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1.
Methods Mol Biol ; 2223: 151-157, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33226593

RESUMO

The regulation of vascular permeability is critical in inflammation. It controls the distribution of water and plasma contents such as immunoglobulins in peripheral tissues. To regulate allergic diseases, it is important to study vascular biology especially in inflammation. Since the vascular permeability changes in minutes upon the exposure to proinflammatory mediators, intravital imaging system is a powerful technique to capture such dynamic responses. We here describe how to evaluate vascular permeability in vivo using multiphoton microscopy. We use various sizes of fluorescence-labeled dextran to visualize how leaky the blood vessels are in the steady state and in inflammation. Using this assay system, we can illustrate the dynamic kinetics of vascular permeability in vivo in real-time. This assay system provides a novel convenient way to study vascular biology that is beneficial in the assessment of various animal models of allergic disease.


Assuntos
Permeabilidade Capilar/efeitos dos fármacos , Dextranos/metabolismo , Fluoresceína-5-Isotiocianato/análogos & derivados , Histamina/farmacologia , Hipersensibilidade Imediata/diagnóstico por imagem , Microscopia Intravital/métodos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Animais , Orelha/irrigação sanguínea , Orelha/diagnóstico por imagem , Fluoresceína-5-Isotiocianato/metabolismo , Corantes Fluorescentes/metabolismo , Hipersensibilidade Imediata/induzido quimicamente , Injeções Intravenosas , Microscopia Intravital/instrumentação , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação , Peso Molecular , Imagem com Lapso de Tempo
2.
PLoS One ; 15(8): e0237230, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32764808

RESUMO

In vivo two-photon microscopy utilizing a nonlinear optical process enables, in living mouse brains, not only the visualization of morphologies and functions of neural networks in deep regions but also their optical manipulation at targeted sites with high spatial precision. Because the two-photon excitation efficiency is proportional to the square of the photon density of the excitation laser light at the focal position, optical aberrations induced by specimens mainly limit the maximum depth of observations or that of manipulations in the microscopy. To increase the two-photon excitation efficiency, we developed a method for evaluating the focal volume in living mouse brains. With this method, we modified the beam diameter of the excitation laser light and the value of the refractive index in the immersion liquid to maximize the excitation photon density at the focal position. These two modifications allowed the successful visualization of the finer structures of hippocampal CA1 neurons, as well as the intracellular calcium dynamics in cortical layer V astrocytes, even with our conventional two-photon microscopy system. Furthermore, it enabled focal laser ablation dissection of both single apical and single basal dendrites of cortical layer V pyramidal neurons. These simple modifications would enable us to investigate the contributions of single cells or single dendrites to the functions of local cortical networks.


Assuntos
Encéfalo/ultraestrutura , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação , Animais , Desenho de Equipamento , Feminino , Masculino , Camundongos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Neurônios/ultraestrutura , Fótons
3.
Nat Methods ; 17(6): 571-581, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32284609

RESUMO

Temporal focusing, with its ability to focus light in time, enables scanless illumination of large surface areas at the sample with micrometer axial confinement and robust propagation through scattering tissue. In conventional two-photon microscopy, widely used for the investigation of intact tissue in live animals, images are formed by point scanning of a spatially focused pulsed laser beam, resulting in limited temporal resolution of the excitation. Replacing point scanning with temporally focused widefield illumination removes this limitation and represents an important milestone in two-photon microscopy. Temporal focusing uses a diffusive or dispersive optical element placed in a plane conjugate to the objective focal plane to generate position-dependent temporal pulse broadening that enables axially confined multiphoton absorption, without the need for tight spatial focusing. Many techniques have benefitted from temporal focusing, including scanless imaging, super-resolution imaging, photolithography, uncaging of caged neurotransmitters and control of neuronal activity via optogenetics.


Assuntos
Imageamento Tridimensional/métodos , Iluminação/métodos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Fótons , Animais , Desenho de Equipamento , Aumento da Imagem/instrumentação , Imageamento Tridimensional/instrumentação , Iluminação/instrumentação , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação
4.
Opt Lett ; 45(4): 909-912, 2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-32058502

RESUMO

In this Letter, we report a low-cost, portable, two-photon excitation fluorescence microscopy imager that uses a fiber-based approach for both femtosecond supercontinuum (SC) generation and light delivery to the optical head. The SC generation is based on a tapered polarization-maintaining photonic crystal fiber that uses pre-chirped femtosecond narrowband pulses to generate a coherent SC spectrum with a bandwidth of approximately 300 nm. Using this approach, high-power, near-transform-limited, wavelength-selectable SC pulses are generated and directly delivered to the imaging optical head. Preliminary testing of this imager on brain slices is presented, demonstrating a high signal-to-noise ratio and sub-cellular imaging capabilities to a depth of approximately 200 µm. These results demonstrate the suitability of the technology for ex vivo and potentially in vivo cellular-level biomedical imaging applications.


