RESUMEN
As a vertebrate model animal, larval zebrafish are widely used in neuroscience and provide a unique opportunity to monitor whole-brain activity at the cellular resolution. Here, we provide an optimized protocol for performing whole-brain imaging of larval zebrafish using three-dimensional fluorescence microscopy, including sample preparation and immobilization, sample embedding, image acquisition, and visualization after imaging. The current protocol enables in vivo imaging of the structure and neuronal activity of a larval zebrafish brain at a cellular resolution for over 1 h using confocal microscopy and custom-designed fluorescence microscopy. The critical steps in the protocol are also discussed, including sample mounting and positioning, preventing bubble formation and dust in the agarose gel, and avoiding motion in images caused by incomplete solidification of the agarose gel and paralyzation of the fish. The protocol has been validated and confirmed in multiple settings. This protocol can be easily adapted for imaging other organs of a larval zebrafish.
Asunto(s)
Encéfalo , Imagenología Tridimensional , Microscopía Intravital , Microscopía Fluorescente , Neuroimagen , Pez Cebra , Animales , Encéfalo/diagnóstico por imagen , Imagenología Tridimensional/instrumentación , Imagenología Tridimensional/métodos , Neuroimagen/instrumentación , Neuroimagen/métodos , Sefarosa , Microscopía Intravital/instrumentación , Microscopía Intravital/métodosRESUMEN
We present an automated method for COVID-19 screening based on reconstructed phase profiles of red blood cells (RBCs) and a highly comparative time-series analysis (HCTSA). Video digital holographic data -was obtained using a compact, field-portable shearing microscope to capture the temporal fluctuations and spatio-temporal dynamics of live RBCs. After numerical reconstruction of the digital holographic data, the optical volume is calculated at each timeframe of the reconstructed data to produce a time-series signal for each cell in our dataset. Over 6000 features are extracted on the time-varying optical volume sequences using the HCTSA to quantify the spatio-temporal behavior of the RBCs, then a linear support vector machine is used for classification of individual RBCs. Human subjects are then classified for COVID-19 based on the consensus of their cells' classifications. The proposed method is tested on a dataset of 1472 RBCs from 24 human subjects (10 COVID-19 positive, 14 healthy) collected at UConn Health Center. Following a cross-validation procedure, our system achieves 82.13% accuracy, with 92.72% sensitivity, and 73.21% specificity (area under the receiver operating characteristic curve: 0.8357). Furthermore, the proposed system resulted in 21 out of 24 human subjects correctly labeled. To the best of our knowledge this is the first report of a highly comparative time-series analysis using digital holographic microscopy data.
Asunto(s)
COVID-19/diagnóstico por imagen , Eritrocitos/clasificación , Holografía/métodos , Microscopía Intravital/métodos , COVID-19/sangre , Estudios de Casos y Controles , Diseño de Equipo , Holografía/instrumentación , Humanos , Microscopía Intravital/instrumentación , Datos Preliminares , Curva ROC , Sensibilidad y EspecificidadRESUMEN
PURPOSE: To evaluate the potential toxicity of operation microscopes with halogen and light emitting diode (LED) light source on the rabbit eyes. MATERIALS AND METHODS: Thirty-two eyes of 16 male New Zealand pigmented rabbits were involved in the study. The rabbits were divided into two groups according to the type of light source applied. Only one eye of each rabbit was exposed to illumination light, unexposed fellow eyes served as the control group. Experimental groups included group 1 exposed to halogen light for 2 h and evaluated 1 day and 1 week after the illumination, group 2 exposed to LED light for two hours and evaluated 1 day and 1 week after the illumination. On the first and seventh days after exposing the light, we evaluated the rabbit corneas using in vivo confocal microscopy (IVCM). At the end of the seventh day, the Hematoxylin-eosin staining and TUNEL staining were performed to investigate the presence of apoptosis in the retina and retina pigment epithelium. RESULTS: Early IVCM findings revealed corneal epithelial cell ovalization and indistinct intercellular borders in the halogen light group. We also observed more increase in the keratocyte density index (23.7% vs 14.1%, p = 0.001, respectively) and the Bowman reflectivity index (12.4% vs 4.1%, p = 0.001, respectively) at first day of the light exposure in halogen light group compared to LED light group. However, late IVCM indicated that these findings disappeared one week later. No apoptosis was observed in the corneal and retinal layers in early and late examination groups. CONCLUSION: The present experimental study demonstrated that both halogen and LED lights, which were commonly used for microscopic eye surgery, had no sustained adverse effect on the cornea and retina of the rabbits; however, halogen light had a temporary adverse effect on corneal epithelium and stroma, which resolved within 1 week.
