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1.
Nat Commun ; 10(1): 4483, 2019 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-31578369

RESUMO

Among optical imaging techniques light sheet fluorescence microscopy is one of the most attractive for capturing high-speed biological dynamics unfolding in three dimensions. The technique is potentially millions of times faster than point-scanning techniques such as two-photon microscopy. However light sheet microscopes are limited by volume scanning rate and/or camera speed. We present speed-optimized Objective Coupled Planar Illumination (OCPI) microscopy, a fast light sheet technique that avoids compromising image quality or photon efficiency. Our fast scan system supports 40 Hz imaging of 700 µm-thick volumes if camera speed is sufficient. We also address the camera speed limitation by introducing Distributed Planar Imaging (DPI), a scaleable technique that parallelizes image acquisition across cameras. Finally, we demonstrate fast calcium imaging of the larval zebrafish brain and find a heartbeat-induced artifact, removable when the imaging rate exceeds 15 Hz. These advances extend the reach of fluorescence microscopy for monitoring fast processes in large volumes.


Assuntos
Encéfalo/diagnóstico por imagem , Diagnóstico por Imagem/instrumentação , Processamento de Imagem Assistida por Computador/instrumentação , Medições Luminescentes/instrumentação , Microscopia/instrumentação , Animais , Diagnóstico por Imagem/métodos , Processamento de Imagem Assistida por Computador/métodos , Larva , Medições Luminescentes/métodos , Microscopia/métodos , Microscopia Confocal/instrumentação , Microscopia Confocal/métodos , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Reprodutibilidade dos Testes , Peixe-Zebra
2.
PLoS One ; 14(4): e0214954, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30947245

RESUMO

Miniaturized microscopes are lightweight imaging devices that allow optical recordings from neurons in freely moving animals over the course of weeks. Despite their ubiquitous use, individual neuronal responses measured with these microscopes have not been directly compared to those obtained with established in vivo imaging techniques such as bench-top two-photon microscopes. To achieve this, we performed calcium imaging in mouse primary visual cortex while presenting animals with drifting gratings. We identified the same neurons in image stacks acquired with both microscopy methods and quantified orientation tuning of individual neurons. The response amplitude and signal-to-noise ratio of calcium transients recorded upon visual stimulation were highly correlated between both microscopy methods, although influenced by neuropil contamination in miniaturized microscopy. Tuning properties, calculated for individual orientation tuned neurons, were strongly correlated between imaging techniques. Thus, neuronal tuning features measured with a miniaturized microscope are quantitatively similar to those obtained with a two-photon microscope.


Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação , Microtecnologia , Neurópilo/metabolismo , Estimulação Luminosa , Córtex Visual/fisiologia , Animais , Camundongos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Neurópilo/citologia , Córtex Visual/citologia
3.
Exp Eye Res ; 182: 194-201, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30822399

RESUMO

The purpose of this study is to provide an intravital noninvasive multiphoton microscopic platform for long-term ocular imaging in transgenic fluorescent mice with subcellular resolution. A multiphoton microscopic system with tunable laser output was employed. We designed a mouse holder incorporated with stereotaxic motorized stage for in vivo three-dimensional imaging of ocular surface in 3 transgenic mouse line with fluorescent protein (FP) expression to visualize distinct structures. With our imaging platform and the expression of FPs, we obtained the three-dimensional images across the whole cornea from epithelium to endothelium and in conjunctiva with subcellular resolution in vivo. Specified EGFP expression in corneal epithelium of K5-H2B-EGFP mice helped to identify both corneal and limbal epithelial cells while ubiquitous nuclear FP expression in R26R-GR mice allowed us to visualized nuclei of all cell types. Universal membrane-localized FP in mT/mG mice outlined all cell boundaries, nerve fibers, and capillaries. The simultaneously collected second harmonic generation signals from collagenous stroma provided architectural contrast. Time-lapsed recording enabled monitoring the mitotic activity of corneal epithelial cells and limbal epithelial cells. We developed an intravital multiphoton microscopic stereotaxic imaging platform and showed that, by incorporating FP-expressing transgenic mice, this platform enables in vivo 4-dimensional ophthalmic study at subcellular resolution.