Assuntos
Luz , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação , Fibras Ópticas , Fenômenos Ópticos , Desenho de Equipamento , Dinâmica não Linear
5.
Nat Commun ; 11(1): 395, 2020 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-31959752

RESUMO

Active nerve cells release vasodilators that increase their energy supply by dilating local blood vessels, a mechanism termed neurovascular coupling and the basis of BOLD functional neuroimaging signals. Here, we reveal a mechanism for cerebral blood flow control, a precapillary sphincter at the transition between the penetrating arteriole and first order capillary, linking blood flow in capillaries to the arteriolar inflow. The sphincters are encircled by contractile mural cells, which are capable of bidirectional control of the length and width of the enclosed vessel segment. The hemodynamic consequence is that precapillary sphincters can generate the largest changes in the cerebrovascular flow resistance of all brain vessel segments, thereby controlling capillary flow while protecting the downstream capillary bed and brain tissue from adverse pressure fluctuations. Cortical spreading depolarization constricts sphincters and causes vascular trapping of blood cells. Thus, precapillary sphincters are bottlenecks for brain capillary blood flow.


Assuntos
Capilares/fisiologia , Córtex Cerebral/irrigação sanguínea , Circulação Cerebrovascular/fisiologia , Contração Muscular/fisiologia , Músculo Liso Vascular/fisiologia , Animais , Capilares/diagnóstico por imagem , Córtex Cerebral/diagnóstico por imagem , Depressão Alastrante da Atividade Elétrica Cortical/fisiologia , Feminino , Neuroimagem Funcional/métodos , Imageamento Tridimensional , Microscopia Intravital/instrumentação , Microscopia Intravital/métodos , Masculino , Camundongos , Microscopia Confocal/instrumentação , Microscopia Confocal/métodos , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Modelos Animais , Modelos Cardiovasculares , Músculo Liso Vascular/diagnóstico por imagem , Fluxo Sanguíneo Regional/fisiologia , Crânio/cirurgia , Trepanação
6.
J Biomed Opt ; 25(1): 1-9, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31994362

RESUMO

Two-photon microscopy (2PM) has revolutionized biomedical imaging by allowing thin optical sectioning in relatively thick biological specimens. Because dispersive microscope components in 2PM, such as objective lens, can alter temporal laser pulse width (typically being broader at the sample plane), for accurate measurements of two-photon absorption properties, it is important to characterize pulse duration at the sample plane. We present a simple modification to a two-photon microscope light path that allows for second-harmonic-generation-based interferometric autocorrelation measurements to characterize ultrafast laser pulse duration at the sample plane using time-correlated single-photon counting (TCSPC). We show that TCSPC can be used as a simple and versatile method to estimate the zero time delay step value between two adjacent ultrafast laser pulses for these measurements. To demonstrate the utility of this modification, we measured the Coherent Chameleon-Ultra II Ti:sapphire laser pulse width at the sample plane using a 10 × air, 40 × air, or 63 × water-immersion objective lens. At 950-nm two-photon excitation, the measured pulse width was 154 ± 32, 165 ± 13, and 218 ± 27 fs (n = 6, mean ± standard deviation), respectively.

.


Assuntos
Interferometria/instrumentação , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação , Eletricidade , Lasers , Microscopia Confocal/métodos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Óptica e Fotônica , Fótons , Processamento de Sinais Assistido por Computador
7.
J Vis Exp ; (153)2019 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-31814618

RESUMO

A protocol is presented to build a custom low-cost yet high-performance femtosecond (fs) fiber laser. This all-normal-dispersion (ANDi) ytterbium-doped fiber laser is built completely using commercially available parts, including $8,000 in fiber optic and pump laser components, plus $4,800 in standard optical components and extra-cavity accessories. Researchers new to fiber optic device fabrication may also consider investing in basic fiber splicing and laser pulse characterization equipment (~$63,000). Important for optimal laser operation, methods to verify true versus apparent (partial or noise-like) mode-locked performance are presented. This system achieves 70 fs pulse duration with a center wavelength of approximately 1,070 nm and a pulse repetition rate of 31 MHz. This fiber laser exhibits the peak performance that may be obtained for an easily assembled fiber laser system, which makes this design ideal for research laboratories aiming to develop compact and portable fs laser technologies that enable new implementations of clinical multiphoton microscopy and fs surgery.


