Name: how light microscopes bring samples into view
Uploaded: 2023-04-30T11:59:33.000Z
Duration: 1 min 18 s
Channel: JoVE

Los microscopios ópticos utilizan fuentes de luz para iluminar las muestras. La luz pasa a través de una serie de lentes; El condensador enfoca la luz en la muestra, mientras que el objetivo y las lentes oculares crean una imagen bidimensional ampliada e invertida.
La apertura numérica de una lente determina su capacidad para recoger la luz. La cantidad de luz recogida depende del ángulo del cono de luz y del índice de refracción del medio que pasa la luz, por ejemplo, aire, agua o aceite.
Los microscopios ópticos tienen un límite de resolución de 0,2 micrómetros. Pueden resolver células procariotas o grandes orgánulos dentro de las células eucariotas, que tienen alrededor de cero punto cinco micrómetros de diámetro. 
La resolución de la lente del objetivo es inversamente proporcional a la longitud de onda de la luz y a la apertura numérica de la lente; cuanto menor sea el valor, mayor será la resolución de la imagen ampliada.
Un microscopio óptico puede producir una imagen con un máximo de 1000 aumentos, calculado multiplicando los aumentos del objetivo y las lentes oculares.

imágenes de muestras biológicas con microscopía óptica

Imaging Biological Samples with Optical Microscopy

La microscopía óptica utiliza principios ópticos para proporcionar imágenes detalladas de las muestras. Antonie van Leeuwenhoek diseñó el primer microscopio óptico compuesto en el siglo XVII para visualizar células sanguíneas, bacterias y levaduras. En 1830, Joseph Jackson Lister creó un microscopio óptico esencialmente moderno. El siglo XX vio el desarrollo de microscopios con mayor aumento y resolución.

En microscopía óptica, la muestra que se va a observar se coloca en un portaobjetos de vidrio y se sujeta al escenario (una plataforma). Una vez que el portaobjetos está asegurado, la muestra en el portaobjetos se coloca sobre la luz usando las perillas mecánicas x-y. Estas perillas mueven la corredera sobre la superficie del escenario pero no suben ni bajan el escenario. Una vez que la muestra está centrada sobre la luz, la posición del escenario se puede subir o bajar para enfocar la imagen. La perilla de enfoque aproximado se utiliza para movimientos a gran escala con lentes objetivos de 4⨯ y 10⨯; La perilla de enfoque fino se utiliza para movimientos de pequeña escala, especialmente con lentes objetivos de 40⨯ o 100⨯.

Cuando las imágenes se amplían, se vuelven más oscuras porque hay menos luz por unidad de área de la imagen. Los microscopios producen imágenes muy ampliadas, por lo que requieren una iluminación intensa. En un microscopio de campo claro, un iluminador proporciona esta luz, generalmente una bombilla de alta intensidad debajo del escenario. La luz del iluminador pasa a través de la lente del condensador (ubicada debajo del escenario), que enfoca todos los rayos de luz en la muestra para maximizar la iluminación. La posición del condensador se puede optimizar utilizando la perilla de enfoque del condensador adjunta; Una vez establecida la distancia óptima, no se debe mover el condensador para ajustar el brillo. Si se necesitan niveles de luz inferiores a los máximos, la cantidad de luz que incide sobre la muestra se puede ajustar fácilmente abriendo o cerrando un diafragma entre el condensador y la muestra. En algunos casos, el brillo también se puede ajustar mediante el reóstato, un regulador de intensidad que controla la intensidad del iluminador.

Un microscopio de campo claro crea una imagen dirigiendo la luz del iluminador hacia la muestra; esta luz se transmite, absorbe, refleja o refracta de manera diferencial por diferentes estructuras. La lente del objetivo forma una imagen real invertida de la muestra. La imagen final visualizada en el ocular u ocular es una imagen virtual ampliada de la formada por el objetivo.

Los diferentes colores pueden comportarse de manera diferente al interactuar con los cromóforos (pigmentos que absorben y reflejan determinadas longitudes de onda de luz) en partes de la muestra. A menudo, los cromóforos se añaden artificialmente a la muestra mediante tinciones, lo que aumenta el contraste y la resolución. En general, las estructuras de la muestra aparecerán más oscuras, en diversos grados, que el fondo brillante, creando imágenes de máxima nitidez con aumentos de hasta aproximadamente 1000⨯. Una mayor ampliación crearía una imagen más grande pero sin mayor resolución. Esto nos permite ver objetos tan pequeños como bacterias, visibles a unos 400° aproximadamente, pero no objetos más pequeños como los virus.