Assuntos
Córnea/diagnóstico por imagem , Técnicas de Diagnóstico Oftalmológico , Imagem Tridimensional/métodos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Animais , Substância Própria/diagnóstico por imagem , Técnicas de Diagnóstico Oftalmológico/instrumentação , Epitélio Anterior/diagnóstico por imagem , Limbo da Córnea/diagnóstico por imagem , Camundongos , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação
4.
Appl Opt ; 58(5): A26-A31, 2019 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-30873988

RESUMO

While simultaneous phase-contrast and two-photon fluorescence imaging in microscopy can bring abundant biomedical information, it is difficult to retrieve phase information from conventional two-photon microscopes. To realize low-cost, in situ phase-contrast and two-photon fluorescence imaging, we propose Schlieren two-photon microscopy, a method that implements phase-contrast imaging on two-photon microscopes. This method involves spatially modulated fluorescence plates, which are made of two-photon fluorescence dyes or upconversion nanoparticles. We demonstrate that the fluorescence intensity fluctuation reflects the phase gradients of the specimen via theoretical analysis, simulations, and experiments. The proposed method is fully compatible with commercial two-photon microscopes, thus enabling widespread applications in live tissue imaging.


Assuntos
Células HeLa/citologia , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação , Microscopia de Contraste de Fase/métodos , Corantes Fluorescentes , Humanos
6.
Methods Mol Biol ; 1929: 15-26, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30710264

RESUMO

Two-photon calcium imaging became in recent years a very popular method for the functional analysis of neural cell populations on a single-cell level in anesthetized or awake behaving animals. Scientific insights about single-cell processing of sensory information but also analyses of higher cognitive functions in healthy or diseased states became thereby feasible. However, two-photon imaging is generally limited to depths of a few hundred micrometers when recording from densely labeled cell populations. Therefore, such recordings are often restricted to the superficial layers 1-3 of the mouse cortex, whereas the deep cell layers 4-6 are hardly accessible with standard two-photon imaging. Here, we provide a protocol for deep two-photon calcium imaging, which allows imaging of neuronal circuits with single-cell resolution in all cortical layers of the mouse primary cortex. This technique can be readily applied to other species. The method includes a reduction of excitation light scattering by the use of a red-shifted calcium indicator and the minimization of background fluorescence by visually guided local application of the fluorescent dye. The technique is similar to previously published protocols for in vivo two-photon calcium imaging with synthetic calcium dyes (Stosiek et al. Proc Natl Acad Sci U S A 100:7319-7324, 2003). Hence, only minor changes of a generic two-photon setup and some adaptations of the experimental procedures are required.


Assuntos
Cálcio/análise , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação , Neurônios/metabolismo , Córtex Visual/citologia , Animais , Sinalização do Cálcio , Corantes Fluorescentes/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Análise de Célula Única/instrumentação , Análise de Célula Única/métodos , Córtex Visual/metabolismo
7.
Methods Mol Biol ; 1880: 529-534, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30610719

RESUMO

Macroautophagy is the process to remove intracellular organelles or proteins by using autophagosome that is composed of autophagy proteins such as atg3, atg7, and atg8/LC3 (Mizushima, et al. Annu Rev Cell Dev Biol. 27:107-132, 2011). Here, we develop a useful method for in vivo imaging of autophagosome under the two-photon microscopy. Time-lapse imaging of LC3-ECFP enables us to quantify the dynamics of number, size, and signal intensity of autophagosomes in neurons or in other types of cells in the brain.


Assuntos
Autofagossomos/metabolismo , Autofagia/fisiologia , Encéfalo/diagnóstico por imagem , Microscopia Intravital/métodos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Animais , Proteínas Relacionadas à Autofagia/metabolismo , Encéfalo/citologia , Encéfalo/metabolismo , Células HEK293 , Humanos , Processamento de Imagem Assistida por Computador/instrumentação , Processamento de Imagem Assistida por Computador/métodos , Microscopia Intravital/instrumentação , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação , Proteínas Associadas aos Microtúbulos/análise , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Neurônios/metabolismo , Técnicas Estereotáxicas/instrumentação , Imagem com Lapso de Tempo/instrumentação , Imagem com Lapso de Tempo/métodos
8.
Methods Mol Biol ; 1862: 227-243, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30315471

RESUMO

We describe here a method for generating mouse orthotopic gliomas in order to follow their progression over time by multi-photon laser scanning microscopy. After craniotomy of the parietal bone, glioma cells are implanted in the brain cortex and a glass window is cemented atop, allowing chronical imaging of the tumor. The expression of different fluorescent proteins in tumor cells and in specific cell types of a number of currently available transgenic mouse strains allows obtaining multicolor 3D images of the tumor over time. This technique is suitable both to evaluate the effect of pharmacological treatments and to unravel basic mechanisms of tumor-host interactions.


