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

Os microscópios ópticos usam fontes de luz para iluminar as amostras. A luz passa por uma série de lentes; O condensador focaliza a luz na amostra, enquanto as lentes objetivas e oculares criam uma imagem bidimensional ampliada e invertida.
A abertura numérica de uma lente determina sua capacidade de coletar luz. A quantidade de luz coletada depende do ângulo do cone de luz e do índice de refração do meio pelo qual a luz passa, por exemplo, ar, água ou óleo.
Os microscópios ópticos têm um limite de resolução de 0,2 micrômetros. Eles podem resolver células procarióticas ou grandes organelas dentro de células eucarióticas, que têm cerca de zero vírgula cinco micrômetros de diâmetro. 
A resolução da lente objetiva é inversamente proporcional ao comprimento de onda da luz e à abertura numérica da lente; quanto menor o valor, maior a resolução da imagem ampliada.
Um microscópio óptico pode produzir uma imagem com uma ampliação máxima de 1000 vezes, calculada multiplicando as ampliações da objetiva e das lentes oculares.

imagens de amostras biológicas com microscopia óptica

Imaging Biological Samples with Optical Microscopy

A microscopia óptica utiliza princípios ópticos para fornecer imagens detalhadas de amostras. Antonie van Leeuwenhoek projetou o primeiro microscópio óptico composto no século 17 para visualizar células sanguíneas, bactérias e células de levedura. Em 1830, Joseph Jackson Lister criou um microscópio óptico essencialmente moderno. O século 20 viu o desenvolvimento de microscópios com ampliação e resolução aprimoradas. Na microscopia óptica, a amostra a ser visualizada é colocada em uma lâmina de vidro e fixada na platina (uma plataforma). Depois que a lâmina estiver fixada, a amostra na lâmina é posicionada sobre a luz usando os botões mecânicos da platina x-y. Esses botões movem o slide na superfície do palco, mas não levantam ou abaixam o palco. Uma vez que a amostra esteja centralizada sobre a luz, a posição da platina pode ser elevada ou abaixada para focar a imagem. O botão de foco grosso é usado para movimentos em grande escala com lentes objetivas de 4⨯ e 10⨯; o botão de foco fino é usado para movimentos de pequena escala, especialmente com lentes objetivas de 40⨯ ou 100⨯. Quando as imagens são ampliadas, elas ficam mais escuras porque há menos luz por unidade de área da imagem. Os microscópios produzem imagens altamente ampliadas, portanto, requerem iluminação intensa. Em um microscópio de campo claro, um iluminador fornece essa luz, normalmente uma lâmpada de alta intensidade abaixo da platina. A luz do iluminador passa através da lente condensadora (localizada abaixo da platina), que concentra todos os raios de luz na amostra para maximizar a iluminação. A posição do condensador pode ser otimizada usando o botão de foco do condensador anexado; uma vez estabelecida a distância ideal, o condensador não deve ser movido para ajustar o brilho. Se forem necessários níveis de luz inferiores ao máximo, a quantidade de luz que atinge a amostra pode ser facilmente ajustada abrindo ou fechando um diafragma entre o condensador e a amostra. Em alguns casos, o brilho também pode ser ajustado usando o reostato, um dimmer que controla a intensidade do iluminador. Um microscópio de campo claro cria uma imagem direcionando a luz do iluminador para a amostra; esta luz é transmitida, absorvida, refletida ou refratada diferencialmente por diferentes estruturas. A lente objetiva forma uma imagem real invertida da amostra. A imagem final visualizada na ocular é uma imagem virtual ampliada daquela formada pela objetiva. Cores diferentes podem se comportar de maneira diferente à medida que interagem com cromóforos (pigmentos que absorvem e refletem comprimentos de onda específicos de luz) em partes da amostra. Frequentemente, os cromóforos são adicionados artificialmente à amostra por meio de manchas, aumentando o contraste e a resolução. Em geral, as estruturas na amostra parecerão mais escuras, em vários graus, do que o fundo brilhante, criando imagens extremamente nítidas em ampliações de até cerca de 1000⨯. Uma ampliação adicional criaria uma imagem maior, mas sem maior resolução. Isso nos permite ver objetos tão pequenos como bactérias, visíveis a cerca de 400⨯ ou mais, mas não objetos menores, como vírus. Em ampliações muito elevadas, a resolução pode ser comprometida quando a luz passa através da pequena quantidade de ar entre a amostra e a lente. Isto se deve à diferença significativa entre os índices de refração do ar e do vidro; o ar dispersa os raios de luz antes que a lente possa focalizá-los. Para resolver este problema, uma gota de óleo pode ser usada para preencher o espaço entre a amostra e uma lente de imersão em óleo, uma lente especial projetada para ser usada com óleos de imersão. Como o óleo tem um índice de refração muito semelhante ao do vidro, ele aumenta o ângulo máximo no qual a luz que sai da amostra pode atingir a lente. Isto aumenta a luz captada e, consequentemente, a resolução da imagem. Uma variedade de óleos pode ser usada para diferentes tipos de luz. Este texto é adaptado de Openstax, Microbiologia 2e, Seção 2.3: Instrumentos de Microscopia.

Imagens de amostras biológicas com microscopia óptica

A microscopia óptica usa princípios ópticos para fornecer imagens detalhadas das amostras. Antonie van Leeuwenhoek projetou o primeiro microscópio óptico composto em ...

imaging-biological-samples-with-optical-microscopy

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Cell Biology

Biologia Celular

Unit 1

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Visualizing Cells, Tissues, and Molecules

Visualizando Células, Tecidos e Moléculas

KEY TERMS AND CONCEPTS

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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

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...

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key terms and concepts

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

JoVE Core - Biologia Celular ilustra processos celulares complexos em vídeos de animação fáceis de entender e, ao mesmo tempo, abrangentes. Esta coleção foca nos componentes e funções essenciais das células, incluindo organelas e membranas, transporte celular e energia celular. É complementado por vídeos de Cientistas em ação que demonstram a aplicação da biologia celular em experimentos de pesquisa do mundo real.

How Light Microscopes Bring Samples Into View

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