Asunto(s)
Epitelio Corneal/efectos de la radiación , Iluminación/efectos adversos , Microcirugia/efectos adversos , Procedimientos Quirúrgicos Oftalmológicos/efectos adversos , Complicaciones Posoperatorias/patología , Epitelio Pigmentado de la Retina/efectos de la radiación , Animales , Apoptosis , Epitelio Corneal/patología , Halógenos , Humanos , Microscopía Intravital/efectos adversos , Microscopía Intravital/instrumentación , Iluminación/instrumentación , Masculino , Microscopía Confocal/instrumentación , Microcirugia/instrumentación , Procedimientos Quirúrgicos Oftalmológicos/instrumentación , Complicaciones Posoperatorias/etiología , Conejos , Epitelio Pigmentado de la Retina/patología , SemiconductoresRESUMEN
Intravital two-photon microscopy enables monitoring of cellular dynamics and communication of complex systems, in genuine environment-the living organism. Particularly, its application in understanding the immune system brought unique insights into pathophysiologic processes in vivo. Here we present a method to achieve multiplexed dynamic intravital two-photon imaging by using a synergistic strategy combining a spectrally broad range of fluorophore emissions, a wave-mixing concept for simultaneous excitation of all targeted fluorophores, and an effective unmixing algorithm based on the calculation of spectral similarities with previously acquired fluorophore fingerprints. Our unmixing algorithm allows us to distinguish 7 fluorophore signals corresponding to various cellular and tissue compartments by using only four detector channels.
Asunto(s)
Técnica del Anticuerpo Fluorescente/métodos , Microscopía Intravital/métodos , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Algoritmos , Animales , Línea Celular , Análisis de Datos , Humanos , Procesamiento de Imagen Asistido por Computador , Microscopía Intravital/instrumentación , Ratones , Microscopía de Fluorescencia por Excitación Multifotónica/instrumentaciónRESUMEN
Calcium imaging is a powerful tool for recording from large populations of neurons in vivo. Imaging in rhesus macaque motor cortex can enable the discovery of fundamental principles of motor cortical function and can inform the design of next generation brain-computer interfaces (BCIs). Surface two-photon imaging, however, cannot presently access somatic calcium signals of neurons from all layers of macaque motor cortex due to photon scattering. Here, we demonstrate an implant and imaging system capable of chronic, motion-stabilized two-photon imaging of neuronal calcium signals from macaques engaged in a motor task. By imaging apical dendrites, we achieved optical access to large populations of deep and superficial cortical neurons across dorsal premotor (PMd) and gyral primary motor (M1) cortices. Dendritic signals from individual neurons displayed tuning for different directions of arm movement. Combining several technical advances, we developed an optical BCI (oBCI) driven by these dendritic signalswhich successfully decoded movement direction online. By fusing two-photon functional imaging with CLARITY volumetric imaging, we verified that many imaged dendrites which contributed to oBCI decoding originated from layer 5 output neurons, including a putative Betz cell. This approach establishes new opportunities for studying motor control and designing BCIs via two photon imaging.
Asunto(s)
Interfaces Cerebro-Computador , Calcio/metabolismo , Dendritas/fisiología , Microscopía Intravital/instrumentación , Microscopía Intravital/métodos , Corteza Motora/diagnóstico por imagen , Imagen Multimodal/métodos , Animales , Proteínas de Unión al Calcio/metabolismo , Dendritas/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Implantes Experimentales , Macaca mulatta , Masculino , Modelos Neurológicos , Actividad Motora/fisiología , Corteza Motora/fisiología , Neuronas/fisiología , FotonesRESUMEN
Intravital microscopy is an imaging technique aimed at the visualization of the dynamics of biological processes in live animals. In the last decade, the development of nonlinear optical microscopy has enormously increased the use of this technique, thus addressing key biological questions in different fields such as immunology, neurobiology and tumor biology. In addition, new upcoming strategies to minimize motion artifacts due to animal respiration and heartbeat have enabled the visualization in real time of biological processes at cellular and subcellular resolution. Recently, intravital microscopy has been applied to analyze different aspect of mucosal immunity in the gut. However, the majority of these studies have been performed on the small intestine. Although crucial aspects of the biology of this organ have been unveiled, the majority of intestinal pathologies in humans occur in the large intestine.Here, we describe a method to surgically expose and stabilize the large intestine in live mice and to perform short-term (up to 2 h) intravital microscopy.
