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

Les microscopes optiques utilisent des sources lumineuses pour éclairer les échantillons. La lumière passe à travers une série de lentilles ; Le condenseur concentre la lumière sur l’échantillon, tandis que l’objectif et les lentilles oculaires créent une image bidimensionnelle agrandie et inversée.
L’ouverture numérique d’un objectif détermine sa capacité à recueillir la lumière. La quantité de lumière recueillie dépend de l’angle du cône lumineux et de l’indice de réfraction du milieu traversé par la lumière, par exemple l’air, l’eau ou l’huile.
Les microscopes optiques ont une limite de résolution de 0,2 micromètre. Ils peuvent résoudre des cellules procaryotes ou de grands organites dans des cellules eucaryotes, qui ont un diamètre d’environ zéro virgule cinq micromètres. 
La résolution de l'objectif est inversement proportionnelle à la longueur d'onde de la lumière et à l'ouverture numérique de l'objectif ; plus la valeur est petite, plus la résolution de l'image agrandie est élevée.
Un microscope optique peut produire une image avec un grossissement maximal de 1000 fois, calculé en multipliant les grossissements de l’objectif et des lentilles oculaires.

imagerie d'échantillons biologiques par microscopie optique

Imaging Biological Samples with Optical Microscopy

La microscopie optique utilise des principes optiques pour fournir des images détaillées des échantillons. Antonie van Leeuwenhoek a conçu le premier microscope optique composé au XVIIe siècle pour visualiser les cellules sanguines, les bactéries et les cellules de levure. En 1830, Joseph Jackson Lister créa un microscope optique essentiellement moderne. Le 20e siècle a vu le développement de microscopes dotés d’un grossissement et d’une résolution améliorés.

En microscopie optique, l'échantillon à visualiser est placé sur une lame de verre et clipsé sur la platine (une plateforme). Une fois la lame sécurisée, l’échantillon sur la lame est positionné au-dessus de la lumière à l’aide des boutons mécaniques x-y. Ces boutons déplacent la glissière sur la surface de la scène mais ne soulèvent pas ni n'abaissent pas la scène. Une fois l’échantillon centré sur la lumière, la position de la platine peut être élevée ou abaissée pour focaliser l’image. Le bouton de mise au point grossière est utilisé pour les mouvements à grande échelle avec des objectifs 4⨯ et 10⨯ ; le bouton de mise au point fine est utilisé pour les mouvements à petite échelle, notamment avec les objectifs 40⨯ ou 100⨯.

Lorsque les images sont agrandies, elles deviennent plus sombres car il y a moins de lumière par unité de surface de l'image. Les microscopes produisent des images très agrandies et nécessitent donc un éclairage intense. Dans un microscope à fond clair, un illuminateur fournit cette lumière, généralement une ampoule à haute intensité située sous la scène. La lumière provenant de l'illuminateur traverse la lentille du condenseur (située sous la scène), qui concentre tous les rayons lumineux sur l'échantillon pour maximiser l'éclairage. La position du condenseur peut être optimisée à l'aide du bouton de mise au point du condenseur ci-joint ; une fois la distance optimale établie, le condenseur ne doit plus être déplacé pour régler la luminosité. Si des niveaux de lumière inférieurs aux niveaux maximaux sont nécessaires, la quantité de lumière frappant l’échantillon peut être facilement ajustée en ouvrant ou en fermant un diaphragme entre le condenseur et l’échantillon. Dans certains cas, la luminosité peut également être réglée à l'aide du rhéostat, un variateur qui contrôle l'intensité de l'illuminateur.

Un microscope à fond clair crée une image en dirigeant la lumière de l'illuminateur vers le spécimen ; cette lumière est transmise, absorbée, réfléchie ou réfractée différemment par différentes structures. L'objectif forme une véritable image inversée de l'échantillon. L'image finale visualisée dans l'oculaire ou l'oculaire est une image virtuelle agrandie de celle formée par l'objectif.

Différentes couleurs peuvent se comporter différemment lorsqu'elles interagissent avec les chromophores (pigments qui absorbent et réfléchissent des longueurs d'onde particulières de la lumière) dans certaines parties du spécimen. Souvent, des chromophores sont ajoutés artificiellement à l’échantillon à l’aide de colorants, augmentant ainsi le contraste et la résolution. En général, les structures de l'échantillon apparaîtront plus sombres, à des degrés divers, que le fond clair, créant des images d'une netteté maximale à des grossissements allant jusqu'à environ 1 000⨯. Un grossissement supplémentaire créerait une image plus grande mais sans augmentation de la résolution. Cela nous permet de voir des objets aussi petits que des bactéries, visibles à environ 400°, mais pas des objets plus petits comme des virus.

À des grossissements très élevés, la résolution peut être compromise lorsque la lumière traverse la petite quantité d'air située entre l'échantillon et la lentille. Cela est dû à la différence significative entre les indices de réfraction de l'air et du verre ; l'air disperse les rayons lumineux avant que la lentille ne puisse les focaliser. Pour résoudre ce problème, une goutte d'huile peut être utilisée pour remplir l'espace entre l'échantillon et une lentille à immersion dans l'huile, une lentille spéciale conçue pour être utilisée avec des huiles à immersion. Étant donné que l’huile a un indice de réfraction très similaire à celui du verre, elle augmente l’angle maximum auquel la lumière sortant de l’échantillon peut frapper la lentille. Cela augmente la lumière collectée et donc la résolution de l’image. Une variété d’huiles peuvent être utilisées pour différents types de lumière.

Ce texte est adapté d'Openstax, Microbiologie 2e, Section 2.3 : Instruments de microscopie.

Imagerie D'échantillons Biologiques par Microscopie Optique

La microscopie optique utilise des principes optiques pour fournir des images détaillées des échantillons. Antonie van Leeuwenhoek a conçu le premier microscope optique composé en ...

imaging-biological-samples-with-optical-microscopy

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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

how-light-microscopes-bring-samples-into-view

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

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How Light Microscopes Bring Samples Into View

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