Name: microscope slide prep and staining
Uploaded: 2023-04-30T11:59:49.000Z
Duration: 1 min 3 s
Channel: JoVE

Większość próbek tkanek biologicznych jest gruba i nieprzezroczysta i musi zostać przetworzona przed obserwacją pod mikroskopem.
Tkanki są najpierw traktowane za pomocą roztworu utrwalającego, który zabija komórki i unieruchamia składniki komórkowe, aby utrzymać ich strukturę.
Utrwalone próbki są przepuszczane przez rosnący gradient alkoholu, który odwadnia próbkę.
Aby zapobiec uszkodzeniu delikatnych próbek, są one zatopiane w materiale hydrofobowym, takim jak wosk parafinowy, i krojone na cienkie sekcje za pomocą mikrotomu.
Cienkie odcinki są przenoszone na szkiełka, gdzie dodaje się kilka kropli rozpuszczalników organicznych w celu usunięcia przylegającego wosku.
Następnie sekcje są barwione i dodawane jest podłoże montażowe z szkiełkiem nakrywkowym na górze. Medium montażowe powinno mieć taki sam współczynnik załamania światła jak szkiełko nakrywkowe i szkiełko, aby uzyskać najlepszą rozdzielczość obrazu.
Próbka jest teraz gotowa do wizualizacji pod mikroskopem.

utrwalanie i cięcie

Fixation and Sectioning

Do wizualizacji preparatów w mikroskopie świetlnym stosuje się dwa podstawowe rodzaje preparatów: preparaty mokre i preparaty utrwalone. Najprostszym rodzajem preparatyki jest mocowanie na mokro, w którym preparat umieszcza się w kropli płynu na szkiełku. Płynną próbkę można umieścić bezpośrednio na szkiełku za pomocą zakraplacza. Próbki stałe, takie jak zeskrobina skóry, można umieścić na szkiełku przed dodaniem kropli płynu w celu przygotowania mokrej oprawy. Czasami płynem jest po prostu woda, ale często dodaje się plamy w celu zwiększenia kontrastu. Po dodaniu płynu do szkiełka, na wierzchu umieszcza się szkiełko nakrywkowe i preparat jest gotowy do badania pod mikroskopem. Drugą metodą przygotowania próbek do mikroskopii świetlnej jest utrwalanie. „Utrwalanie” próbki odnosi się do procesu mocowania komórek do szkiełka. Utrwalanie często osiąga się poprzez ogrzewanie (utrwalanie termiczne) lub obróbkę chemiczną próbki. Oprócz mocowania próbki do szkiełka, utrwalenie zabija również mikroorganizmy w próbce, zatrzymując ich ruch i metabolizm, zachowując jednocześnie integralność ich składników komórkowych na potrzeby obserwacji. Aby utrwalić próbkę na gorąco, na szkiełku rozprowadza się cienką warstwę próbki (tzw. rozmaz), a następnie szkiełko krótko podgrzewa się nad źródłem ciepła. W przypadku próbek tkanek często preferowane jest utrwalanie chemiczne zamiast ogrzewania. Czynniki chemiczne, takie jak kwas octowy, etanol, metanol, formaldehyd (formalina) i aldehyd glutarowy, mogą powodować denaturację białek, zatrzymywać reakcje biochemiczne i stabilizować struktury komórkowe w próbkach tkanek. Oprócz utrwalenia, prawie zawsze stosuje się barwienie w celu zabarwienia pewnych cech próbki przed zbadaniem jej pod mikroskopem świetlnym. Plamy lub barwniki zawierają sole składające się z jonu dodatniego i jonu ujemnego. W zależności od rodzaju barwnika jonem dodatnim lub ujemnym może być chromofor (jon kolorowy); a drugi bezbarwny jon nazywany jest przeciwjonem. Jeśli chromofor jest jonem naładowanym dodatnio, plamę klasyfikuje się jako barwnik zasadowy; jeśli jonem ujemnym jest chromofor, plamę uważa się za barwnik kwasowy. Barwniki dobiera się do barwienia na podstawie właściwości chemicznych barwnika i obserwowanej próbki, które określają, w jaki sposób barwnik będzie oddziaływać z próbką. W większości przypadków lepiej jest użyć bejcy pozytywowej, barwnika, który zostanie wchłonięty przez obserwowane komórki lub organizmy, dodając koloru interesującym obiektom, aby wyróżniały się na tle. Istnieją jednak scenariusze, w których korzystne jest zastosowanie barwnika negatywowego wchłoniętego przez tło, ale nie przez komórki lub organizmy w próbce. Barwienie negatywowe pozwala uzyskać zarys lub sylwetkę organizmów na kolorowym tle. Ponieważ komórki zazwyczaj mają ujemnie naładowane ściany komórkowe, dodatnie chromofory w barwnikach zasadowych mają tendencję do przyklejania się do ścian komórkowych, powodując powstawanie pozytywnych plam. Zatem barwniki zasadowe, takie jak zasadowa fuksyna, fiolet krystaliczny, zieleń malachitowa, błękit metylenowy i safranina, zazwyczaj służą jako plamy pozytywowe. Z drugiej strony ujemnie naładowane chromofory w barwnikach kwasowych są odpychane przez ujemnie naładowane ściany komórkowe, co powoduje powstawanie plam ujemnych. Powszechnie stosowane barwniki kwasowe obejmują kwaśną fuksynę, eozynę i róż bengalski. Niektóre techniki barwienia obejmują nałożenie na próbkę tylko jednego barwnika; inne wymagają więcej niż jednego barwnika. W przypadku prostego barwienia stosuje się pojedynczy barwnik w celu uwypuklenia poszczególnych struktur próbki. Proste plamienie zazwyczaj powoduje, że wszystkie organizmy w próbce mają ten sam kolor, nawet jeśli próbka zawiera więcej niż jeden typ organizmów. Natomiast barwienie różnicowe rozróżnia organizmy na podstawie ich interakcji z wieloma plamami. Innymi słowy, dwa organizmy w różnie zabarwionej próbce mogą mieć różne kolory. Techniki barwienia różnicowego powszechnie stosowane w warunkach klinicznych obejmują barwienie metodą Grama, barwienie kwasoodporne, barwienie endospor, barwienie wici i barwienie torebki. This text is adapted from <a href="https://openstax.org/books/microbiology/pages/2-4-staining-microscopic-specimens">Openstax, Microbiology 2e, Section 2.4: </a><a href="https://openstax.org/books/microbiology/pages/2-4-staining-microscopic-specimens">Staining microscopic specimens</a>