Asunto(s)
Intestino Grueso/diagnóstico por imagen , Microscopía Intravital/métodos , Animales , Femenino , Intestino Grueso/cirugía , Microscopía Intravital/instrumentación , Masculino , Ratones , Microscopía de Fluorescencia por Excitación MultifotónicaRESUMEN
Structured Illumination Microscopy enables live imaging with sub-diffraction resolution. Unfortunately, optical aberrations can lead to loss of resolution and artifacts in Structured Illumination Microscopy rendering the technique unusable in samples thicker than a single cell. Here we report on the combination of Adaptive Optics and Structured Illumination Microscopy enabling imaging with 150 nm lateral and 570 nm axial resolution at a depth of 80 µm through Caenorhabditis elegans. We demonstrate that Adaptive Optics improves the three-dimensional resolution, especially along the axial direction, and reduces artifacts, successfully realizing 3D-Structured Illumination Microscopy in a variety of biological samples.
Asunto(s)
Imagenología Tridimensional/métodos , Microscopía Intravital/métodos , Iluminación/instrumentación , Animales , Artefactos , Ascomicetos , Caenorhabditis elegans , Línea Celular , Imagenología Tridimensional/instrumentación , Microscopía Intravital/instrumentación , Ratones , Microscopía Confocal/instrumentación , Microscopía Confocal/métodos , Microscopía Fluorescente/instrumentación , Microscopía Fluorescente/métodos , Oryza/microbiología , Reproducibilidad de los ResultadosRESUMEN
When exploring new environments animals form spatial memories that are updated with experience and retrieved upon re-exposure to the same environment. The hippocampus is thought to support these memory processes, but how this is achieved by different subnetworks such as CA1 and CA3 remains unclear. To understand how hippocampal spatial representations emerge and evolve during familiarization, we performed 2-photon calcium imaging in mice running in new virtual environments and compared the trial-to-trial dynamics of place cells in CA1 and CA3 over days. We find that place fields in CA1 emerge rapidly but tend to shift backwards from trial-to-trial and remap upon re-exposure to the environment a day later. In contrast, place fields in CA3 emerge gradually but show more stable trial-to-trial and day-to-day dynamics. These results reflect different roles in CA1 and CA3 in spatial memory processing during familiarization to new environments and constrain the potential mechanisms that support them.
Asunto(s)
Región CA1 Hipocampal/fisiología , Región CA3 Hipocampal/fisiología , Células de Lugar/fisiología , Percepción Espacial/fisiología , Memoria Espacial/fisiología , Animales , Técnicas de Observación Conductual , Conducta Animal/fisiología , Región CA1 Hipocampal/citología , Región CA1 Hipocampal/diagnóstico por imagen , Región CA3 Hipocampal/citología , Región CA3 Hipocampal/diagnóstico por imagen , Craneotomía , Microscopía Intravital/instrumentación , Microscopía Intravital/métodos , Masculino , Ratones , Microscopía Confocal/instrumentación , Microscopía Confocal/métodos , Modelos Animales , Imagen Óptica/instrumentación , Imagen Óptica/métodosRESUMEN
Conventional standing-wave (SW) fluorescence microscopy uses a single wavelength to excite fluorescence from the specimen, which is normally placed in contact with a first surface reflector. The resulting excitation SW creates a pattern of illumination with anti-nodal maxima at multiple evenly-spaced planes perpendicular to the optical axis of the microscope. These maxima are approximately 90 nm thick and spaced 180 nm apart. Where the planes intersect fluorescent structures, emission occurs, but between the planes are non-illuminated regions which are not sampled for fluorescence. We evaluate a multi-excitation-wavelength SW fluorescence microscopy (which we call TartanSW) as a method for increasing the density of sampling by using SWs with different axial periodicities, to resolve more of the overall cell structure. The TartanSW method increased the sampling density from 50 to 98% over seven anti-nodal planes, with no notable change in axial or lateral resolution compared to single-excitation-wavelength SW microscopy. We demonstrate the method with images of the membrane and cytoskeleton of living and fixed cells.