Assuntos
Neoplasias Encefálicas/diagnóstico por imagem , Glioma/diagnóstico por imagem , Microscopia Intravital/métodos , Animais , Encéfalo/diagnóstico por imagem , Encéfalo/patologia , Neoplasias Encefálicas/patologia , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Linhagem Celular Tumoral/transplante , Craniotomia , Modelos Animais de Doenças , Progressão da Doença , Glioma/patologia , Humanos , Imagem Tridimensional/instrumentação , Imagem Tridimensional/métodos , Microscopia Intravital/instrumentação , Proteínas Luminescentes/química , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Microscopia Confocal/instrumentação , Microscopia Confocal/métodos , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Ensaios Antitumorais Modelo de Xenoenxerto/instrumentação , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
9.
J Biomed Opt ; 23(12): 1-12, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30574695

RESUMO

Frequency-doubled femtosecond Er-doped fiber laser is a low-cost and portable excitation source suitable for multiphoton endoscopy. The frequency-doubled wavelength at 780 nm is used to excite the intrinsic fluorescence signal. The frequency-doubling with a periodically poled MgO : LiNbO3 (PPLN) is integrated in the distal end of the imaging head to achieve fiber connection. The imaging speed is further improved by optimizing the excitation laser source. A 0.3-mm length of PPLN crystal is selected and the Er-doped fiber laser is manipulated to match its bandwidth with the acceptance bandwidth of the PPLN. Through this optimization, a reduced pulsewidth of 80 fs of the frequency-doubled pulse is achieved. All-fiber dispersion compensation and pulse compression by single mode fiber is conducted, which makes the fiber laser directly fiber-coupled to the imaging head. An imaging speed of 4 frames / s is demonstrated on ex vivo imaging of unstained biological tissues, which is 10 times faster than our previous study using a 1-mm-long PPLN. The results show that miniature multiphoton endoscopy using frequency-doubled Er-doped fiber laser has great potential for clinical applications.


Assuntos
Endoscopia/instrumentação , Lasers , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação , Miniaturização/instrumentação , Animais , Endoscopia/métodos , Desenho de Equipamento , Camundongos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Pele/diagnóstico por imagem
10.
J Biomed Opt ; 23(11): 1-8, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30444085

RESUMO

Conventional temporal focusing-based multiphoton excitation microscopy (TFMPEM) can offer widefield optical sectioning with an axial excitation confinement of a few microns. To improve the axial confinement of TFMPEM, a binary computer-generated Fourier hologram (CGFH) via a digital-micromirror-device (DMD) was implemented to intrinsically improve the axial confinement by filling the back-focal aperture of the objective lens. Experimental results show that the excitation focal volume can be condensed and the axial confinement improved about 24% according to the DMD holography. In addition, pseudouniform MPE can be achieved using two complementary CGFHs with rapid pulse-width modulation switching via the DMD. Furthermore, bioimaging of CV-1 in origin with SV40 genes-7 cells demonstrates that the TFMPEM with binary DMD holography can improve image quality by enhancing axial excitation confinement and rejecting out-of-focus excitation.


Assuntos
Holografia/métodos , Processamento de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Animais , Células COS , Desenho de Equipamento , Holografia/instrumentação , Lasers , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação
11.
Nat Commun ; 9(1): 4878, 2018 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-30451853

RESUMO

Flow of cerebrospinal fluid (CSF) through perivascular spaces (PVSs) in the brain is important for clearance of metabolic waste. Arterial pulsations are thought to drive flow, but this has never been quantitatively shown. We used particle tracking to quantify CSF flow velocities in PVSs of live mice. CSF flow is pulsatile and driven primarily by the cardiac cycle. The speed of the arterial wall matches that of the CSF, suggesting arterial wall motion is the principal driving mechanism, via a process known as perivascular pumping. Increasing blood pressure leaves the artery diameter unchanged but changes the pulsations of the arterial wall, increasing backflow and thereby reducing net flow in the PVS. Perfusion-fixation alters the normal flow direction and causes a 10-fold reduction in PVS size. We conclude that particle tracking velocimetry enables the study of CSF flow in unprecedented detail and that studying the PVS in vivo avoids fixation artifacts.


