanning, is developed for quantitative characterization of fast reaction-transport processes in solution and in live cells. The method is based on massively parallel fluorescence correlation spectroscopy (FCS). Simultaneous excitation of fluorescent molecules in multiple spots in the focal plane is achieved using a diffractive optical element (DOE). Fluorescence from the DOE-generated 1024 illuminated spots is detected in a confocal arrangement by a matching matrix detector comprising 32 × 32 single-photon avalanche photodiodes (SPADs). Software for data acquisition and fast auto- and cross-correlation analysis by parallel signal processing using a graphic processing unit (GPU) allows temporal autocorrelation across all pixels in the image frame in 4 s and cross-correlation between first- and second-order neighbor pixels in 45 s. We present here this quantitative, time-resolved imaging method with single-molecule sensitivity and demonstrate its usefulness for mapping in live cell location-specific differences in the concentration and translational diffusion of molecules in different subcellular compartments. In particular, we show that molecules without a specific biological function, e.g., the enhanced green fluorescent protein (eGFP), exhibit uniform diffusion. In contrast, molecules that perform specialized biological functions and bind specifically to their molecular targets show location-specific differences in their concentration and diffusion, exemplified here for two transcription factor molecules, the glucocorticoid receptor (GR) before and after nuclear translocation and the Sex combs reduced (Scr) transcription factor in the salivary gland of Drosophila ex vivo.

4.
J Synchrotron Radiat ; 26(Pt 3): 700-707, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-31074433

RESUMO

The THz beamline at FLASH, DESY, provides both tunable (1-300 THz) narrow-bandwidth (∼10%) and broad-bandwidth intense (up to 150 uJ) THz pulses delivered in 1 MHz bursts and naturally synchronized with free-electron laser X-ray pulses. Combination of these pulses, along with the auxiliary NIR and VIS ultrashort lasers, supports a plethora of dynamic investigations in physics, material science and biology. The unique features of the FLASH THz pulses and the accelerator source, however, bring along a set of challenges in the diagnostics of their key parameters: pulse energy, spectral, temporal and spatial profiles. Here, these challenges are discussed and the pulse diagnostic tools developed at FLASH are presented. In particular, a radiometric power measurement is presented that enables the derivation of the average pulse energy within a pulse burst across the spectral range, jitter-corrected electro-optical sampling for the full spectro-temporal pulse characterization, spatial beam profiling along the beam transport line and at the sample, and a lamellar grating based Fourier transform infrared spectrometer for the on-line assessment of the average THz pulse spectra. Corresponding measurement results provide a comprehensive insight into the THz beamline capabilities.

5.
Mater Sci Eng C Mater Biol Appl ; 91: 597-605, 2018 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-30033292

RESUMO

Lanthanide-doped fluoride up-converting nanoparticles (UCNPs) represent the new class of imaging contrast agents which hold great potential for overcoming existing problems associated with traditionally used dyes, proteins and quantum dots. In this study, a new kind of hybrid NaYF4:Yb,Er/PLGA nanoparticles for efficient biolabeling were prepared through one-pot solvothermal synthesis route. Morphological and structural characteristics of the as-designed particles were obtained using X-ray powder diffraction (XRPD), scanning and transmission electron microscopy (SEM/TEM), energy dispersive spectroscopy (EDS), Fourier transform infrared (FTIR) and photoluminescence (PL) spectroscopy, while their cytotoxicity as well as up-conversion (UC) labeling capability were tested in vitro toward human gingival cells (HGC) and oral squamous cell carcinoma (OSCC). The results revealed coexistence of the cubic (Fm-3m) and hexagonal (P63/m) phase in spherical and irregularly shaped nanoparticles, respectively. PLGA [Poly(lactic-co-glycolic acid)] ligands attached at the surface of UCNPs particles provide their enhanced cellular uptake and enable high-quality cells imaging through a near-infrared (NIR) laser scanning microscopy (λex = 980 nm). Moreover, the fact that NaYF4:Yb,Er/PLGA UCNPs show low cytotoxicity against HGC over the whole concentration range (10-50 µg/mL) while a dose dependent viability of OSCC is obtained indicates that these might be a promising candidates for targeted cancer cell therapy.