Asunto(s)
Membrana Celular , Citoesqueleto , Aumento de la Imagen/métodos , Microscopía Intravital/métodos , Animales , Línea Celular Tumoral , Humanos , Aumento de la Imagen/instrumentación , Microscopía Intravital/instrumentación , Ratones , Microscopía Fluorescente/instrumentación , Microscopía Fluorescente/métodosRESUMEN
Intravital microscopy (IVM) is an essential experimental approach for evaluating, in real time, cell interactions in the blood and rheological parameters in the microcirculation of the living animals. Different tissues are surgically exposed to the visualization of the microvascular network in optical microscopies connected to video cameras and image software. By evaluating in situ microcirculatory network, IVM allows the visualization and quantification of physiological and pathological processes in the blood or in the adjacent tissues considering the whole system. Therefore, IVM has been used to evaluate the effects and mechanisms of actions in the microvascular network caused by pharmacological or toxic chemical agents. In this chapter, different experimental approaches are described to study the toxic effects and mechanisms of xenobiotics in the microcirculatory network.
Asunto(s)
Microscopía Intravital/métodos , Microvasos/efectos de los fármacos , Nanoconjugados/toxicidad , Pruebas de Toxicidad/métodos , Xenobióticos/toxicidad , Animales , Microscopía Intravital/instrumentación , Microvasos/diagnóstico por imagen , Reología/métodos , Xenobióticos/farmacocinéticaRESUMEN
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.
Asunto(s)
Permeabilidad Capilar/efectos de los fármacos , Dextranos/metabolismo , Fluoresceína-5-Isotiocianato/análogos & derivados , Histamina/farmacología , Hipersensibilidad Inmediata/diagnóstico por imagen , Microscopía Intravital/métodos , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Animales , Oído/irrigación sanguínea , Oído/diagnóstico por imagen , Fluoresceína-5-Isotiocianato/metabolismo , Colorantes Fluorescentes/metabolismo , Hipersensibilidad Inmediata/inducido químicamente , Inyecciones Intravenosas , Microscopía Intravital/instrumentación , Ratones Endogámicos BALB C , Microscopía de Fluorescencia por Excitación Multifotónica/instrumentación , Peso Molecular , Imagen de Lapso de TiempoRESUMEN
It is well recognized that isolated cardiac muscle cells beat in a periodic manner. Recently, evidence indicates that other, non-muscle cells, also perform periodic motions that are either imperceptible under conventional lab microscope lens or practically not easily amenable for analysis of oscillation amplitude, frequency, phase of movement and its direction. Here, we create a real-time video analysis tool to visually magnify and explore sub-micron rhythmic movements performed by biological cells and the induced movements in their surroundings. Using this tool, we suggest that fibroblast cells perform small fluctuating movements with a dominant frequency that is dependent on their surrounding substrate and its stiffness.
Asunto(s)
Movimiento Celular/fisiología , Procesamiento de Imagen Asistido por Computador/métodos , Microscopía Intravital/métodos , Microscopía por Video/métodos , Imagen de Lapso de Tiempo/métodos , Células 3T3 , Animales , Procesamiento de Imagen Asistido por Computador/instrumentación , Microscopía Intravital/instrumentación , Ratones , Microscopía por Video/instrumentación , Imagen de Lapso de Tiempo/instrumentaciónRESUMEN
Understanding the structure and function of vasculature in the brain requires us to monitor distributed hemodynamics at high spatial and temporal resolution in three-dimensional (3D) volumes in vivo. Currently, a volumetric vasculature imaging method with sub-capillary spatial resolution and blood flow-resolving speed is lacking. Here, using two-photon laser scanning microscopy (TPLSM) with an axially extended Bessel focus, we capture volumetric hemodynamics in the awake mouse brain at a spatiotemporal resolution sufficient for measuring capillary size and blood flow. With Bessel TPLSM, the fluorescence signal of a vessel becomes proportional to its size, which enables convenient intensity-based analysis of vessel dilation and constriction dynamics in large volumes. We observe entrainment of vasodilation and vasoconstriction with pupil diameter and measure 3D blood flow at 99 volumes/second. Demonstrating high-throughput monitoring of hemodynamics in the awake brain, we expect Bessel TPLSM to make broad impacts on neurovasculature research.