Assuntos
Artérias/diagnóstico por imagem , Líquido Cefalorraquidiano/diagnóstico por imagem , Cisterna Magna/diagnóstico por imagem , Sistema Glinfático/diagnóstico por imagem , Análise de Onda de Pulso/métodos , Animais , Artérias/fisiologia , Líquido Cefalorraquidiano/fisiologia , Cisterna Magna/anatomia & histologia , Cisterna Magna/fisiologia , Corantes Fluorescentes/química , Sistema Glinfático/anatomia & histologia , Sistema Glinfático/fisiologia , Frequência Cardíaca/fisiologia , Processamento de Imagem Assistida por Computador , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Microesferas , Tamanho da Partícula , Fluxo Pulsátil/fisiologia , Análise de Onda de Pulso/instrumentação , Reologia/instrumentação , Reologia/métodos
12.
Org Lett ; 20(20): 6425-6429, 2018 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-30295496

RESUMO

A fluorescence microscopy-based distinguishment between biotin receptor (BiR) positive and negative cell lines via receptor-mediated endocytosis has been demonstrated. A water-soluble, three-component, two-photon (2P) active solvatofluorochromic probe has been designed and synthesized. The applicability of the probe for 2P microscopy and 3D-spheroid was also assessed.


Assuntos
Biotina/metabolismo , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/metabolismo , Células HEK293 , Células HeLa , Humanos , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Imagem Óptica/instrumentação , Receptores de Fatores de Crescimento/metabolismo
13.
Nat Commun ; 9(1): 2746, 2018 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-30013228

RESUMO

The vasculature undergoes changes in diameter, permeability and blood flow in response to specific stimuli. The dynamics and interdependence of these responses in different vessels are largely unknown. Here we report a non-invasive technique to study dynamic events in different vessel categories by multi-photon microscopy and an image analysis tool, RVDM (relative velocity, direction, and morphology) allowing the identification of vessel categories by their red blood cell (RBC) parameters. Moreover, Claudin5 promoter-driven green fluorescent protein (GFP) expression is used to distinguish capillary subtypes. Intradermal injection of vascular endothelial growth factor A (VEGFA) is shown to induce leakage of circulating dextran, with vessel-type-dependent kinetics, from capillaries and venules devoid of GFP expression. VEGFA-induced leakage in capillaries coincides with vessel dilation and reduced flow velocity. Thus, intravital imaging of non-invasive stimulation combined with RVDM analysis allows for recording and quantification of very rapid events in the vasculature.


Assuntos
Velocidade do Fluxo Sanguíneo/fisiologia , Vasos Sanguíneos/diagnóstico por imagem , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Imagem Molecular/métodos , Animais , Velocidade do Fluxo Sanguíneo/efeitos dos fármacos , Vasos Sanguíneos/anatomia & histologia , Vasos Sanguíneos/efeitos dos fármacos , Vasos Sanguíneos/fisiologia , Claudina-5/genética , Claudina-5/metabolismo , Feminino , Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Injeções Intradérmicas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação , Imagem Molecular/instrumentação , Regiões Promotoras Genéticas , Fator A de Crescimento do Endotélio Vascular/administração & dosagem
14.
Methods Mol Biol ; 1820: 179-219, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29884947

RESUMO

This chapter describes how to apply two-photon neuroimaging to study the insect olfactory system in vivo. It provides a complete protocol for insect brain functional imaging, with some additional remarks on the acquisition of morphological information from the living brain. We discuss the most important choices to make when buying or building a two-photon laser-scanning microscope. We illustrate different possibilities of animal preparation and brain tissue labeling for in vivo imaging. Finally, we give an overview of the main methods of image data processing and analysis, followed by a short description of pioneering applications of this imaging modality.


