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

Optische Mikroskope verwenden Lichtquellen, um Proben zu beleuchten. Das Licht dringt durch eine Reihe von Linsen; Der Kondensor fokussiert das Licht auf die Probe, während das Objektiv und die Okularlinse ein vergrößertes und invertiertes zweidimensionales Bild erzeugen.
Die numerische Apertur eines Objektivs bestimmt seine Fähigkeit, Licht zu sammeln. Die Menge des gesammelten Lichts hängt vom Winkel des Lichtkegels und dem Brechungsindex des Mediums ab, durch das das Licht fällt, z. B. Luft, Wasser oder Öl.
Optische Mikroskope haben eine Auflösungsgrenze von 0,2 Mikrometern. Sie können prokaryotische Zellen oder große Organellen innerhalb eukaryotischer Zellen auflösen, die einen Durchmesser von etwa null Komma und fünf Mikrometern haben. 
Die Auflösung des Objektivs ist umgekehrt proportional zur Wellenlänge des Lichts und der numerischen Apertur des Objektivs; je kleiner der Wert, desto höher die Auflösung des vergrößerten Bildes.
Ein optisches Mikroskop kann ein Bild mit maximal 1000-facher Vergrößerung erzeugen, berechnet durch Multiplikation der Vergrößerungen der Objektiv- und Okularlinsen.

abbildung biologischer proben mit optischer mikroskopie

Imaging Biological Samples with Optical Microscopy

Die optische Mikroskopie nutzt optische Prinzipien, um detaillierte Bilder von Proben zu liefern. Antonie van Leeuwenhoek entwarf im 17. Jahrhundert das erste zusammengesetzte optische Mikroskop zur Visualisierung von Blutzellen, Bakterien und Hefezellen. Im Jahr 1830 entwickelte Joseph Jackson Lister ein im Wesentlichen modernes Lichtmikroskop. Im 20. Jahrhundert wurden Mikroskope mit verbesserter Vergrößerung und Auflösung entwickelt.

Bei der optischen Mikroskopie wird die zu betrachtende Probe auf einen Glasobjektträger gelegt und auf dem Tisch (einer Plattform) befestigt. Sobald der Objektträger befestigt ist, wird die Probe auf dem Objektträger mithilfe der x-y-Knöpfe des mechanischen Tisches über dem Licht positioniert. Diese Knöpfe bewegen den Schlitten auf der Oberfläche der Bühne, heben oder senken die Bühne jedoch nicht. Sobald die Probe über dem Licht zentriert ist, kann die Tischposition angehoben oder abgesenkt werden, um das Bild zu fokussieren. Der Grobfokussierungsknopf wird für großflächige Bewegungen mit 4⨯- und 10⨯-Objektiven verwendet; Der Feinfokussierungsknopf wird für kleine Bewegungen verwendet, insbesondere bei 40⨯- oder 100⨯-Objektiven.

Wenn Bilder vergrößert werden, werden sie dunkler, da pro Flächeneinheit des Bildes weniger Licht vorhanden ist. Mikroskope erzeugen stark vergrößerte Bilder und erfordern daher eine intensive Beleuchtung. In einem Hellfeldmikroskop liefert eine Beleuchtungseinrichtung dieses Licht, typischerweise eine Hochleistungslampe unter dem Objekttisch. Das Licht des Illuminators gelangt durch die Kondensorlinse (unterhalb des Tisches), die alle Lichtstrahlen auf die Probe fokussiert, um die Beleuchtung zu maximieren. Die Position des Kondensors kann mit dem angebrachten Kondensor-Fokusknopf optimiert werden; Sobald der optimale Abstand erreicht ist, sollte der Kondensor nicht mehr bewegt werden, um die Helligkeit anzupassen. Wenn weniger als die maximale Lichtmenge benötigt wird, kann die auf die Probe treffende Lichtmenge einfach durch Öffnen oder Schließen einer Blende zwischen dem Kondensor und der Probe angepasst werden. In manchen Fällen lässt sich die Helligkeit auch mit dem Rheostat regulieren, einem Dimmerschalter, der die Intensität der Beleuchtung steuert.

Ein Hellfeldmikroskop erzeugt ein Bild, indem es Licht von der Beleuchtung auf die Probe richtet; Dieses Licht wird von verschiedenen Strukturen unterschiedlich durchgelassen, absorbiert, reflektiert oder gebrochen. Die Objektivlinse erzeugt ein reales invertiertes Bild der Probe. Das im Okular oder Okular sichtbare endgültige Bild ist ein vergrößertes virtuelles Bild des vom Objektiv erzeugten Bildes.

Verschiedene Farben können sich unterschiedlich verhalten, da sie in Teilen der Probe mit Chromophoren (Pigmenten, die bestimmte Wellenlängen des Lichts absorbieren und reflektieren) interagieren. Oftmals werden der Probe durch Färben Chromophore künstlich hinzugefügt, um den Kontrast und die Auflösung zu erhöhen. Im Allgemeinen erscheinen Strukturen in der Probe in unterschiedlichem Maße dunkler als der helle Hintergrund, wodurch maximal scharfe Bilder bei Vergrößerungen bis zu etwa 1000⨯ entstehen. Eine weitere Vergrößerung würde ein größeres Bild erzeugen, jedoch ohne höhere Auflösung. Dies ermöglicht es uns, so kleine Objekte wie Bakterien zu sehen, die in etwa 400⨯ sichtbar sind, aber keine kleineren Objekte wie Viren.