Utrwalanie i cięcie

Do wizualizacji preparatów za pomocą mikroskopu świetlnego stosuje się dwa podstawowe rodzaje preparacji: mocowania mokre i próbki stałe.
Najprostszy rodzaj przygotowania ...

Education

JoVE Core

Cell Biology

Biologia komórki

Unit 1

Jednostka 1

Visualizing Cells, Tissues, and Molecules

Wizualizacja komórek, tkanek i cząsteczek

KEY TERMS AND CONCEPTS

Kluczowe terminy i pojęcia

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 water, but stains are often mixed in to improve contrast. A coverslip is placed on top, and the sample is then ready for viewing.

The other main method is fixation, which means attaching cells to the slide. Fixation can be done with heat or with chemicals. Heat fixing uses a thin smear of the specimen that is briefly heated over a heat source. This method also kills microorganisms, stops movement and metabolism, and helps preserve cell structures for observation. Chemical fixatives are often better for tissue samples. Common chemical agents include acetic acid, ethanol, methanol, formaldehyde, and glutaraldehyde. These chemicals denature proteins, stop biochemical reactions, and stabilize cell structures.

Staining is usually used along with fixation to add color before light microscopy. Stains, or dyes, are salts with a positive ion and a negative ion. The colored ion is called the chromophore, and the uncolored ion is the counterion. If the chromophore is positive, the dye is a basic dye. If the chromophore is negative, the dye is an acidic dye. Dyes are chosen based on the chemical properties of both the dye and the specimen.

Most of the time, a positive stain is preferred because it is absorbed by the cells or organisms being observed. This makes the objects of interest stand out from the background. In some cases, a negative stain works better. A negative stain colors the background but not the cells, so the organisms appear as outlines or silhouettes. Because cell walls are usually negatively charged, basic dyes tend to stick to them and act as positive stains. Examples include basic fuchsin, crystal violet, malachite green, methylene blue, and safranin. Acidic dyes have negatively charged chromophores and are repelled by negatively charged cell walls. Common acidic dyes include acid fuchsin, eosin, and rose bengal.

Some staining methods use one dye, while others use more than one. Simple staining uses a single dye to make certain structures easier to see. It usually makes all organisms in a sample appear the same color, even when more than one type is present. Differential staining uses multiple stains to tell organisms apart. In a differentially stained sample, two organisms may appear different colors. Common differential stains used in clinical settings include Gram staining, acid-fast staining, endospore staining, flagella staining, and capsule staining.

Microscope Slide Prep and Staining

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...

phase contrast and differential interference contrast microscopy

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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...

fixation and sectioning

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 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

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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

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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

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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

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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

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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 wet mounts, fixation, and staining prepare specimens for light microscopy, including positive and negative stains and simple versus differential staining.

Microscope Slide Prep and Staining | 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.

Microscope Slide Prep and Staining

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