RESUMO
We present a combination of light-sheet excitation and two-dimensional fluorescence intensity ratio (FIR) measurements as a simple and promising technique for three-dimensional temperature mapping. The feasibility of this approach is demonstrated with samples fabricated with sodium yttrium fluoride nanoparticles co-doped with rare-earth ytterbium and erbium ions (NaYF4:Yb3+/Er3+) incorporated into polydimethylsiloxane (PDMS) as a host material. In addition, we also evaluate the technique using lipid-coated NaYF4:Yb3+/Er3+ nanoparticles immersed in agar. The composite materials show upconverted (UC) fluorescence bands when excited by a 980 nm near-infrared laser light-sheet. Using a single CMOS camera and a pair of interferometric optical filters to specifically image the two thermally-coupled bands (at 525 and 550 nm), the two-dimensional FIR and, hence, the temperature map can be readily obtained. The proposed method can take optically sectioned (confocal-like) images with good optical resolution over relatively large samples (up to the millimetric scale) for further 3D temperature reconstruction.
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We present a Silicon-based Charge-Coupled Device (Si-CCD) sensor applied as a cost-effective spectrometer for femtosecond pulse characterization in the Near Infrared region in two different configurations: two-Fourier and Czerny-Turner setups. To test the spectrometer's performance, a femtosecond Optical Parametric Oscillator with a tuning range between 1100 and 1700 nm and a femtosecond Erbium-Doped Fiber Amplifier at 1582 nm were employed. The nonlinear spectrometer operation is based on the Two-Photon Absorption effect generated in the Si-CCD sensor. The achieved spectrometer resolution was 0.6 ± 0.1 nm with a threshold peak intensity of 2×106Wcm2. An analysis of the nonlinear response as a function of the wavelength, the response saturation, and the criteria to prevent it are also presented.
RESUMO
Light-sheet fluorescence microscopy (LSFM) is useful for developmental biology studies, which require a simultaneous visualization of dynamic microstructures over large fields of views (FOVs). A comparative study between multicolor Bessel and Gaussian-based LSFM systems is presented. Discussing the chromatic implications to achieve colocalized and large FOVs when both optical arrays are implemented under the same excitation objective is the purpose of this work. The light-sheets FOVs, optical sectioning, and resolution are experimentally characterized and discussed. The advantages of using Bessel beams and the main drawbacks of using Gaussian beams for multicolor imaging are highlighted. Multiple Bessel excitation minimizes the FOV's mismatch's effects due to the beams chromatic defocusing and alleviates the aside object blurring obtained with multiple Gaussian beams. It also offers a fair homogeneous axial resolution and optical sectioning over a larger effective FOV. Imaging over perithecia samples of the fungus Sordaria macrospora demonstrates such advantages. This work complements previous comparative studies that discuss only single wavelengths light-sheets excitations.
Assuntos
Técnicas Histológicas , Microscopia de Fluorescência/métodos , Distribuição NormalRESUMO
Cortical dysplasias are alterations in the organization of the layers of the brain cortex due to problems in neuronal migration during development. The neuronal component has been widely studied in experimental models of cortical dysplasias. In contrast, little is known about how glia are affected. In the cerebellum, Bergmann glia (BG) are essential for neuronal migration during development, and in adult they mediate the control of fine movements through glutamatergic transmission. The aim of this study was to characterize the morphology and intracellular calcium dynamics of BG and astrocytes from mouse cerebellum and their modifications in a model of cortical dysplasia induced by carmustine (BCNU). Carmustine-treated mice were affected in their motor coordination and balance. Cerebellar dysplasias and heterotopias were more frequently found in lobule X. Morphology of BG cells and astrocytes was affected, as were their spontaneous [Ca2+]i transients in slice preparation and in vitro.
Assuntos
Sinalização do Cálcio , Cerebelo/patologia , Malformações do Desenvolvimento Cortical/metabolismo , Malformações do Desenvolvimento Cortical/patologia , Neuroglia/metabolismo , Neuroglia/patologia , Animais , Astrócitos/patologia , Carmustina , Células Cultivadas , Malformações do Desenvolvimento Cortical/induzido quimicamente , Camundongos Transgênicos , Atividade MotoraRESUMO
We present a study of the optical second-order nonlinearity of type I collagen fibers grown in vitro via second harmonic generation (SHG) experiments and analyze the observed polarization-resolved SHG signal using previously reported SHG analytical expressions obtained for anisotropic tissue. Our results indicate that the effective second-order nonlinearity measured in the grown fibers is one order of magnitude lower than that of native collagen fibers. This is attributed to the formation of loose and dispersive fibrillar networks of thinner collagen fibrils that constitute the reassembled collagen fibers. This is confirmed by scanning electronic microscopy (SEM) imaging and the polarization dependence of the SHG signal. The measured values of the anisotropy parameter ρ of the reassembled collagen fibers are found to be similar to that obtained for native fibers on the relevant sub-µm scale.
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We present a multicolor fluorescence microscope system, under a selective plane illumination microscopy (SPIM) configuration, using three continuous wave-lasers and a single-channel-detection camera. The laser intensities are modulated with three time-delayed pulse trains that operate synchronously at one third of the camera frame rate, allowing a sequential excitation and an image acquisition of up to three different biomarkers. The feasibility of this imaging acquisition mode is demonstrated by acquiring single-plane multicolor images of living hyphae of Neurospora crassa. This allows visualizing simultaneously the localization and dynamics of different cellular components involved in apical growth in living hyphae. The configuration presented represents a noncommercial, cost-effective alternative microscopy system for the rapid and simultaneous acquisition of multifluorescent images and can be potentially useful for three-dimensional imaging of large biological samples.
Assuntos
Processamento de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/métodos , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Neurospora crassa/metabolismo , Biomarcadores/metabolismo , Cor , Desenho de Equipamento , Corantes Fluorescentes/química , Proteínas de Fluorescência Verde/química , Lasers , Luz , Proteínas Luminescentes/química , Rodaminas/química , Proteína Vermelha FluorescenteRESUMO
Light sheet optical microscopy on strontium aluminate nanoparticles (SrAl2 O4 NPs)1 codoped with Eu2+ and Dy3+ was used for cancer cell tagging and tracking. The nanoparticles were synthesized by urea-assisted combustion with optimized percentage values of the 2 codoping rare-earth ions for cell viability and for lower cytotoxic effects. The optical properties of these materials showed an excitation wide range of wavelengths (λexc = 254-460 nm), a broad emission band (λem = 475-575 nm) with the maximum centered wavelength at 525 nm and a half lifetime within the seconds regime. The feasibility to measure the nanoparticle luminescence under the selective plane illumination configuration was studied by immersing the nanoparticles in 1% Agarose. The potential applicability of the synthesized nanophosphors for cancer cell tagging was demonstrated by using in vitro experiments with human breast adenocarcinoma MCF-7 cells. A single MCF-7 cell observed by the use of light sheet microscopy with UV excitation. The cell has been bio-labeled with FA-SrAl2 04 : Eu2+ , Dy3+ NPs and 4',6-diamidino-2-phenylindole, dihydrochloride for nucleus identification.