Bei sehr hohen Vergrößerungen kann die Auflösung beeinträchtigt werden, wenn Licht durch die kleine Luftmenge zwischen Probe und Objektiv dringt. Dies ist auf den erheblichen Unterschied zwischen den Brechungsindizes von Luft und Glas zurückzuführen; Die Luft streut die Lichtstrahlen, bevor die Linse sie fokussieren kann. Um dieses Problem zu lösen, kann ein Tropfen Öl verwendet werden, um den Raum zwischen der Probe und einer Ölimmersionslinse zu füllen, einer speziellen Linse, die für die Verwendung mit Immersionsölen entwickelt wurde. Da das Öl einen Brechungsindex hat, der dem von Glas sehr ähnlich ist, erhöht es den maximalen Winkel, in dem Licht, das die Probe verlässt, auf die Linse treffen kann. Dies erhöht das gesammelte Licht und damit die Auflösung des Bildes. Für unterschiedliche Lichtarten können verschiedene Öle verwendet werden.

This text is adapted from Openstax, Microbiology 2e, Section 2.3: Instruments of Microscopy.

Abbildung biologischer Proben mit optischer Mikroskopie

Die optische Mikroskopie nutzt optische Prinzipien, um detaillierte Bilder von Proben zu liefern. Antonie van Leeuwenhoek entwarf das erste zusammengesetzte optische Mikroskop in ...

imaging-biological-samples-with-optical-microscopy

Education

Lehre

JoVE Core

Cell Biology

Zellbiologie

Unit 1

Einheit 1

Visualizing Cells, Tissues, and Molecules

Visualisierung von Zellen, Geweben und Molekülen

KEY TERMS AND CONCEPTS

KERNBEGRIFFE UND KONZEPTE

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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Microscope Contrast for Live, Unstained Cells

Phase-contrast and differential interference contrast microscopy help scientists see live cells without stains. Both methods boost contrast so thin, transparent structures become easier to study under the microscope. Phase-contrast microscopes use interference between light that passes straight through a cell and light that is refracted by cell parts. The microscope uses an annular stop in the condenser to make a hollow cone of light. The objective lens also contains a phase plate with a phase...

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Microscope slide preparation includes wet mounts, fixation, and staining. These steps help make specimens easier to see under a light microscope. They also help preserve cells and highlight important structures. A wet mount is the simplest slide preparation. The specimen is placed in a drop of liquid on the slide. A liquid sample can be added directly with a dropper, and a solid sample, such as a skin scraping, can be placed on the slide before the liquid is added. The liquid is sometimes...

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Fluorescent Antibody Staining in Cells

Fluorescent antibody staining uses a fluorescence microscope to make cells and microbes visible in bright colors against a dark background. The microscope works with fluorochromes, which are fluorescent dyes that absorb energy from a light source and then give off visible light. Some fluorochromes are natural, such as chlorophylls. Others are added to the specimen to improve contrast. Common examples of fluorochromes include Texas red and FITC. The method can also use nucleic acid dyes such as...

immunocytochemistry and immunohistochemistry

immunocytochemistry-and-immunohistochemistry

Antibody Staining in Cells and Tissues

Immunocytochemistry (ICC) and immunohistochemistry (IHC) use antibodies to detect specific proteins or antigens in samples. ICC focuses on individual cells, such as blood cells and stem cells. IHC is used for tissue samples. These methods were first published by Albert Coons in 1941, when they were used to detect pneumococcal antigen in mouse tissue sections from mice infected with Pneumococcus. Both techniques can produce either a colored signal or a fluorescent signal. Enzyme-conjugated...

confocal fluorescence microscopy

Confocal Microscopy for Sharp Cell Images

Confocal microscopy is an advanced imaging method used to capture sharp fluorescence images. Its main advantage is that it blocks out-of-focus light from the sample with pinholes. That helps produce high-resolution images with strong contrast. Unlike a standard optical microscope, a confocal microscope uses a focused laser beam to scan the sample surface at different z-planes. This makes it especially useful for thick specimens, such as biofilms. These samples can often be examined alive and...

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Fluorescent Methods for Protein Movement

Protein movement in living cells can be measured with fluorescence-based methods such as FRAP, FRET, and PET. These techniques help scientists track how proteins move, interact, and change distance inside the cell. Fluorescent recovery after photobleaching, or FRAP, measures protein movement rates within the cell. In this method, a small region is exposed to a strong laser beam. The beam permanently bleaches fluorophore-tagged proteins in that area. Over time, bleached proteins diffuse away...

