Name: fluorescent antibody staining in cells
Uploaded: 2023-04-30T11:59:57.000Z
Duration: 1 min 12 s
Channel: JoVE

In der Immunfluoreszenzmikroskopie emittieren Fluorophor-markierte Antikörper, wenn sie an ein bestimmtes Ziel oder ein Antigen binden.
Die Immunfluoreszenzmikroskopie nutzt Licht, um Elektronen des Fluorophors in einen höheren Energiezustand anzuregen. Wenn sie in den Grundzustand zurückkehren, geben die Elektronen eine längere Wellenlänge des Lichts ab.
Diese Emission oder Fluoreszenz ermöglicht die Visualisierung bestimmter Zellen innerhalb von Geweben oder bestimmter Proteine innerhalb von Zellen.
Die Immunfluoreszenz kann direkt sein, wenn der primäre Fluorophor-markierte Antikörper an das Zielprotein bindet. Oder es kann indirekt sein, indem Sekundärantikörper, die mit Fluorophoren markiert sind, einen spezifischen Primärantikörper binden, der an das gewünschte Protein gebunden ist. Die resultierende Fluoreszenz ist stärker als die der direkten Immunfluoreszenz.
Die direkte Immunfluoreszenz wird verwendet, um eine abnormale Proteinaggregation in Geweben nachzuweisen, während die indirekte Immunfluoreszenz die zirkulierenden Antikörper im Serum bei der Diagnose von Autoimmunerkrankungen nachweisen kann.

<ol>
	<li>Odell, I.D., und Cook, D., 2013. Immunfluoreszenz-Techniken. Das Journal für investigative Dermatologie, 133(1), S.e4.</li>
	<li>Sanderson, M.J., Smith, I., Parker, I. und Bootman, M.D., 2014. Fluoreszenzmikroskopie. Cold Spring Harbor Protocols, 2014(10), pp.pdb-top071795.</li>
</ol>

immunfluoreszenzmikroskopie

Immunofluorescence Microscopy

Ein Fluoreszenzmikroskop verwendet fluoreszierende Chromophore, sogenannte Fluorochrome, die Energie von einer Lichtquelle absorbieren und diese Energie dann als sichtbares Licht abgeben können. Fluorochrome umfassen natürlich fluoreszierende Substanzen (z. B. Chlorophylle) und fluoreszierende Farbstoffe, die der Probe hinzugefügt werden, um einen Kontrast zu erzeugen. FarbSubstanzenwie Texasrot und FITC sind Beispiele für Fluorochrome. Weitere Beispiele sind die NukleinsäurefarbSubstanzen4’,6’-Diamidino-2-phenylindol (DAPI) und Acridinorange.

Das Mikroskop bestrahlt die Probe mit kurzwelliger Anregung, beispielsweise ultraviolettem oder blauem Licht. Die Chromophore absorbieren das Anregungslicht und emittieren sichtbares Licht längerer Wellenlänge. Das Anregungslicht wird dann herausgefiltert (unter anderem weil ultraviolettes Licht für die Augen schädlich ist), sodass nur sichtbares Licht durch die Okularlinse gelangt und ein Bild der Probe in hellen Farben vor einem dunklen Hintergrund entsteht.

Fluoreszenzmikroskope können Krankheitserreger identifizieren, bestimmte Arten in einer Umgebung finden oder die Positionen bestimmter Moleküle und Strukturen innerhalb einer Zelle ermitteln. Es wurden auch Ansätze entwickelt, lebende von toten Zellen anhand der Aufnahme bestimmter Fluorochrome zu unterscheiden. Manchmal werden mehrere Fluorochrome auf derselben Probe verwendet, um unterschiedliche Strukturen oder Merkmale darzustellen.