Assuntos
Lasers , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Desenho de Equipamento , Tecnologia de Fibra Óptica , Microscopia de Fluorescência por Excitação Multifotônica/economia , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação , Fótons , Itérbio
8.
J Biomed Opt ; 25(1): 1-17, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31833280

RESUMO

The excited state lifetime of a fluorophore together with its fluorescence emission spectrum provide information that can yield valuable insights into the nature of a fluorophore and its microenvironment. However, it is difficult to obtain both channels of information in a conventional scheme as detectors are typically configured either for spectral or lifetime detection. We present a fiber-based method to obtain spectral information from a multiphoton fluorescence lifetime imaging (FLIM) system. This is made possible using the time delay introduced in the fluorescence emission path by a dispersive optical fiber coupled to a detector operating in time-correlated single-photon counting mode. This add-on spectral implementation requires only a few simple modifications to any existing FLIM system and is considerably more cost-efficient compared to currently available spectral detectors.


Assuntos
Microscopia de Fluorescência por Excitação Multifotônica/instrumentação , Fibras Ópticas , Imagem Óptica/instrumentação , Animais , Bovinos , Células Cultivadas , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Desenho de Equipamento , Corantes Fluorescentes , Microscopia de Fluorescência por Excitação Multifotônica/estatística & dados numéricos , Imagem Óptica/estatística & dados numéricos , Fenômenos Ópticos
9.
J Biomed Opt ; 25(1): 1-8, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31729201

RESUMO

Multiphoton microscopy provides a suitable technique for imaging biological tissues with submicrometer resolution. Usually a Gaussian beam (GB) is used for illumination, leading to a reduced power efficiency in the multiphoton response and vignetting for a square-shaped imaging area. A flat-top beam (FTB) provides a uniform spatial intensity distribution that equalizes the probability of a multiphoton effect across the imaging area. We employ a customized widefield multiphoton microscope to compare the performance of a square-shaped FTB illumination with that based on using a GB, for both two-photon fluorescence (TPF) and second-harmonic generation (SHG) imaging. The variation in signal-to-noise ratio across TPF images of fluorescent dyes spans ∼5.6 dB for the GB and ∼1.2 dB for the FTB illumination, respectively. For the GB modality, TPF images of mouse colon and Convallaria root, and SHG images of chicken tendon and human breast biopsy tissue showcase ∼20 % area that are not imaged due to either insufficient or lack of illumination. For quantitative analysis that depends on the illuminated area, this effect can potentially lead to inaccuracies. This work emphasizes the applicability of FTB illumination to multiphoton applications.


Assuntos
Microscopia de Fluorescência por Excitação Multifotônica/métodos , Animais , Mama/anatomia & histologia , Galinhas , Colo/anatomia & histologia , Simulação por Computador , Convallaria/anatomia & histologia , Desenho de Equipamento , Feminino , Corantes Fluorescentes , Humanos , Conceitos Matemáticos , Camundongos , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação , Microscopia de Fluorescência por Excitação Multifotônica/estatística & dados numéricos , Imagem Óptica/instrumentação , Imagem Óptica/métodos , Imagem Óptica/estatística & dados numéricos , Fenômenos Ópticos , Razão Sinal-Ruído , Tendões/anatomia & histologia
10.
J Biomed Opt ; 25(1): 1-5, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31691550

RESUMO

Two-photon excitation microscopy is one of the key techniques used to observe three-dimensional (3-D) structures in biological samples. We utilized a visible-wavelength laser beam for two-photon excitation in a multifocus confocal scanning system to improve the spatial resolution and image contrast in 3-D live-cell imaging. Experimental and numerical analyses revealed that the axial resolution has improved for a wide range of pinhole sizes used for confocal detection. We observed the 3-D movements of the Golgi bodies in living HeLa cells with an imaging speed of 2 s per volume. We also confirmed that the time-lapse observation up to 8 min did not cause significant cell damage in two-photon excitation experiments using wavelengths in the visible light range. These results demonstrate that multifocus, two-photon excitation microscopy with the use of a visible wavelength can constitute a simple technique for 3-D visualization of living cells with high spatial resolution and image contrast.


