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

במיקרוסקופ אימונופלואורסצנטי, נוגדנים המסומנים בפלואורופור פולטים פלואורסצנטיות בעת קשירת מטרה מסוימת או אנטיגן.
מיקרוסקופ אימונופלואורסצנטי משתמש באור כדי לעורר אלקטרונים של הפלואורופור למצב אנרגיה גבוה יותר. כאשר הם חוזרים למצב הקרקע, האלקטרונים משחררים אורך גל ארוך יותר של אור.
פליטה או פלואורסצנטיות זו מאפשרת הדמיה של תאים ספציפיים בתוך רקמות או חלבונים מסוימים בתוך תאים.
אימונופלואורסנציה יכולה להיות ישירה כאשר הנוגדן העיקרי המתויג פלואורופור נקשר לחלבון המטרה. לחלופין, זה יכול להיות עקיף, שבו נוגדנים משניים המתויגים עם פלואורופורים קושרים נוגדן ראשוני ספציפי המחובר לחלבון המעניין. הפלואורסצנטיות המתקבלת חזקה יותר מזו הנפלטת מאימונופלואורסנציה ישירה.
אימונופלואורסנציה ישירה משמשת לזיהוי צבירת חלבונים חריגה ברקמות, בעוד שאימונופלואורסנציה עקיפה יכולה לזהות את הנוגדנים במחזור בסרום במהלך אבחון מחלות אוטואימוניות.

<ol>
	<li>אודל, א.ד. וקוק, ד., 2013. טכניקות immunofluorescence. כתב העת לדרמטולוגיה חוקרת, 133 (1), עמ 'ה4.</li>
	<li>סנדרסון, מ.ג., סמית, א., פרקר, א. ובוטמן, מ.ד., 2014. מיקרוסקופ פלואורסצנטי. פרוטוקולי קולד ספרינג הארבור, 2014(10), pp.pdb-top071795.</li>
</ol>

מיקרוסקופיה אימונופלואורסצנטית

Immunofluorescence Microscopy

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מיקרוסקופ פלואורסצנטי משתמש בכרומופורים פלואורסצנטיים הנקראים פלואורכרומים, שיכולים לספוג אנרגיה ממקור אור ואז לפלוט אנרגיה זו כאור נראה. פלואורכרומים כוללים חומרים ניאון טבעיים (כגון כלורופילים) וכתמים פלורסנטים שמתווספים לדגימה כדי ליצור ניגודיות. צבעים כגון אדום טקסס ו-FITC הם דוגמאות של פלואורכרומים. דוגמאות אחרות כוללות את צבעי חומצת הגרעין 4',6'-diamidino-2-phenylindole (DAPI), וכתום אקרידין.

המיקרוסקופ מקרין את המדגם עם עירור באורך גל קצר, כגון אור אולטרה סגול או כחול. הכרומופורים סופגים את אור העירור ופולטים אור נראה באורכי גל ארוכים יותר. לאחר מכן, אור העירור מסונן החוצה (בין השאר בגלל שאור אולטרה סגול מזיק לעיניים) כך שרק אור נראה עובר דרך עדשת העין, ומייצר תמונה של הדגימה בצבעים בהירים על רקע כהה.

מיקרוסקופים פלואורסצנטיים יכולים לזהות פתוגנים, למצוא מינים מסוימים בתוך סביבה, או למצוא את המיקומים של מולקולות ומבנים מסוימים בתוך תא. פותחו גם גישות להבדיל בין תאים חיים לבין תאים מתים על סמך האם הם קולטים פלואורכרומים מסוימים. לפעמים, משתמשים במספר פלואורכרומים על אותה דגימה כדי להראות מבנים או תכונות שונות.

אחד היישומים החשובים ביותר של מיקרוסקופ פלואורסצנטי הוא אימונופלואורסצנטי, המשמש לזיהוי חיידקים מסוימים על ידי התבוננות האם נוגדנים נקשרים אליהם. (נוגדנים הם מולקולות חלבון שהמערכת החיסונית מייצרת שמתחברות לפתוגנים ספציפיים כדי להרוג או לעכב אותם.) לטכניקה זו יש שתי גישות: בדיקת אימונופלוארסצנציה ישירה (DFA) ובדיקת אימונופלוארסצנציה עקיפה (IFA). ב-DFA, נוגדנים ספציפיים (למשל, אלו המכוונים לנגיף הכלבת) נצבעים בפלואורכרום. אם הדגימה מכילה את הפתוגן הממוקד, ניתן לראות את הנוגדנים הנקשרים לפתוגן תחת מיקרוסקופ פלואורסצנטי. זהו כתם נוגדן ראשוני מכיוון שהנוגדנים המוכתמים מתחברים ישירות לפתוגן.

ב-IFA, נוגדנים משניים נצבעים עם פלואורכרום ולא בנוגדנים ראשוניים. נוגדנים משניים אינם מתחברים ישירות לאורגניזם אלא נקשרים לנוגדנים ראשוניים. כאשר הנוגדנים הראשוניים הלא מוכתמים נקשרים לפתוגן, ניתן לראות את הנוגדנים המשניים הניאון נקשרים לנוגדנים הראשוניים. לפיכך, הנוגדנים המשניים מחוברים בעקיפין לפתוגן. מכיוון שלעתים קרובות נוגדנים משניים יכולים להיצמד לנוגדן ראשוני, IFA מגדיל את מספר הנוגדנים הפלורסנטים המחוברים לדגימה, מה שמקל על הצגת תכונותיו.

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מיקרוסקופ פלואורסצנטי משתמש בכרומופורים פלואורסצנטיים הנקראים פלואורוכרומים, אשר יכולים לספוג אנרגיה ממקור אור ואז לפלוט אנרגיה זו כ...

Education

JoVE Core

Cell Biology

ביולוגיה של התא

Unit 1

יחידה 1

Visualizing Cells, Tissues, and Molecules

ויזואליזציה של תאים, רקמות ומולקולות

KEY TERMS AND CONCEPTS

מונחים ומושגים מרכזיים

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

imaging biological samples with optical microscopy

imaging-biological-samples-with-optical-microscopy

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...

phase contrast and differential interference contrast microscopy

phase-contrast-and-differential-interference-contrast-microscopy

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...

fixation and sectioning

Microscope Slide Prep and Staining

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...

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...

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...

protein dynamics in living cells

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...

total internal reflection fluorescence microscopy

total-internal-reflection-fluorescence-microscopy

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...

super-resolution fluorescence microscopy

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...

immunogold electron microscopy

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 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

JoVE Core Cell Biology illustrates complex cellular processes in easy-to-understand yet comprehensive animated video lessons. This collection focuses on the essential components and functions of cells, including organelles and membranes, cellular transport, and cell energetics. It is complemented by scientist-in-action videos that demonstrate the application of cell biology in real-world research experiments.

Fluorescent Antibody Staining in Cells

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