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Surface-Only Imaging with TIRF Microscopy

Total internal reflection fluorescence microscopy, or TIRF, is a microscope method used to view fluorophores near a solid surface with a higher refractive index, such as a glass coverslip. It is useful when scientists want to study signals close to the cell surface. TIRF works when light from a medium with a lower refractive index, such as air, hits the glass coverslip at a critical angle. Instead of entering the glass, the light undergoes total internal reflection. This reflection creates an...

atomic force microscopy

Atomic Force Microscopy for Surface Mapping

Atomic force microscopy is used to map the surface details of materials. It can examine ceramics, glass, polymers, and biological samples. The method gives far more resolution than an optical imaging system and produces three-dimensional surface profiles instead of flat 2D images. The probe is the key part of an AFM setup. It includes the cantilever and tip assembly, and it is also the part that is replaced most often. Constant contact with samples wears down the tip, so probe choice depends...

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Sharper Cell Images with Fluorescence Microscopy

Super-resolution fluorescence microscopy gives sharper images than standard fluorescence microscopy by reducing the point spread function, or PSF. The PSF is the light pattern from a point source that makes it look blurred. Because of this blur, each fluorescent spot appears larger than it really is. Nearby fluorophores can also overlap through their PSFs and make the image less clear. Several super-resolution methods have been developed to improve image detail. Photoactivated localization...

overview of electron microscopy

Electron Microscopy: TEM vs SEM

Electron microscopy uses electrons instead of visible light to make images with much higher detail than a light microscope. Visible light limits the resolution of light microscopes, which usually magnify up to 1000X, and sometimes up to 1500X. Electrons have a much shorter wavelength, about 0.005 nm, so they can produce resolution as fine as 0.05 nm. An electron microscope can also magnify images up to 2,000,000X and reveal subcellular structures and some molecular structures, including single...

scanning electron microscopy

How SEM Reveals Surface Details

A scanning electron microscope (SEM) reveals the surface details of a sample with an electron beam. The beam scans across the sample in a two-dimensional pattern. SEM can image areas from about 1 centimeter down to 5 micrometers wide. SEM is useful for studying bacteria, viruses, tissues, and larger samples such as insects. Conventional SEM can produce magnification from 20X to 30,000X. It also offers spatial resolution of about 50 to 100 nanometers. The process starts when the electron gun...

transmission electron microscopy

How TEM Reveals Tiny Structures

Transmission electron microscopy uses electrons to reveal very small structures inside a specimen. The technique grew from the first electron microscope prototype developed in 1931 by physicist Ernst Ruska. He built on the idea that magnetic fields can direct an electron beam, just as lenses direct light in an optical microscope. In a transmission electron microscope, a hot tungsten element produces electrons. An electron gun then accelerates them with a potential difference, giving the beam...

preparation of samples for electron microscopy

preparation-of-samples-for-electron-microscopy

Electron Microscopy Sample Prep Methods

Electron microscopy sample prep methods help biological samples survive imaging in TEM and SEM. Before a specimen can be viewed, it must be fixed, or stabilized, so the electron beam does not damage it. It also must be dried well, or desiccated, because the microscope uses a vacuum. Fixation should happen as quickly as possible. Once a sample leaves its natural environment, its properties begin to change. In tissue samples, oxygen levels can drop and change the appearance of mitochondria. That...

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Immunogold Labeling in Electron Microscopy

Immunogold labeling in electron microscopy uses antibodies and tiny gold particles to find specific proteins in cells and tissues. The gold particles are very electron-dense, which means they show up clearly in electron microscope images. This gives researchers a high-resolution way to see where proteins are located and to compare their amounts under different stimulation conditions. The method can reveal proteins on the cell surface and inside cells. It has been used to study proteins...

cryo-electron microscopy

Cryo-EM for Native Biomolecule Imaging

Cryo-EM, or cryo-electron microscopy, lets scientists image biomolecules in their frozen, native state. It avoids the dehydration, fixation, and staining used in conventional electron microscopy, which can distort samples and create artifacts. It also reduces damage from the electron beam by using gentler imaging conditions. This method is useful when other structure tools have limits. X-ray diffraction needs a molecule to form a crystal, and that is not always possible. Even when...

electron microscope tomography and single-particle reconstruction

electron-microscope-tomography-and-single-particle-reconstruction

3D Imaging with Electron Microscopy

Transmission electron microscopy (TEM) can build 3D images of biological samples. It does this with electron microscope tomography and single-particle reconstruction. Tomography can show cell parts or small cells in vivo, while single-particle reconstruction is used for macromolecules and macromolecular complexes in vitro only. Electron tomography can be done in TEM or in STEM, which stands for scanning transmission electron microscopy. STEM is mainly used for thick biological specimens. It...

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 Zellbiologie veranschaulicht komplexe zelluläre Prozesse in leicht verständlichen und dennoch umfassenden animierten Videolektionen. Diese Sammlung konzentriert sich auf die wesentlichen Bestandteile und Funktionen von Zellen, einschließlich Organellen und Membranen, Zelltransport und Zellenergie. Ergänzt wird sie durch Videos von Wissenschaftlern in Aktion, die die Anwendung der Zellbiologie in realen Forschungsexperimenten demonstrieren.

How Light Microscopes Bring Samples Into View

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