Assuntos
Microscopia de Fluorescência por Excitação Multifotônica/métodos , Corantes Fluorescentes , Complexo de Golgi/fisiologia , Complexo de Golgi/ultraestrutura , Células HeLa , Humanos , Imageamento Tridimensional/métodos , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação , Movimento/fisiologia , Fenômenos Ópticos , Análise de Célula Única/métodos , Imagem com Lapso de Tempo/instrumentação , Imagem com Lapso de Tempo/métodos
11.
J Neurosci ; 39(46): 9042-9052, 2019 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-31578235

RESUMO

Multiphoton microscopy (MPM) has emerged as one of the most powerful and widespread technologies to monitor the activity of neuronal networks in awake, behaving animals over long periods of time. MPM development spanned across decades and crucially depended on the concurrent improvement of calcium indicators that report neuronal activity as well as surgical protocols, head fixation approaches, and innovations in optics and microscopy technology. Here we review the last decade of MPM development and highlight how in vivo imaging has matured and diversified, making it now possible to concurrently monitor thousands of neurons across connected brain areas or, alternatively, small local networks with sampling rates in the kilohertz range. This review includes different laser scanning approaches, such as multibeam technologies as well as recent developments to image deeper into neuronal tissues using new, long-wavelength laser sources. As future development will critically depend on our ability to resolve and discriminate individual neuronal spikes, we will also describe a simple framework that allows performing quantitative comparisons between the reviewed MPM instruments. Finally, we provide our own opinion on how the most recent MPM developments can be leveraged at scale to enable the next generation of discoveries in brain function.


Assuntos
Encéfalo/fisiologia , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Neurônios/fisiologia , Animais , Encéfalo/citologia , Processamento de Imagem Assistida por Computador , Microscopia Confocal/instrumentação , Microscopia Confocal/métodos , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação , Neurônios/citologia
12.
J Biomed Opt ; 24(10): 1-7, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31625323

RESUMO

The silicon photomultiplier (SIPM) is an emerging detector technology that enables both high sensitivity and high dynamic range detection of visible and near-infrared light at a fraction of the cost of conventional vacuum tube photomultiplier tubes (PMTs). A low-cost detection circuit is presented and the performance of a commercial SIPM is evaluated for high-speed laser scanning microscopy applications. For moderate-to-high-speed fluorescent imaging applications, the measurements and imaging results indicate that the SIPM exceeds the sensitivity of GaAsP PMTs, while providing higher dynamic range and better saturation behavior. For low speed or applications requiring large detector areas, the GaAsP PMT retains a sensitivity advantage due to large area and lower dark counts. The calculations presented show that, above a critical detection bandwidth, the SIPM sensitivity exceeds that of a GaAsP PMT.


Assuntos
Microscopia Confocal/instrumentação , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação , Processamento de Sinais Assistido por Computador/instrumentação , Silício/química , Desenho de Equipamento , Humanos , Fótons , Razão Sinal-Ruído , Pele/diagnóstico por imagem
13.
J Biomed Opt ; 24(10): 1-12, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31646840

RESUMO

Multiphoton microscopy (MPM) has the capacity to record second-harmonic generation (SHG) and endogenous two-photon excitation fluorescence (2PEF) signals emitted from biological tissues. The development of fiber-based miniaturized endomicroscopes delivering pulses in the femtosecond range will allow the transfer of MPM to clinical endoscopy. We present real-time SHG and 2PEF ex vivo images using an endomicroscope, which totally complies with clinical endoscopy regulations. This system is based on the proximal scanning of a commercial multicore image guide (IG). For understanding the inhomogeneities of the recorded images, we quantitatively characterize the IG at the single-core level during nonlinear excitation. The obtained results suggest that these inhomogeneities originate from the variable core geometries that, therefore, exhibit variable nonlinear and dispersive properties. Finally, we propose a method based on modulation of dispersion precompensation to address the image inhomogeneity issue and, as a proof of concept, we demonstrate its capability to improve the nonlinear image quality.