Asunto(s)
Encéfalo/diagnóstico por imagen , Circulación Cerebrovascular/fisiología , Microscopía Intravital/métodos , Animales , Velocidad del Flujo Sanguíneo/fisiología , Encéfalo/irrigación sanguínea , Estudios de Factibilidad , Microscopía Intravital/instrumentación , Ratones , Microscopía Confocal/métodos , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Modelos Animales , Pupila/fisiología , Técnicas Estereotáxicas , Vasoconstricción/fisiología , Vasodilatación/fisiología , Vigilia/fisiologíaRESUMEN
In the field of ischemic cerebral injury, precise characterization of neurovascular hemodynamic is required to select candidates for reperfusion treatments. It is thus admitted that advanced imaging-based approaches would be able to better diagnose and prognose those patients and would contribute to better clinical care. Current imaging modalities like MRI allow a precise diagnostic of cerebral injury but suffer from limited availability and transportability. The recently developed ultrafast ultrasound could be a powerful tool to perform emergency imaging and long term follow-up of cerebral perfusion, which could, in combination with MRI, improve imaging solutions for neuroradiologists. Methods: In this study, in a model of in situ thromboembolic stroke in mice, we compared a control group of non-treated mice (N=10) with a group receiving the gold standard pharmacological stroke therapy (N=9). We combined the established tool of magnetic resonance imaging (7T MRI) with two innovative ultrafast ultrasound methods, ultrafast Doppler and Ultrasound Localization Microscopy, to image the cerebral blood volumes at early and late times after stroke onset and compare with the formation of ischemic lesions.Results: Our study shows that ultrafast ultrasound can be used through the mouse skull to monitor cerebral perfusion during ischemic stroke. In our data, the monitoring of the reperfusion following thrombolytic within the first 2 h post stroke onset matches ischemic lesions measured 24 h. Moreover, similar results can be made with Ultrasound Localization Microscopy which could make it applicable to human patients in the future. Conclusion: We thus provide the proof of concept that in a mouse model of thromboembolic stroke with an intact skull, early ultrafast ultrasound can be indicative of responses to treatment and cerebral tissue fates following stroke. It brings new tools to study ischemic stroke in preclinical models and is the first step prior translation to the clinical settings.
Asunto(s)
Circulación Cerebrovascular , Microscopía Intravital/métodos , Arteria Cerebral Media/diagnóstico por imagen , Accidente Cerebrovascular Trombótico/diagnóstico , Ultrasonografía Doppler/métodos , Animales , Encéfalo/irrigación sanguínea , Encéfalo/diagnóstico por imagen , Modelos Animales de Enfermedad , Fibrinolíticos/administración & dosificación , Humanos , Microscopía Intravital/instrumentación , Imagen por Resonancia Magnética , Masculino , Ratones , Prueba de Estudio Conceptual , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/toxicidad , Terapia Trombolítica , Accidente Cerebrovascular Trombótico/inducido químicamente , Accidente Cerebrovascular Trombótico/tratamiento farmacológico , Factores de Tiempo , Activador de Tejido Plasminógeno/administración & dosificación , Activador de Tejido Plasminógeno/toxicidad , Ultrasonografía Doppler/instrumentaciónRESUMEN
In vivo calcium imaging in zebrafish provides the ability to investigate calcium dynamics within neurons. Utilizing genetically encoded calcium sensors it is possible to monitor calcium signals within a single axon during axon injury and degeneration with high temporal and spatial resolution. Here we will describe in vivo, time-lapse confocal imaging methods of calcium imaging. Imaging of calcium dynamics with genetically encoded calcium sensors (GECS) within living axons can serve as a method to assess axonal physiology and effects of pharmacologic and genetic manipulation, as well as characterize responses to different injury models.