Assuntos
Abelhas , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Neurônios Receptores Olfatórios , Olfato/fisiologia , Coloração e Rotulagem , Animais , Abelhas/citologia , Abelhas/metabolismo , Cálcio/metabolismo , Drosophila , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação , Neurônios Receptores Olfatórios/citologia , Neurônios Receptores Olfatórios/metabolismo
15.
Elife ; 72018 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-29932052

RESUMO

Rewiring neural circuits by the formation and elimination of synapses is thought to be a key cellular mechanism of learning and memory in the mammalian brain. Dendritic spines are the postsynaptic structural component of excitatory synapses, and their experience-dependent plasticity has been extensively studied in mouse superficial cortex using two-photon microscopy in vivo. By contrast, very little is known about spine plasticity in the hippocampus, which is the archetypical memory center of the brain, mostly because it is difficult to visualize dendritic spines in this deeply embedded structure with sufficient spatial resolution. We developed chronic 2P-STED microscopy in mouse hippocampus, using a 'hippocampal window' based on resection of cortical tissue and a long working distance objective for optical access. We observed a two-fold higher spine density than previous studies and measured a spine turnover of ~40% within 4 days, which depended on spine size. We thus provide direct evidence for a high level of structural rewiring of synaptic circuits and new insights into the structure-dynamics relationship of hippocampal spines. Having established chronic super-resolution microscopy in the hippocampus in vivo, our study enables longitudinal and correlative analyses of nanoscale neuroanatomical structures with genetic, molecular and behavioral experiments.


Assuntos
Espinhas Dendríticas/ultraestrutura , Hipocampo/ultraestrutura , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Imagem Molecular/métodos , Rede Nervosa/ultraestrutura , Células Piramidais/ultraestrutura , Sinapses/ultraestrutura , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Córtex Cerebral/cirurgia , Espinhas Dendríticas/fisiologia , Feminino , Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hipocampo/anatomia & histologia , Hipocampo/fisiologia , Processamento de Imagem Assistida por Computador/estatística & dados numéricos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Memória/fisiologia , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação , Imagem Molecular/instrumentação , Rede Nervosa/anatomia & histologia , Rede Nervosa/fisiologia , Plasticidade Neuronal/fisiologia , Células Piramidais/fisiologia , Sinapses/fisiologia
16.
Methods Mol Biol ; 1782: 171-186, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29851000

RESUMO

The concentration of calcium ions in the mitochondria has been shown to affect its function, modulating respiratory activity at low levels and causing lethal damage at high concentrations. The rhodamine series of dyes can be used to measure mitochondrial calcium concentration, but the reliability of measurements depends upon correct partitioning of the dye within to the mitochondria. Methods are described to aid verification and quantification of the mitochondrial calcium concentration using single- or two-photon confocal microscopy. The method of linear unmixing to separate fluorescent signals based on either differing excitation or emission spectra is outlined and for the purposes of illustration is applied to the separation of rhod-2 signals originating from the dye within the mitochondria and nucleoli.


Assuntos
Cálcio/metabolismo , Microscopia Intravital/métodos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Mitocôndrias/metabolismo , Fótons , Animais , Cálcio/análise , Linhagem Celular , Corantes Fluorescentes/química , Compostos Heterocíclicos com 3 Anéis/química , Microscopia Intravital/instrumentação , Camundongos , Microscopia Confocal/instrumentação , Microscopia Confocal/métodos , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação , Reprodutibilidade dos Testes
17.
Curr Protoc Cytom ; 85(1): e40, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29944202

RESUMO

Multiphoton intravital calcium imaging is a powerful technique that enables high-resolution longitudinal monitoring of cellular and subcellular activity hundreds of microns deep in the living organism. This unit addresses the application of 2-photon microscopy to imaging of genetically encoded calcium indicators (GECIs) in the mouse brain. The protocols in this unit enable real-time intravital imaging of intracellular calcium concentration simultaneously in hundreds of neurons, or at the resolution of single synapses, as mice respond to sensory stimuli or perform behavioral tasks. Protocols are presented for implantation of a cranial imaging window to provide optical access to the brain and for 2-photon image acquisition. Protocols for implantation of both open skull and thinned skull windows for single or multi-session imaging are described. © 2018 by John Wiley & Sons, Inc.


