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

ほとんどの生体組織サンプルは厚くて不透明であり、顕微鏡で観察する前に処理する必要があります。
組織は最初に、細胞を殺し、細胞成分を固定してその構造を維持する固定溶液を使用して処理されます。
固定されたサンプルは、アルコールの勾配が増加するため、サンプルが脱水されます。
壊れやすいサンプルが損傷するのを防ぐために、パラフィンワックスなどの撥水素材に埋め込まれ、ミクロトームで薄いセクションにスライスされます。
薄い切片をスライドに移し、そこで数滴の有機溶剤を加えて付着したワックスを除去します。
次に、切片を染色し、封入剤を添加し、その上にカバーガラスを敷きます。封入剤は、最高の画像解像度を得るために、カバーガラスおよびスライドと同じ屈折率を持つ必要があります。
これで、サンプルを顕微鏡で視覚化する準備が整いました。

固定と切断

Fixation and Sectioning

光学顕微鏡で標本を視覚化するには、ウェットマウントと固定標本という 2 つの基本的な準備方法が使用されます。

最も簡単な準備方法はウェットマウントです。この方法では、標本をスライド上の液体滴の中に置きます。 スポイトを使用して液体試料をスライド上に直接滴下できます。 皮膚の削り取りなどの固体標本は、液体を一滴加えてウェットマウントを準備する前にスライド上に置くことができます。 液体が単なる水の場合もありますが、コントラストを高めるために汚れが追加されることもよくあります。 液体をスライドに加えたら、カバースリップを上に置き、標本を顕微鏡で検査する準備が整います。

光学顕微鏡用に標本を準備する 2 番目の方法は固定です。 サンプルの「固定」とは、細胞をスライドに付着させるプロセスを指します。 固定は、多くの場合、標本を加熱する (熱固定) か化学処理することによって行われます。 標本をスライドに付着させることに加えて、固定により標本内の微生物が死滅し、細胞成分の完全性が保たれた状態で観察できるようにしながら、微生物の動きや代謝が停止されます。

サンプルを熱固定するには、標本の薄い層をスライド上に広げ（スメアと呼ばれます）、次にスライドを熱源で短時間加熱します。 多くの場合、組織標本を加熱するよりも化学固定剤の方が適しています。 酢酸、エタノール、メタノール、ホルムアルデヒド (ホルマリン)、グルタルアルデヒドなどの化学薬品は、組織サンプル内のタンパク質を変性し、生化学反応を停止し、細胞構造を安定化させることができます。

固定に加えて、ほとんどの場合、光学顕微鏡で検査する前に、標本の特定の特徴に色を付けるために染色が適用されます。 汚れ、つまり染料には、陽イオンと陰イオンからなる塩が含まれています。 染料の種類に応じて、正イオンまたは負イオンが発色団 (着色イオン) となる場合があります。 もう一方の無色のイオンは対イオンと呼ばれます。 発色団が正に帯電したイオンの場合、その汚れは塩基性染料として分類されます。 マイナスイオンが発色団の場合、その汚れは酸性染料とみなされます。

染色用の染料は、染料と観察される標本の化学的特性に基づいて選択され、染料が標本とどのように相互作用するかが決まります。 ほとんどの場合、観察対象の細胞や生物に吸収され、対象物に色を加えて背景から目立たせるポジティブ染色を使用することが望ましいです。 ただし、標本内の細胞や生物には吸収されず、背景には吸収されるネガティブ染色を使用することが有利なシナリオもあります。 ネガティブ染色では、カラフルな背景に対して生物の輪郭またはシルエットが生成されます。

通常、細胞は負に帯電した細胞壁を持っているため、塩基性色素中の正の発色団は細胞壁に付着する傾向があり、細胞壁を正の染色とします。 したがって、塩基性フクシン、クリスタル バイオレット、マラカイト グリーン、メチレン ブルー、サフラニンなどの塩基性染料は、通常、陽性染色として機能します。 一方、酸性染料の負に帯電した発色団は、負に帯電した細胞壁によって反発され、負の汚れとなります。 一般的に使用される酸性染料には、酸性フクシン、エオシン、ローズベンガルなどがあります。

一部の染色技術では、サンプルに 1 つの染料のみを適用します。 他のものは複数の染料を必要とします。 単純な染色では、標本の特定の構造を強調するために単一の染料が使用されます。 サンプルに複数の種類の微生物が含まれている場合でも、単純な染色では通常、サンプル内のすべての微生物が同じ色に見えます。 対照的に、示差染色では、複数の染色との相互作用に基づいて生物を区別します。 言い換えれば、異なって染色されたサンプル内の 2 つの生物は、異なる色に見える可能性があります。 臨床現場で一般的に使用される鑑別染色技術には、グラム染色、抗酸性染色、内生胞子染色、鞭毛染色、莢膜染色などがあります。

このテキストは <a href="Openstax, Microbiology 2e, Section 2.4: Staining microscopic specimens">Openstax, Microbiology 2e, Section 2.4: Staining microscopic specimens</a>

光学顕微鏡で試料を可視化するには、ウェットマウントと固定試料の2つの基本的な試料作製が使用されます。
最もシンプルなタイプの準備...

Education

JoVE Core

Cell Biology

細胞生物学

Unit 1

ユニット1

Visualizing Cells, Tissues, and Molecules

細胞・組織・分子の可視化

KEY TERMS AND CONCEPTS

主要用語と概念

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

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

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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 細胞生物学は、複雑な細胞プロセスを、わかりやすく包括的なアニメーション ビデオ レッスンで説明します。このコレクションは、細胞小器官や膜、細胞輸送、細胞のエネルギー学など、細胞の必須コンポーネントと機能に焦点を当てています。これは、実際の研究実験における細胞生物学の応用を実証する科学者による実際のビデオによって補足されています。

Microscope Slide Prep and Staining

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