Assuntos
Diagnóstico por Imagem , Érbio/química , Ácido Láctico/química , Neoplasias Bucais/diagnóstico por imagem , Neoplasias de Células Escamosas/diagnóstico por imagem , Ácido Poliglicólico/química , Espectroscopia de Luz Próxima ao Infravermelho , Itérbio/química , Adulto , Morte Celular , Linhagem Celular Tumoral , Gengiva/patologia , Humanos , Neoplasias Bucais/patologia , Nanopartículas/química , Nanopartículas/ultraestrutura , Neoplasias de Células Escamosas/patologia , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Espectroscopia de Infravermelho com Transformada de Fourier , Difração de Raios X , Adulto Jovem
6.
Tumour Biol ; 39(7): 1010428317711654, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28718368

RESUMO

In recent years, it has been demonstrated that malignancy arises and advances through the molecular interplay between tumor cells and non-malignant elements of the tumor stroma, that is, fibroblasts and extracellular matrix. However, in contrast to the mounting evidence about the role of tumor stroma in the genesis and progression of the malignant disease, there are very few data regarding the uninvolved stromal tissue in the remote surrounding of the tumor. Using the objective morphometric approach in patients with adenocarcinoma, we demonstrate the remodeling of extracellular matrix of the lamina propria in the uninvolved rectal mucosa 10 and 20 cm away from the neoplasm. We show that the representation of basic extracellular matrix constituents (reticular and collagen fibers and ground substance) is decreased. Also, the diameter of empty spaces that appear within the extracellular matrix of the lamina propria is increased. These spaces do not represent the blood or lymphatic vessel elements. Very likely, they reflect the development of tissue edema in the remote, uninvolved lamina propria of the mucosa in patients with the malignant tumor of the rectum. We hypothesize that the remodeling of extracellular matrix in lamina propria of the rectal mucosa may increase its stiffness, modulating the mechano-signal transduction, and thus promote the progression of the malignant disease.


Assuntos
Adenocarcinoma/patologia , Matriz Extracelular/patologia , Membrana Mucosa/patologia , Neoplasias Retais/patologia , Idoso , Vasos Sanguíneos/patologia , Carcinogênese/patologia , Progressão da Doença , Feminino , Humanos , Mucosa Intestinal , Masculino
7.
Phys Rev E ; 95(3-1): 032405, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28415223

RESUMO

Here we report how interference and scattering-enhanced absorption act together to produce the golden wing patches of the burnished brass moth. The key mechanism is scattering on rough internal surfaces of the wing scales, accompanied by a large increase of absorption in the UV-blue spectral range. Unscattered light interferes and efficiently reflects from the multilayer composed of the scales and the wing membranes. The resulting spectrum is remarkably similar to the spectrum of metallic gold. Subwavelength morphology and spectral and absorptive properties of the wings are described. Theories of subwavelength surface scattering and local intensity enhancement are used to quantitatively explain the observed reflectance spectrum.


Assuntos
Cor , Mariposas , Asas de Animais , Animais , Microscopia , Microscopia Eletrônica de Varredura , Modelos Biológicos , Mariposas/anatomia & histologia , Mariposas/efeitos da radiação , Espalhamento de Radiação , Asas de Animais/anatomia & histologia , Asas de Animais/efeitos da radiação
8.
J Biomed Opt ; 22(2): 26003, 2017 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-28301654

RESUMO

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.