Assuntos
Encéfalo , Sinalização do Cálcio/fisiologia , Microscopia Intravital/métodos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Sinapses/fisiologia , Animais , Encéfalo/citologia , Encéfalo/fisiologia , Microscopia Intravital/instrumentação , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação
18.
Sci Rep ; 8(1): 8108, 2018 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-29802371

RESUMO

We present a miniature head mounted two-photon fiber-coupled microscope (2P-FCM) for neuronal imaging with active axial focusing enabled using a miniature electrowetting lens. We show three-dimensional two-photon imaging of neuronal structure and record neuronal activity from GCaMP6s fluorescence from multiple focal planes in a freely-moving mouse. Two-color simultaneous imaging of GFP and tdTomato fluorescence is also demonstrated. Additionally, dynamic control of the axial scanning of the electrowetting lens allows tilting of the focal plane enabling neurons in multiple depths to be imaged in a single plane. Two-photon imaging allows increased penetration depth in tissue yielding a working distance of 450 µm with an additional 180 µm of active axial focusing. The objective NA is 0.45 with a lateral resolution of 1.8 µm, an axial resolution of 10 µm, and a field-of-view of 240 µm diameter. The 2P-FCM has a weight of only ~2.5 g and is capable of repeatable and stable head-attachment. The 2P-FCM with dynamic axial scanning provides a new capability to record from functionally distinct neuronal layers, opening new opportunities in neuroscience research.


Assuntos
Encéfalo/diagnóstico por imagem , Imagem Tridimensional/instrumentação , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação , Movimento , Animais , Cor , Camundongos
19.
J Biomed Opt ; 23(5): 1-8, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29740994

RESUMO

A spatiotemporal phase modulator (STPM) is theoretically investigated using the vectorial diffraction theory. The STPM is equivalent to a time-dependent phase-only pupil filter that alternates between a homogeneous filter and a stripe-shaped filter with a sinusoidal phase distribution. It is found that two-photon focal modulation microscopy (TPFMM) using this STPM can significantly suppress the background contribution from out-of-focus ballistic excitation and achieve almost the same resolution as two-photon microscopy. The modulation depth is also evaluated and a compromise exists between the signal-to-background ratio and signal-to-noise ratio. The theoretical investigations provide important insights into future implementations of TPFMM and its potential to further extend the penetration depth of nonlinear microscopy in imaging multiple-scattering biological tissues.


Assuntos
Microscopia de Fluorescência por Excitação Multifotônica/métodos , Processamento de Sinais Assistido por Computador , Desenho de Equipamento , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação , Modelos Teóricos , Razão Sinal-Ruído
20.
Methods Mol Biol ; 1755: 223-232, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29671273

RESUMO

Noninvasive imaging of reporter gene expression by two-photon excitation (2PE) laser scanning microscopy is uniquely suited to perform dynamic and multidimensional imaging down to single-cell detection sensitivity in vivo in deep tissues. Here we used 2PE microscopy to visualize green fluorescent protein (GFP) as a reporter gene in human melanoma cells implanted into the dermis of the mouse ear skin. We first provide a step-by-step methodology to set up a 2PE imaging model of the mouse ear's skin and then apply it for the observation of the primary tumor and its associated vasculature in vivo. This approach is minimally invasive and allows repeated imaging over time and continuous visual monitoring of malignant growth within intact animals. Imaging fluorescence reporter gene expression in small living animals by 2PE provides a unique tool to investigate critical pathways and molecular events in cancer biology such as tumorigenesis and metastasis in vivo with high-spatial and temporal resolutions.


Assuntos
Genes Reporter/genética , Microscopia Intravital/métodos , Melanoma/patologia , Neoplasias Cutâneas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Linhagem Celular Tumoral , Derme/citologia , Derme/diagnóstico por imagem , Orelha Externa , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Humanos , Injeções Intradérmicas , Microscopia Intravital/instrumentação , Melanoma/diagnóstico por imagem , Camundongos , Camundongos Nus , Microscopia Confocal/instrumentação , Microscopia Confocal/métodos , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Neoplasias Cutâneas/diagnóstico por imagem , Ensaios Antitumorais Modelo de Xenoenxerto/instrumentação
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