33.11
Descripción general de la microscopía electrónica
Las longitudes de onda de la luz visible limitan en última instancia la resolución teórica máxima de las imágenes creadas por los microscopios ópticos…
Los microscopios ópticos utilizan luz visible para iluminar un objeto, y sus lentes pueden ampliar una imagen hasta 2000 veces. En lugar de luz, los microscopios electrónicos utilizan un haz de electrones con una longitud de onda muy corta que permite ampliar los objetos de 2 a 50 millones de veces.
Un filamento emisor de electrones genera el haz de electrones, y las lentes electrostáticas y electromagnéticas enfocan el haz en una muestra. El entorno interno de un microscopio electrónico se mantiene bajo el vacío, ya que las moléculas de aire pueden desviar el haz de electrones.
Dependiendo de cómo interactúa el haz de electrones con la muestra, los microscopios electrónicos se pueden dividir en dos tipos: el microscopio electrónico de transmisión, o TEM, y el microscopio electrónico de barrido, o SEM.
En TEM, los electrones se transmiten a través de una muestra delgada y luego se usan para generar una imagen 2D. A medida que el haz de electrones pasa a través de la muestra, se puede obtener información sobre su composición interna y estructura utilizando TEM.
En SEM, el haz de electrones escanea la superficie de una muestra y, a continuación, los electrones reflejados se utilizan para generar información sobre la topografía 3D y la composición de la superficie.
View the full transcript and gain access to JoVE Core videos
Q1: How do electron microscopes achieve much higher magnification than light microscopes?
Electron microscopes use an electron beam with a wavelength of 0.005 nanometers instead of visible light, which has a wavelength of 500 nanometers. This shorter wavelength allows electron microscopes to magnify objects up to 2 to 50 million times, compared to light microscopes' maximum of 2,000 times. This superior magnification enables visualization of subcellular structures and some molecular structures like DNA strands.
Q2: What is the main difference between transmission and scanning electron microscopes?
Transmission electron microscopes (TEM) transmit electrons through an ultrathin sample to generate 2D images revealing internal composition and structure. Scanning electron microscopes (SEM) scan the sample's surface with an electron beam and use reflected electrons to create 3D topography information. TEM requires samples 20-100 nanometers thick, while SEM can image larger objects and surface details of smaller samples.
Q3: Why must electron microscopes operate in a vacuum environment?
Air molecules can deflect the electron beam, disrupting image formation and reducing resolution. Maintaining a vacuum inside the electron microscope ensures the electron beam travels unobstructed from the electron-emitting filament through the electromagnetic lenses to the sample. This controlled environment is essential for achieving the high magnification and resolution that electron microscopes provide.
Q4: How are samples prepared differently for transmission versus scanning electron microscopy?
TEM samples must be ultrathin (20-100 nanometers), dehydrated, and often stained with electron-dense heavy metals to enhance contrast. SEM samples are typically dried, treated with fixatives to reduce artifacts, and sputter-coated with a thin metal layer such as gold. These preparation methods reflect each technique's requirements: TEM needs thin sections for electron transmission, while SEM requires conductive surfaces for surface imaging.
Q5: What role do electromagnetic lenses play in electron microscopes?
Electromagnetic lenses focus the electron beam onto the sample, similar to how glass lenses focus light in optical microscopes. An electron-emitting filament generates the electron beam, which is then directed and concentrated by these magnetic lenses. This focusing capability is critical for achieving the precise magnification and resolution needed to visualize subcellular and molecular structures.
Q6: What information can be obtained from transmission electron microscopy images?
TEM images reveal internal composition and ultrastructure of samples by showing how electrons are transmitted through different regions. Varying opacity in different sample areas creates contrast in the 2D image, allowing visualization of organelles, membrane interiors, and molecular structures. Heavy metal staining enhances this contrast, making internal cellular details clearly distinguishable in the final image.
Q7: How does scanning electron microscopy create three-dimensional surface images?
SEM scans the sample's surface with an electron beam, which knocks electrons off the specimen. These reflected electrons are detected and used to generate detailed 3D surface topography information displayed on a monitor. This technique is particularly useful for examining surface features of larger objects like pollen grains or the detailed surfaces of very small samples.