Assuntos
Endoscopia/instrumentação , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação , Processamento de Sinais Assistido por Computador/instrumentação , Desenho de Equipamento , Humanos , Processamento de Imagem Assistida por Computador , Pulmão/diagnóstico por imagem , Fibras Ópticas , Fótons
14.
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
15.
Methods Cell Biol ; 154: 85-107, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31493823

RESUMO

Fluorescence microscopy techniques are powerful tools to study tissue dynamics, cellular function and biology both in vivo and in vitro. These tools allow for functional assessment and quantification along with qualitative analysis, thus providing a comprehensive understanding of various cellular processes under normal physiological and disease conditions. The main focus of this chapter is the recently developed method of serial intravital multiphoton microscopy that has helped shed light on the dynamic alterations of the spatial distribution and fate of single renal cells or cell populations and their migration patterns in the same tissue region over several days in response to various stimuli within the living kidney. This technique is very useful for studying in vivo the molecular and cellular mechanisms of tissue remodeling and repair after injury. In addition, complementary in vitro imaging tools are also described and discussed, like tissue clearing techniques and protein synthesis measurement in tissues in situ that provide an in depth assessment of changes at the cellular level. Thus, these novel fluorescence techniques can be effectively leveraged for different tissue types, experimental conditions as well as disease models to improve our understanding of renal cell biology.


Assuntos
Células Epiteliais/ultraestrutura , Microscopia Intravital/métodos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Nefrite/fisiopatologia , Podócitos/ultraestrutura , Obstrução Ureteral/fisiopatologia , Animais , Movimento Celular , Modelos Animais de Doenças , Doxorrubicina/administração & dosagem , Células Epiteliais/metabolismo , Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Microscopia Intravital/instrumentação , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação , Nefrite/induzido quimicamente , Nefrite/metabolismo , Podócitos/metabolismo , Análise de Célula Única/métodos , Obstrução Ureteral/metabolismo
16.
Methods Cell Biol ; 153: 43-67, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31395384

RESUMO

Renal tubular epithelial cells are consistently exposed to flow of glomerular filtrate that creates fluid shear stress at the apical cell surface. This biophysical stimulus regulates several critical renal epithelial cell functions, including transport, protein uptake, and barrier function. Defining the in vivo mechanical conditions in the kidney tubule is important for accurately recapitulating these conditions in vitro. Here we provide a summary of the fluid flow conditions in the kidney and how this translates into different levels of fluid shear stress down the length of the nephron. A detailed method is provided for measuring fluid flow in the proximal tubule by intravital microscopy. Devices to mimic in vivo fluid shear stress for in vitro studies are discussed, and we present two methods for culture and analysis of renal tubule epithelial cells exposed physiological levels of fluid shear stress. The first is a microfluidic device that permits application of controlled shear stress to cells cultured on porous membranes. The second is culture of renal tubule cells on an orbital shaker. Each method has advantages and disadvantages that should be considered in the context of the specific experimental objectives.


Assuntos
Células Epiteliais/fisiologia , Microscopia Intravital/métodos , Túbulos Renais Proximais/citologia , Técnicas Analíticas Microfluídicas/métodos , Estresse Mecânico , Administração Intravenosa , Animais , Membrana Celular/fisiologia , Células Cultivadas , Células Epiteliais/citologia , Corantes Fluorescentes/administração & dosagem , Taxa de Filtração Glomerular/fisiologia , Microscopia Intravital/instrumentação , Túbulos Renais Proximais/fisiologia , Técnicas Analíticas Microfluídicas/instrumentação , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Ratos , Resistência ao Cisalhamento
17.
Scanning ; 2019: 5192875, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31341525

RESUMO

Blood vessels are the important components of the circulatory systems that transport blood throughout the human body and maintain the homeostasis of physiological tissues. Pathologically, blood vessels are often affected by diseases, leading to the formation of unstable, irregular, and hyperpermeable blood vessels. In the tumor microenvironment, abnormal leakage of tumor blood vessels is related to the histological grade and malignant potential of tumors and may also facilitate metastasis of cancer. Visual diagnosis of blood vessels is very important for us to understand the occurrence and development of diseases. Multiphoton microscopy (MPM) is a potential label-free diagnostic tool based on second harmonic generation (SHG) and two-photon excited fluorescence (TPEF). MPM can effectively observe the morphological changes of biological tissues at the molecular and cellular levels. In this work, we demonstrate that label-free MPM can be used to visualize the microstructure of blood vessels in human normal breast and breast tumor tissue. Moreover, MPM can monitor the changes of blood vessels in tumor microenvironment. These results show that the MPM will become a promising technique for clinicians to study the properties of the microstructure of the blood vessels.


