RESUMEN
Matrix MAPS provides an intuitive interface for acquiring light microscopy data during a correlative light and electron microscopy experiment either at room or cryogenic temperatures. First, the user graphically defines the geometry of the acquisition region on top of preview images. Multiple independent regions can then be imaged in an automated way, each with individual settings. The same interface allows the user to mark and select points or regions of interest whose coordinates can subsequently be transferred directly to the electron microscope.
Asunto(s)
Microscopía Electrónica/métodos , Microscopía Fluorescente/métodos , Programas InformáticosRESUMEN
Correlative light and electron microscopy allows features of interest defined by fluorescence signals to be located in an electron micrograph of the same sample. Rare dynamic events or specific objects can be identified, targeted and imaged by electron microscopy or tomography. To combine it with structural studies using cryo-electron microscopy or tomography, fluorescence microscopy must be performed while maintaining the specimen vitrified at liquid-nitrogen temperatures and in a dry environment during imaging and transfer. Here we present instrumentation, software and an experimental workflow that improves the ease of use, throughput and performance of correlated cryo-fluorescence and cryo-electron microscopy. The new cryo-stage incorporates a specially modified high-numerical aperture objective lens and provides a stable and clean imaging environment. It is combined with a transfer shuttle for contamination-free loading of the specimen. Optimized microscope control software allows automated acquisition of the entire specimen area by cryo-fluorescence microscopy. The software also facilitates direct transfer of the fluorescence image and associated coordinates to the cryo-electron microscope for subsequent fluorescence-guided automated imaging. Here we describe these technological developments and present a detailed workflow, which we applied for automated cryo-electron microscopy and tomography of various specimens.
Asunto(s)
Microscopía por Crioelectrón/instrumentación , Microscopía por Crioelectrón/métodos , Tomografía con Microscopio Electrónico/instrumentación , Tomografía con Microscopio Electrónico/métodos , Microscopía/instrumentación , Microscopía/métodos , Microscopía Fluorescente/instrumentación , Microscopía Fluorescente/métodos , Programas InformáticosRESUMEN
A high-content colocalization RNA interference screen based on automatic three-color confocal fluorescence microscopy was developed to analyze the alternative lengthening of telomeres (ALT) pathway. Via this pathway telomerase-negative cancer cells can maintain their telomeres and with it their unlimited proliferative potential. A hallmark of ALT cells is the colocalization of promyelocytic leukemia (PML) nuclear bodies with telomeres to form ALT-associated PML nuclear bodies (APBs). In our screen, the presence of APBs was used as a marker to identify proteins required for the ALT mechanism. A cell-based assay and an automatic confocal image acquisition procedure were established. Using automatic image analysis based on 3D parametric intensity models to identify APBs, we conducted an unbiased and quantitative analysis of nine different candidate genes. A comparison with the literature and manual analysis of the gene knockdown demonstrates the reliability of our approach. It extends the available repertoire of high-content screening to studies of cellular colocalizations and allows the identification of candidate genes for the ALT mechanism that represent possible targets for cancer therapy.
Asunto(s)
Imagenología Tridimensional/métodos , Microscopía Confocal/métodos , Microscopía Fluorescente/métodos , Interferencia de ARN , Telómero/genética , Telómero/metabolismo , Ciclo Celular/genética , Línea Celular Tumoral , Técnicas de Silenciamiento del Gen/métodos , Células HeLa , Humanos , Cuerpos de Inclusión Intranucleares/genética , Cuerpos de Inclusión Intranucleares/metabolismo , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Transducción de Señal/genética , Telomerasa/genética , Telomerasa/metabolismoRESUMEN
Quantitative microscopy relies on imaging of large cell numbers but is often hampered by time-consuming manual selection of specific cells. The 'Micropilot' software automatically detects cells of interest and launches complex imaging experiments including three-dimensional multicolor time-lapse or fluorescence recovery after photobleaching in live cells. In three independent experimental setups this allowed us to statistically analyze biological processes in detail and is thus a powerful tool for systems biology.
Asunto(s)
Recuperación de Fluorescencia tras Fotoblanqueo/métodos , Microscopía Fluorescente/métodos , Programas Informáticos , Biología de Sistemas/métodos , Homólogo de la Proteína Chromobox 5 , Proteínas Cromosómicas no Histona/análisis , Proteínas Fluorescentes Verdes/análisis , Células HeLa , HumanosRESUMEN
Despite our rapidly growing knowledge about the human genome, we do not know all of the genes required for some of the most basic functions of life. To start to fill this gap we developed a high-throughput phenotypic screening platform combining potent gene silencing by RNA interference, time-lapse microscopy and computational image processing. We carried out a genome-wide phenotypic profiling of each of the approximately 21,000 human protein-coding genes by two-day live imaging of fluorescently labelled chromosomes. Phenotypes were scored quantitatively by computational image processing, which allowed us to identify hundreds of human genes involved in diverse biological functions including cell division, migration and survival. As part of the Mitocheck consortium, this study provides an in-depth analysis of cell division phenotypes and makes the entire high-content data set available as a resource to the community.