A aumentos muy altos, la resolución puede verse comprometida cuando la luz pasa a través de la pequeña cantidad de aire entre la muestra y la lente. Esto se debe a la diferencia significativa entre los índices de refracción del aire y del vidrio; el aire dispersa los rayos de luz antes de que la lente pueda enfocarlos. Para resolver este problema, se puede usar una gota de aceite para llenar el espacio entre la muestra y una lente de inmersión en aceite, una lente especial diseñada para usarse con aceites de inmersión. Dado que el aceite tiene un índice de refracción muy similar al del vidrio, aumenta el ángulo máximo en el que la luz que sale de la muestra puede incidir en la lente. Esto aumenta la luz captada y, por tanto, la resolución de la imagen. Se puede utilizar una variedad de aceites para diferentes tipos de luz.

Imágenes de muestras biológicas con microscopía óptica

La microscopía óptica utiliza principios ópticos para proporcionar imágenes detalladas de las muestras. Antonie van Leeuwenhoek diseñó el primer microscopio óptico compuesto ...

imaging-biological-samples-with-optical-microscopy

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JoVE Core

Cell Biology

Biología Celular

Unit 1

Unidad 1

Visualizing Cells, Tissues, and Molecules

Visualización de células, tejidos y moléculas

KEY TERMS AND CONCEPTS

TÉRMINOS Y CONCEPTOS CLAVE

how light microscopes bring samples into view

Optical microscopy uses light and lenses to make tiny biological samples visible. Antonie van Leeuwenhoek built the first compound optical microscope in the 17th century to view blood cells, bacteria, and yeast cells. In 1830, Joseph Jackson Lister developed an essentially modern light microscope. During the 20th century, microscopes gained better magnification and resolution.

A sample is placed on a glass slide and clipped onto the stage, which is the flat platform of the microscope. The slide is then moved over the light with the x-y mechanical stage knobs. These knobs move the slide across the stage but do not lift it. After the specimen is centered over the light, the stage is raised or lowered to bring the image into focus.

The coarse focus knob is used for large adjustments with 4x and 10x objective lenses. The fine focus knob makes small adjustments, especially with 40x and 100x lenses. As images become more magnified, they also become dimmer because less light reaches each part of the image. For that reason, microscopes need strong lighting.

In a brightfield microscope, a high-intensity bulb below the stage acts as the illuminator. Light from the illuminator passes through the condenser lens, which focuses the light onto the specimen. The condenser can be set with the condenser focus knob. After the best distance is found, the condenser should not be moved just to change brightness. If less light is needed, the diaphragm between the condenser and the specimen can be opened or closed. In some microscopes, the rheostat, or dimmer switch, also changes the illuminator intensity.

A brightfield microscope forms an image because the specimen transmits, absorbs, reflects, or refracts light in different ways. The objective lens makes a real inverted image. The eyepiece, also called the ocular, then shows a magnified virtual image of that picture. Chromophores, which are pigments that absorb and reflect certain wavelengths of light, can affect how colors appear. Stains are often added to increase contrast and resolution. This makes structures in the sample appear darker than the bright background and allows sharp images at magnifications up to about 1000x.

Higher magnification does not keep improving resolution. It can make the image larger without making it clearer. This is why bacteria can be seen at about 400x, but smaller objects such as viruses cannot. At very high magnifications, resolution can drop because light passes through the thin air gap between the specimen and the lens. Air and glass have very different refractive indices, so air scatters light before it can be focused.

Oil immersion solves this problem. A drop of oil fills the space between the specimen and an oil immersion lens, which is designed to work with immersion oils. Because the oil has a refractive index very close to glass, it lets light leave the specimen at a wider angle and reach the lens more easily. This increases the amount of light collected and improves image resolution. Different oils can be used for different types of light.

How Light Microscopes Bring Samples Into View

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imaging biological samples with optical microscopy

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Learn how optical and brightfield microscopes use light, lenses, stains, and oil immersion to view cells, bacteria, and other tiny samples clearly.

How Light Microscopes Bring Samples Into View | JoVE K12

cell biology

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How Light Microscopes Bring Samples Into View

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