Assuntos
Vasos Sanguíneos/diagnóstico por imagem , Neoplasias da Mama/diagnóstico por imagem , Glândulas Mamárias Humanas/diagnóstico por imagem , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Neovascularização Patológica/diagnóstico por imagem , Vasos Sanguíneos/patologia , Neoplasias da Mama/irrigação sanguínea , Neoplasias da Mama/patologia , Neoplasias da Mama/cirurgia , Amarelo de Eosina-(YS) , Feminino , Hematoxilina , Humanos , Glândulas Mamárias Humanas/irrigação sanguínea , Glândulas Mamárias Humanas/patologia , Glândulas Mamárias Humanas/cirurgia , Mastectomia , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação , Neovascularização Patológica/patologia , Neovascularização Patológica/cirurgia , Inclusão em Parafina , Fixação de Tecidos , Microambiente Tumoral
18.
Clin Cancer Res ; 25(17): 5376-5387, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31175091

RESUMO

PURPOSE: Cancer treatment is limited by inaccurate predictors of patient-specific therapeutic response. Therefore, some patients are exposed to unnecessary side effects and delays in starting effective therapy. A clinical tool that predicts treatment sensitivity for individual patients is needed. EXPERIMENTAL DESIGN: Patient-derived cancer organoids were derived across multiple histologies. The histologic characteristics, mutation profile, clonal structure, and response to chemotherapy and radiation were assessed using bright-field and optical metabolic imaging on spheroid and single-cell levels, respectively. RESULTS: We demonstrate that patient-derived cancer organoids represent the cancers from which they were derived, including key histologic and molecular features. These cultures were generated from numerous cancers, various biopsy sample types, and in different clinical settings. Next-generation sequencing reveals the presence of subclonal populations within the organoid cultures. These cultures allow for the detection of clonal heterogeneity with a greater sensitivity than bulk tumor sequencing. Optical metabolic imaging of these organoids provides cell-level quantification of treatment response and tumor heterogeneity allowing for resolution of therapeutic differences between patient samples. Using this technology, we prospectively predict treatment response for a patient with metastatic colorectal cancer. CONCLUSIONS: These studies add to the literature demonstrating feasibility to grow clinical patient-derived organotypic cultures for treatment effectiveness testing. Together, these culture methods and response assessment techniques hold great promise to predict treatment sensitivity for patients with cancer undergoing chemotherapy and/or radiation.


Assuntos
Ensaios de Seleção de Medicamentos Antitumorais/métodos , Neoplasias/tratamento farmacológico , Neoplasias/radioterapia , Organoides/efeitos dos fármacos , Organoides/efeitos da radiação , Humanos , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação , Neoplasias/metabolismo , Neoplasias/patologia , Organoides/metabolismo , Organoides/patologia , Medicina de Precisão/métodos , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Esferoides Celulares/efeitos da radiação
19.
J Vis Exp ; (148)2019 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-31233031

RESUMO

Maintenance of normal brain function requires a sufficient and efficient supply of oxygen and nutrition by a complex network of vessels. However, the regulation of cerebral blood flow (CBF) is incompletely understood, especially at the capillary level. Two-photon microscopy is a powerful tool widely used to study CBF and its regulation. Currently, this field is limited by the lack of in vivo two-photon microscopy studies examining (1) CBF responses in three-dimensions, (2) conducted vascular responses, and (3) localized interventions within the vascular network. Here, we describe a 3D in vivo method using two-photon microscopy to study conducted vascular responses elicited by local ejection of ATP with a glass micro-pipette. Our method uses fast and repetitive hyperstack two-photon imaging providing precise diameter measurements by maximal intensity projection of the obtained images. Furthermore, we show that this method can also be used to study 3D astrocytic calcium responses. We also discuss the advantages and limitations of glass micro-pipette insertion and two-photon hyperstack imaging.


Assuntos
Trifosfato de Adenosina/metabolismo , Circulação Cerebrovascular , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação , Radioterapia Conformacional/instrumentação , Astrócitos/metabolismo , Encéfalo/irrigação sanguínea , Encéfalo/citologia , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Cálcio/metabolismo , Humanos
20.
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
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