RESUMEN
Quantitative optical microscopy-an emerging, transformative approach to single-cell biology-has seen dramatic methodological advancements over the past few years. However, its impact has been hampered by challenges in the areas of data generation, management, and analysis. Here we outline these technical and cultural challenges and provide our perspective on the trajectory of this field, ushering in a new era of quantitative, data-driven microscopy. We also contrast it to the three decades of enormous advances in the field of genomics that have significantly enhanced the reproducibility and wider adoption of a plethora of genomic approaches.
Asunto(s)
Genómica/tendencias , Microscopía/tendencias , Imagen Óptica/tendencias , Análisis de la Célula Individual/tendencias , Animales , Difusión de Innovaciones , Genómica/historia , Ensayos Analíticos de Alto Rendimiento/tendencias , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Microscopía/historia , Imagen Óptica/historia , Reproducibilidad de los Resultados , Proyectos de Investigación/tendencias , Análisis de la Célula Individual/historiaRESUMEN
In 1980, Roger Tsien published a paper, in this journal [Tsien, R. Y. (1980) Biochemistry, 19 (11), 2396], titled "New calcium indicators and buffers with high selectivity against magnesium and protons: design, synthesis, and properties of prototype structures". These new buffers included 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, or BAPTA, which is still widely used today. And so, the world was set alight with new ways in which to visualize Ca2+. The ability to watch fluctuations in intracellular Ca2+ revolutionized the life sciences, although the fluorescent indicators used today, particularly in neurobiology, no longer rely exclusively on BAPTA but on genetically encoded fluorescent Ca2+ indicators. In this Perspective, we reflect on the origins of Ca2+ imaging with a special focus on the contributions made by Roger Tsien, from the early concept of selective Ca2+ binding described in Biochemistry to optical Ca2+ indicators based on chemically synthesized fluorophores to genetically encoded fluorescent Ca2+ indicators.
Asunto(s)
Calcio/metabolismo , Colorantes Fluorescentes/química , Microscopía Intravital/métodos , Imagen Óptica/métodos , Calcio/química , Ácido Egtácico/análogos & derivados , Ácido Egtácico/química , Historia del Siglo XX , Microscopía Intravital/historia , Imagen Óptica/historiaAsunto(s)
Imagen Molecular/métodos , Imagen Óptica/métodos , Animales , Transferencia Resonante de Energía de Fluorescencia/métodos , Colorantes Fluorescentes/química , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Imagen por Resonancia Magnética/historia , Imagen por Resonancia Magnética/métodos , Imagen Molecular/historia , Imagen Multimodal/historia , Imagen Multimodal/métodos , Neoplasias/diagnóstico por imagen , Premio Nobel , Imagen Óptica/historia , Tomografía de Emisión de Positrones/historia , Tomografía de Emisión de Positrones/métodos , Ultrasonografía/historia , Ultrasonografía/métodosAsunto(s)
Carcinoma/historia , Imagen Óptica/historia , Neoplasias de la Vejiga Urinaria/historia , Carcinoma/diagnóstico por imagen , Carcinoma/patología , Cistoscopía , Fluorescencia , Historia del Siglo XX , Humanos , Imagen Óptica/métodos , Fármacos Fotosensibilizantes , Tetraciclina , Rayos Ultravioleta , Neoplasias de la Vejiga Urinaria/diagnóstico por imagen , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
Light-sheet-based fluorescence microscopy features optical sectioning in the excitation process. This reduces phototoxicity and photobleaching by up to four orders of magnitude compared with that caused by confocal fluorescence microscopy, simplifies segmentation and quantification for three-dimensional cell biology, and supports the transition from on-demand to systematic data acquisition in developmental biology applications.
Asunto(s)
Procesamiento de Imagen Asistido por Computador/métodos , Procesamiento de Imagen Asistido por Computador/tendencias , Microscopía Fluorescente/métodos , Imagen Óptica/métodos , Animales , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Procesamiento de Imagen Asistido por Computador/historia , Microscopía Fluorescente/historia , Microscopía Fluorescente/tendencias , Imagen Óptica/historia , Imagen Óptica/tendenciasRESUMEN
Electron cryotomography (ECT) provides three-dimensional views of macromolecular complexes inside cells in a native frozen-hydrated state. Over the last two decades, ECT has revealed the ultrastructure of cells in unprecedented detail. It has also allowed us to visualize the structures of macromolecular machines in their native context inside intact cells. In many cases, such machines cannot be purified intact for in vitro study. In other cases, the function of a structure is lost outside the cell, so that the mechanism can be understood only by observation in situ. In this review, we describe the technique and its history and provide examples of its power when applied to cell biology. We also discuss the integration of ECT with other techniques, including lower-resolution fluorescence imaging and higher-resolution atomic structure determination, to cover the full scale of cellular processes.
Asunto(s)
Microscopía por Crioelectrón/métodos , Tomografía con Microscopio Electrónico/métodos , Fimbrias Bacterianas/ultraestructura , Poro Nuclear/química , Imagen Óptica/métodos , Células Procariotas/ultraestructura , Archaea/metabolismo , Archaea/ultraestructura , Bacterias/metabolismo , Bacterias/ultraestructura , Sistemas de Secreción Bacterianos/metabolismo , Sistemas de Secreción Bacterianos/ultraestructura , Microscopía por Crioelectrón/historia , Microscopía por Crioelectrón/instrumentación , Tomografía con Microscopio Electrónico/historia , Tomografía con Microscopio Electrónico/instrumentación , Fimbrias Bacterianas/metabolismo , Flagelos/metabolismo , Flagelos/ultraestructura , Historia del Siglo XX , Historia del Siglo XXI , Modelos Moleculares , Poro Nuclear/metabolismo , Poro Nuclear/ultraestructura , Imagen Óptica/historia , Imagen Óptica/instrumentación , Células Procariotas/metabolismo , Dominios Proteicos , Estructura Secundaria de ProteínaRESUMEN
In the more than 100 years since its discovery, our knowledge of Toxoplasma biology has improved enormously. The evolution of molecular biology, immunology and genomics has had profound influences on our understanding of this ubiquitous bug. However, it could be argued that in science today the adage "seeing is believing" has never been truer. Images are highly influential and in the time since the first description of T. gondii, advances in microscopy and imaging technology have been and continue to be dramatic. In this review we recount the discovery of T. gondii and the contribution of imaging techniques to elucidating its life cycle, biology and the immune response of its host.
Asunto(s)
Imagen Óptica/métodos , Imagen Óptica/tendencias , Toxoplasma/citología , Historia del Siglo XX , Historia del Siglo XXI , Procesamiento de Imagen Asistido por Computador , Microscopía , Imagen Óptica/historia , Toxoplasma/fisiologíaRESUMEN
Optical imaging has a long history in physiology and in neurophysiology in particular. Over the past 15 years or so, new methodologies have emerged that combine genetic engineering with light-based imaging methods. This merger has resulted in a tool box of genetically encoded optical indicators that enable nondestructive and long-lasting monitoring of neuronal activities at the cellular, circuit, and system level. This review describes the historical roots and fundamental concepts underlying these new approaches, evaluates current progress in this field, and concludes with a forward-looking perspective on current work and potential future developments in this field.