Asunto(s)
Axones/ultraestructura , Calcio/análisis , Microscopía Intravital/métodos , Imagen de Lapso de Tiempo/métodos , Degeneración Walleriana/patología , Animales , Animales Modificados Genéticamente , Axones/química , Axones/fisiología , Señalización del Calcio , Proteínas de Unión al Calcio/análisis , Proteínas de Unión al Calcio/genética , Citoplasma/química , Femenino , Genes Reporteros , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Proteínas Sensoras del Calcio Intracelular/análisis , Microscopía Intravital/instrumentación , Proteínas Luminiscentes , Masculino , Mitocondrias/química , Imagen de Lapso de Tiempo/instrumentación , Degeneración Walleriana/metabolismo , Pez Cebra/embriologíaRESUMEN
Axonal transport, which is the process mediating the active shuttling of a variety cargoes from one end of an axon to the other, is essential for the development, function, and survival of neurons. Impairments in this dynamic process are linked to diverse nervous system diseases and advanced ageing. It is thus essential that we quantitatively study the kinetics of axonal transport to gain an improved understanding of neuropathology as well as the molecular and cellular mechanisms regulating cargo trafficking. One of the best ways to achieve this goal is by imaging individual, fluorescent cargoes in live systems and analyzing the kinetic properties of their progression along the axon. We have therefore developed an intravital technique to visualize different organelles, such as signaling endosomes and mitochondria, being actively transported in the axons of both motor and sensory neurons in live, anesthetized rodents. In this chapter, we provide step-by-step instructions on how to deliver specific organelle-targeting, fluorescent probes using several routes of administration to image individual cargoes being bidirectionally transported along axons within the exposed sciatic nerve. This method can provide detailed, physiologically relevant information on axonal transport, and is thus poised to elucidate mechanisms regulating this process in both health and disease.
Asunto(s)
Transporte Axonal/fisiología , Microscopía Intravital/métodos , Degeneración Nerviosa/patología , Nervios Periféricos/fisiología , Animales , Endosomas/ultraestructura , Colorantes Fluorescentes/administración & dosificación , Colorantes Fluorescentes/análisis , Colorantes Fluorescentes/farmacocinética , Genes Reporteros , Inyecciones Intramusculares , Microscopía Intravital/instrumentación , Cinesinas/fisiología , Músculo Esquelético , Orgánulos/ultraestructura , Nervios Periféricos/ultraestructura , Roedores , Nervio Ciático/fisiología , Nervio Ciático/ultraestructuraRESUMEN
The invention of two-photon excitation microscopes widens the potential application of intravital microscopy (IVM) to the broad field of experimental pathology. Moreover, the recent development of fluorescent protein-based, genetically encoded biosensors provides an ideal tool to visualize the cell function in live animals. We start from a brief review of IVM with two-photon excitation microscopes and genetically encoded biosensors based on the principle of Förster resonance energy transfer (FRET). Then, we describe how IVM using biosensors has revealed the pathogenesis of several disease models.
Asunto(s)
Técnicas Biosensibles/métodos , Microscopía Intravital/métodos , Microscopía Fluorescente/métodos , Patología/métodos , Animales , Modelos Animales de Enfermedad , Transferencia Resonante de Energía de Fluorescencia/métodos , Microscopía Intravital/instrumentación , Microscopía Fluorescente/instrumentaciónRESUMEN
Motility analysis of microswimmers has long been limited to a few model cell types and broadly restricted by technical challenges of high-resolution in vivo microscopy. Recently, interdisciplinary interest in detailed analysis of the motile behavior of various species has gained momentum. Here we describe a basic protocol for motility analysis of an important, highly diverse group of eukaryotic flagellate microswimmers, using high spatiotemporal resolution videomicroscopy. Further, we provide a special, time-dependent tomographic approach for the proof of rotational locomotion of periodically oscillating microswimmers, using the same data. Taken together, the methods describe part of an integrative approach to generate decisive information on three-dimensional in vivo motility from standard two-dimensional videomicroscopy data.
Asunto(s)
Microscopía Intravital/métodos , Microscopía por Video/métodos , Parasitología/métodos , Trypanosoma brucei brucei/fisiología , Flagelos/metabolismo , Imagenología Tridimensional , Microscopía Intravital/instrumentación , Estadios del Ciclo de Vida/fisiología , Microscopía por Video/instrumentación , Análisis Espacio-Temporal , Trypanosoma brucei brucei/citologíaRESUMEN
The recent introduction by Carl Zeiss Ltd. of the Airyscan detector module for their LSM880 confocal laser-scanning microscope has enabled routine superresolution microscopy to be combined with the advantages of confocal-based fluorescence imaging. Resulting enhanced spatial resolution in X, Y, and Z provides tractable opportunity to derive new insight into protein localization(s), organelle dynamics, and thence protein function within trypanosomatids or other organisms. Here, we describe methods for preparing slides, cells, and basic microscope setup for fluorescence imaging of trypanosomatids using the LSM-880 with Airyscan platform.