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

Optical microscopes use light sources to illuminate samples. The light passes through a series of lenses; the condenser focuses the light on the sample, while the objective and ocular lenses create a magnified and inverted two-dimensional image.
The numerical aperture of a lens determines its ability to gather light. The amount of light gathered depends on the angle of the light cone and the refractive index of the medium the light passes through, for example,&#160; air, water, or oil.
Optical microscopes have a resolution limit of 0.2 micrometers. They can resolve prokaryotic cells or large organelles within eukaryotic cells, which are around zero point five micrometers in diameter.&#160;
The resolution of the objective lens is inversely proportional to the wavelength of light and the lens&#39;s numerical aperture; the smaller the value, the higher the resolution of the magnified image.
An optical microscope can produce an image with a maximum of 1000 times magnification, calculated by multiplying the magnifications of the objective and ocular lenses.

beeldvorming van biologische monsters met optische microscopie

Imaging Biological Samples with Optical Microscopy

Optische microscopie maakt gebruik van optische principes om gedetailleerde beelden van monsters te verkrijgen. Antonie van Leeuwenhoek ontwierp in de 17e eeuw de eerste samengestelde optische microscoop om bloedcellen, bacteriën en gistcellen zichtbaar te maken. In 1830 creëerde Joseph Jackson Lister een in wezen moderne lichtmicroscoop. In de 20e eeuw zagen we de ontwikkeling van microscopen met verbeterde vergroting en resolutie.

Bij optische microscopie wordt het te bekijken preparaat op een glasplaatje geplaatst en op het podium (een platform) geklikt. Zodra het objectglaasje is vastgezet, wordt het preparaat op het objectglaasje over het licht geplaatst met behulp van de x-y mechanische podiumknoppen. Deze knoppen verplaatsen de schuif op het oppervlak van het podium, maar verhogen of verlagen het podium niet. Zodra het preparaat boven het licht is gecentreerd, kan de podiumpositie omhoog of omlaag worden gebracht om het beeld scherp te stellen. De grove scherpstelknop wordt gebruikt voor grootschalige bewegingen met 4⨯ en 10⨯ objectieflenzen; de fijne scherpstelknop wordt gebruikt voor kleinschalige bewegingen, vooral bij 40⨯ of 100⨯ objectieflenzen.

Wanneer afbeeldingen worden vergroot, worden ze zwakker omdat er minder licht is per oppervlakte-eenheid van de afbeelding. Microscopen produceren sterk vergrote beelden en vereisen daarom intensieve verlichting. In een helderveldmicroscoop zorgt een illuminator voor dit licht, meestal een lamp met hoge intensiteit onder het podium. Licht van de illuminator gaat door de condensorlens (die zich onder de tafel bevindt), die alle lichtstralen op het preparaat focust om de verlichting te maximaliseren. De positie van de condensor kan worden geoptimaliseerd met behulp van de bijgevoegde condensorfocusknop; zodra de optimale afstand is vastgesteld, mag de condensor niet meer worden verplaatst om de helderheid aan te passen. Als er minder dan maximale lichtniveaus nodig zijn, kan de hoeveelheid licht die op het preparaat valt eenvoudig worden aangepast door een diafragma tussen de condensor en het preparaat te openen of te sluiten. In sommige gevallen kan de helderheid ook worden aangepast met behulp van de reostaat, een dimmerschakelaar die de intensiteit van de verlichting regelt.

Een helderveldmicroscoop creëert een beeld door het licht van de illuminator op het preparaat te richten; dit licht wordt op verschillende manieren doorgelaten, geabsorbeerd, gereflecteerd of gebroken door verschillende structuren. De objectieflens vormt een reëel omgekeerd beeld van het preparaat. Het uiteindelijke beeld dat in het oculair of oculair wordt gevisualiseerd, is een vergroot virtueel beeld van het beeld dat door het objectief wordt gevormd.

Verschillende kleuren kunnen zich anders gedragen als ze interageren met chromoforen (pigmenten die bepaalde golflengten van licht absorberen en reflecteren) in delen van het monster. Vaak worden chromoforen kunstmatig aan het monster toegevoegd met behulp van vlekken, waardoor het contrast en de resolutie toenemen. Over het algemeen zullen structuren in het preparaat in verschillende mate donkerder lijken dan de heldere achtergrond, waardoor maximaal scherpe beelden ontstaan bij vergrotingen tot ongeveer 1000⨯. Verdere vergroting zou een groter beeld creëren, maar zonder verhoogde resolutie. Hierdoor kunnen we objecten zien die zo klein zijn als bacteriën, zichtbaar op ongeveer 400⨯ of zo, maar geen kleinere objecten zoals virussen.

Bij zeer hoge vergrotingen kan de resolutie in gevaar komen wanneer licht door de kleine hoeveelheid lucht tussen het preparaat en de lens valt. Dit komt door het significante verschil tussen de brekingsindices van lucht en glas; de lucht verstrooit de lichtstralen voordat de lens ze kan focusseren. Om dit probleem op te lossen, kan een druppel olie worden gebruikt om de ruimte tussen het monster en een olie-immersielens te vullen, een speciale lens die is ontworpen voor gebruik met immersie-oliën. Omdat de olie een brekingsindex heeft die sterk lijkt op die van glas, vergroot het de maximale hoek waaronder licht dat het monster verlaat de lens kan raken. Dit verhoogt het opgevangen licht en daarmee de resolutie van het beeld. Voor verschillende soorten licht kunnen verschillende oliën worden gebruikt.

Beeldvorming van biologische monsters met optische microscopie

Optical microscopy uses optic principles to provide detailed images of samples. Antonie van Leeuwenhoek designed the first compound optical microscope in ...

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

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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

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How Light Microscopes Bring Samples Into View

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