Eine der wichtigsten Anwendungen der Fluoreszenzmikroskopie ist die Immunfluoreszenz, mit der bestimmte Mikroben identifiziert werden, indem beobachtet wird, ob Antikörper an sie binden. (Antikörper sind Proteinmoleküle, die das Immunsystem produziert und die sich an bestimmte Krankheitserreger binden, um diese abzutöten oder zu hemmen.) Bei dieser Technik gibt es zwei Ansätze: den direkten Immunfluoreszenztest (DFA) und den indirekten Immunfluoreszenztest (IFA). Bei der DFA werden spezifische Antikörper (z. B. solche, die gegen das Tollwutvirus gerichtet sind) mit einem Fluorochrom angefärbt. Wenn die Probe den Zielerreger enthält, kann man unter dem Fluoreszenzmikroskop beobachten, wie sich Antikörper an den Erreger binden. Hierbei handelt es sich um eine primäre Antikörperfärbung, da die gefärbten Antikörper direkt an den Erreger binden.

Bei der IFA werden Sekundärantikörper anstelle von Primärantikörpern mit einem Fluorochrom gefärbt. Sekundärantikörper heften sich nicht direkt an den Organismus, sondern binden an Primärantikörper. Wenn die ungefärbten Primärantikörper an den Erreger binden, kann beobachtet werden, dass die fluoreszierenden Sekundärantikörper an die Primärantikörper binden. Dadurch werden die Sekundärantikörper indirekt an den Erreger gebunden. Da sich häufig mehrere sekundäre Antikörper an einen primären Antikörper binden können, erhöht IFA die Anzahl der an der Probe haftenden fluoreszierenden Antikörper, wodurch ihre Merkmale leichter sichtbar gemacht werden können.

Dieser Text ist adaptiert von Openstax, Microbiology 2e, Section 2.4: Staining Microscopic Specimens.,

Immunfluoreszenzmikroskopie

Ein Fluoreszenzmikroskop verwendet fluoreszierende Chromophore, sogenannte Fluorochrome, die Energie von einer Lichtquelle absorbieren und diese Energie dann als ...

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

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 DAPI, which stands for 4',6'-diamidino-2-phenylindole, and acridine orange. The sample is exposed to short-wavelength light, such as ultraviolet or blue light. The fluorochromes absorb this excitation light and emit light at a longer wavelength.

The microscope then filters out the excitation light. This is done partly because ultraviolet light can harm the eyes. Only the visible light reaches the ocular lens. That is why the specimen appears in bright colors on a dark background. Fluorescence microscopy can help identify pathogens, locate particular species in an environment, and show where molecules or structures are found inside a cell.

Some fluorescence methods can also help tell living cells from dead cells. They do this by using fluorochromes that are taken up differently by the cells. In some specimens, more than one fluorochrome is used at the same time. This lets scientists show different structures or features in the same sample.

A major application of this technique is immunofluorescence. Immunofluorescence identifies certain microbes by checking whether antibodies bind to them. Antibodies are protein molecules made by the immune system that attach to specific pathogens and help kill or block them. The method has two forms: direct immunofluorescence assay, or DFA, and indirect immunofluorescence assay, or IFA.

In DFA, the specific antibodies are stained with a fluorochrome before they are added to the specimen. For example, antibodies that target the rabies virus can be used this way. If the pathogen is present, the stained antibodies bind directly to it under the fluorescent microscope. This is called a primary antibody stain because the stained antibodies attach straight to the pathogen.

In IFA, the primary antibodies are not stained. Instead, the secondary antibodies carry the fluorochrome and bind to the primary antibodies. After the unstained primary antibodies attach to the pathogen, the fluorescent secondary antibodies can be seen attached to them. This makes the pathogen appear indirectly labeled. Because several secondary antibodies can bind to one primary antibody, IFA often produces a stronger fluorescent signal and makes the specimen easier to see.

Fluorescent Antibody Staining in Cells

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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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immunofluorescence microscopy

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...

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Surface-Only Imaging with TIRF Microscopy

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key terms and concepts

Learn how fluorescence microscopes use fluorochromes and antibody staining to reveal pathogens, cells, and cell structures through direct and indirect immunofluorescence.

Fluorescent Antibody Staining in Cells | JoVE K12

cell biology

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Fluorescent Antibody Staining in Cells

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