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

En microscopie d’immunofluorescence, les anticorps marqués au fluorophore émettent une fluorescence lors de la liaison d’une cible spécifique ou d’un antigène.
La microscopie à immunofluorescence utilise la lumière pour exciter les électrons du fluorophore à un état d’énergie plus élevé. Lorsqu’ils reviennent à l’état fondamental, les électrons libèrent une longueur d’onde de lumière plus longue.
Cette émission ou fluorescence permet de visualiser des cellules spécifiques dans les tissus ou des protéines particulières dans les cellules.
L’immunofluorescence peut être directe lorsque l’anticorps primaire marqué au fluorophore se lie à la protéine cible. Ou, elle peut être indirecte, où des anticorps secondaires marqués avec des fluorophores se lient à un anticorps primaire spécifique attaché à la protéine d’intérêt. La fluorescence résultante est plus forte que celle émise par l’immunofluorescence directe.
L’immunofluorescence directe est utilisée pour détecter l’agrégation anormale des protéines dans les tissus, tandis que l’immunofluorescence indirecte peut détecter les anticorps circulants dans le sérum lors du diagnostic de maladies auto-immunes.

<ol>
	<li>Odell, I.D., et Cook, D., 2013. Techniques d’immunofluorescence. Le Journal de dermatologie d’investigation, 133(1), p.e4.</li>
	<li>Sanderson, M.J., Smith, I., Parker, I. et Bootman, M.D., 2014. Microscopie à fluorescence. Protocoles de Cold Spring Harbor, 2014(10), pp.pdb-top071795.</li>
</ol>

microscopie à immunofluorescence

Immunofluorescence Microscopy

Un microscope à fluorescence utilise des chromophores fluorescents appelés fluorochromes, qui peuvent absorber l'énergie d'une source lumineuse puis émettre cette énergie sous forme de lumière visible. Les fluorochromes comprennent des substances naturellement fluorescentes (telles que les chlorophylles) et des colorants fluorescents qui sont ajoutés à l'échantillon pour créer un contraste. Les colorants tels que le Texas red et le FITC sont des exemples de fluorochromes. D’autres exemples incluent les colorants d’acide nucléique 4’,6’-diamidino-2-phénylindole (DAPI) et l’acridine orange.

Le microscope irradie l’échantillon avec une excitation à courte longueur d’onde, telle que la lumière ultraviolette ou bleue. Les chromophores absorbent la lumière d'excitation et émettent de la lumière visible de longueurs d'onde plus longues. La lumière d'excitation est ensuite filtrée (en partie parce que la lumière ultraviolette est nocive pour les yeux) de sorte que seule la lumière visible passe à travers la lentille oculaire, produisant une image de l'échantillon dans des couleurs vives sur un fond sombre.

Les microscopes à fluorescence peuvent identifier des agents pathogènes, trouver des espèces particulières dans un environnement ou trouver l'emplacement de molécules et de structures particulières dans une cellule. Des approches ont également été développées pour distinguer les cellules vivantes des cellules mortes selon qu'elles absorbent ou non des fluorochromes particuliers. Parfois, plusieurs fluorochromes sont utilisés sur le même spécimen pour montrer différentes structures ou caractéristiques.

L’une des applications les plus importantes de la microscopie à fluorescence est l’immunofluorescence, qui permet d’identifier certains microbes en observant si les anticorps s’y lient. (Les anticorps sont des molécules protéiques produites par le système immunitaire qui s'attachent à des agents pathogènes spécifiques pour les tuer ou les inhiber.) Cette technique comporte deux approches : le test d'immunofluorescence directe (DFA) et le test d'immunofluorescence indirecte (IFA). Dans le DFA, des anticorps spécifiques (par exemple ceux qui ciblent le virus de la rage) sont colorés avec un fluorochrome. Si l’échantillon contient l’agent pathogène ciblé, on peut observer les anticorps se liant à l’agent pathogène au microscope à fluorescence. Il s’agit d’une coloration d’anticorps primaire car les anticorps colorés se fixent directement sur l’agent pathogène.

En IFA, les anticorps secondaires sont colorés avec un fluorochrome plutôt que des anticorps primaires. Les anticorps secondaires ne s'attachent pas directement à l'organisme mais se lient aux anticorps primaires. Lorsque les anticorps primaires non colorés se lient à l’agent pathogène, on peut observer que les anticorps secondaires fluorescents se lient aux anticorps primaires. Ainsi, les anticorps secondaires se fixent indirectement sur l’agent pathogène. Étant donné que plusieurs anticorps secondaires peuvent souvent s’attacher à un anticorps primaire, l’IFA augmente le nombre d’anticorps fluorescents attachés à l’échantillon, ce qui facilite la visualisation de ses caractéristiques.

Ce texte est adapté d'Openstax, Microbiologie 2e, Section 2.4 : Coloration des échantillons microscopiques.

Microscopie à Immunofluorescence

Un microscope à fluorescence utilise des chromophores fluorescents appelés fluorochromes, qui peuvent absorber l’énergie d’une source lumineuse, puis émettre cette énergie sous forme de ...

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Cell Biology

Biologie cellulaire

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Visualizing Cells, Tissues, and Molecules

Visualisation des cellules, des tissus et des molécules

KEY TERMS AND CONCEPTS

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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 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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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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