Assuntos
Eritrócitos/química , Eritrócitos/citologia , Hemoglobinas/análise , Microscopia de Fluorescência , Animais , Membrana Eritrocítica/química , Hemoglobinas/metabolismo , Humanos , Suínos
9.
Mech Dev ; 138 Pt 2: 218-225, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26428533

RESUMO

Hox genes encode transcription factors that control the formation of body structures, segment-specifically along the anterior-posterior axis of metazoans. Hox transcription factors bind nuclear DNA pervasively and regulate a plethora of target genes, deploying various molecular mechanisms that depend on the developmental and cellular context. To analyze quantitatively the dynamics of their DNA-binding behavior we have used confocal laser scanning microscopy (CLSM), single-point fluorescence correlation spectroscopy (FCS), fluorescence cross-correlation spectroscopy (FCCS) and bimolecular fluorescence complementation (BiFC). We show that the Hox transcription factor Sex combs reduced (Scr) forms dimers that strongly associate with its specific fork head binding site (fkh250) in live salivary gland cell nuclei. In contrast, dimers of a constitutively inactive, phospho-mimicking variant of Scr show weak, non-specific DNA-binding. Our studies reveal that nuclear dynamics of Scr is complex, exhibiting a changing landscape of interactions that is difficult to characterize by probing one point at a time. Therefore, we also provide mechanistic evidence using massively parallel FCS (mpFCS). We found that Scr dimers are predominantly formed on the DNA and are equally abundant at the chromosomes and an introduced multimeric fkh250 binding-site, indicating different mobilities, presumably reflecting transient binding with different affinities on the DNA. Our proof-of-principle results emphasize the advantages of mpFCS for quantitative characterization of fast dynamic processes in live cells.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Genes Homeobox/genética , Proteínas de Homeodomínio/metabolismo , Ligação Proteica/fisiologia , Fatores de Transcrição/metabolismo , Animais , Sítios de Ligação/genética , Núcleo Celular/metabolismo , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Drosophila/genética , Drosophila/metabolismo , Fluorescência , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Homeodomínio/genética , Espectrometria de Fluorescência/métodos
10.
J Biomed Opt ; 20(1): 016010, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25574994

RESUMO

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-um 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.


Assuntos
Quitina/química , Besouros/química , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Animais , Artrópodes/química , Cavernas , Desenho de Equipamento , Imagem Tridimensional/métodos , Dinâmica não Linear
11.
Lasers Surg Med ; 42(4): 338-47, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20432283

RESUMO

BACKGROUND AND OBJECTIVE: Laser phototherapy could be potentially used for cancer treatment, but the mechanisms of laser-induced cell death are not completely understood. Autophagy is the process in which the damaged cellular proteins and organelles are engulfed by and destroyed in acidified multiple-membrane vesicles. The aim of the present study was to investigate the role of autophagy in laser-induced tumor cell death in vitro. STUDY DESIGN/MATERIALS AND METHODS: The monolayers of U251 human glioma tumor cells were exposed to 532 nm laser light from a single mode frequency-doubled Nd-YVO4 laser. A flattened Gaussian radial profile of laser beam (0.5-4 W) was used to uniformly illuminate entire colony of cells for various amounts of time (15-120 seconds) in the absence of cell culture medium. The cells were grown for 24 hours and the cell viability was determined by crystal violet or MTT assay. The presence of autophagy was assessed after 16 hours by fluorescence microscopy/flow cytometric analysis of acridine orange-stained autophagolysosomes and Western blot analysis of the autophagosome-associated LC3-II protein. The concentration of the principal pro-autophagic protein beclin-1 was determined after 6 hours by cell-based ELISA. RESULTS: The intracytoplasmic accumulation of autophagic vesicles, increase in LC3-II and up-regulation of beclin-1 expression were clearly observed under irradiation conditions that caused approximately 50% cytotoxicity. Post-irradiation addition of three different autophagy inhibitors (bafilomycin A1, chloroquine, or wortmannin) further increased the laser-induced cytotoxicity, without affecting non-irradiated cells. CONCLUSIONS: These data indicate that beclin-1-dependent induction of autophagy can protect glioma cells from laser-mediated cytotoxicity.


Assuntos
Autofagia , Glioma/patologia , Terapia a Laser/métodos , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Proteína Beclina-1 , Western Blotting , Proliferação de Células , Sobrevivência Celular , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Hipertermia Induzida , Técnicas In Vitro , Terapia a Laser/instrumentação , Lasers de Estado Sólido , Microscopia de Fluorescência , Necrose , Células Tumorais Cultivadas , Regulação para Cima
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