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

대부분의 생체 조직 샘플은 두껍고 불투명하므로 현미경으로 관찰하기 전에 처리해야 합니다.
조직은 먼저 세포를 죽이고 세포 구성 요소를 고정시켜 구조를 유지하는 고정 용액을 사용하여 치료합니다.
고정된 샘플은 증가하는 알코올 구배를 통과하여 샘플을 탈수시킵니다.
깨지기 쉬운 샘플이 손상되는 것을 방지하기 위해 파라핀 왁스와 같은 발수 물질에 삽입하고 마이크로톰으로 얇은 부분으로 자릅니다.
얇은 부분은 슬라이드로 옮겨져 부착 된 왁스를 제거하기 위해 몇 방울의 유기 용매가 추가됩니다.
다음으로, 섹션을 염색하고 상단에 커버 슬립을 사용하여 장착 매체를 추가합니다. 장착 매체는 최상의 이미지 해상도를 얻기 위해 커버슬립 및 슬라이드와 동일한 굴절률을 가져야 합니다.
이제 샘플을 현미경으로 시각화할 준비가 되었습니다.

고정 및 절편

Fixation and Sectioning

광학 현미경으로 표본을 시각화하는 데 두 가지 기본 유형의 준비, 즉 습식 마운트와 고정 표본이 사용됩니다.

가장 간단한 준비 유형은 표본을 슬라이드의 액체 한 방울에 넣는 습식 마운트입니다. 드로퍼를 사용하여 액체 시료를 슬라이드에 직접 올려 놓을 수 있습니다. 피부 긁힘과 같은 고체 표본을 슬라이드 위에 놓은 후 액체 한 방울을 추가하여 습식 마운트를 준비할 수 있습니다. 때로는 액체가 단순히 물일 때도 있지만 대비를 높이기 위해 얼룩이 추가되는 경우가 많습니다. 액체가 슬라이드에 추가되면 커버슬립을 위에 놓고 표본을 현미경으로 검사할 준비가 됩니다.

광학 현미경 검사를 위한 표본을 준비하는 두 번째 방법은 고정입니다. 표본의 "고정"은 슬라이드에 세포를 부착하는 과정을 의미합니다. 고정은 종종 표본을 가열(가열 고정)하거나 화학적으로 처리하여 달성됩니다. 표본을 슬라이드에 부착하는 것 외에도 고정은 표본에 있는 미생물을 죽이고 관찰을 위해 세포 구성 요소의 무결성을 보존하면서 미생물의 움직임과 대사를 중지합니다.

표본을 열 고정하기 위해 표본의 얇은 층을 슬라이드 위에 펴고(도말이라고 함) 슬라이드를 열원 위에서 잠시 가열합니다. 조직 표본의 경우 가열보다 화학적 고정제가 더 선호되는 경우가 많습니다. 아세트산, 에탄올, 메탄올, 포름알데히드(포르말린) 및 글루타르알데히드와 같은 화학 물질은 단백질을 변성시키고 생화학 반응을 중지하며 조직 표본의 세포 구조를 안정화시킬 수 있습니다.

고정 외에도 염색은 광학 현미경으로 검사하기 전에 표본의 특정 특징을 색칠하기 위해 거의 항상 적용됩니다. 얼룩이나 염료에는 양이온과 음이온으로 구성된 염이 포함되어 있습니다. 염료의 유형에 따라 양이온 또는 음이온이 발색단(유색 이온)이 될 수 있습니다. 다른 무색 이온을 반대이온이라고 합니다. 발색단이 양전하를 띤 이온인 경우 얼룩은 염기성 염료로 분류됩니다. 음이온이 발색단인 경우 얼룩은 산성 염료로 간주됩니다.

염료와 관찰되는 표본의 화학적 특성을 기반으로 염색을 위해 염료가 선택되며, 이는 염료가 표본과 상호 작용하는 방식을 결정합니다. 대부분의 경우, 관찰 중인 세포나 유기체에 흡수되는 염료인 포지티브 염료를 사용하여 관심 대상에 색상을 추가하여 배경에서 눈에 띄게 만드는 것이 바람직합니다. 그러나 배경에는 흡수되지만 표본의 세포나 유기체에는 흡수되지 않는 음성 염료를 사용하는 것이 유리한 시나리오가 있습니다. 음성 염색은 다채로운 배경에 대비하여 유기체의 윤곽이나 실루엣을 생성합니다.

세포는 일반적으로 음전하를 띤 세포벽을 가지고 있기 때문에 염기성 염료의 양성 발색단은 세포벽에 달라붙어 양성 얼룩이 되는 경향이 있습니다. 따라서 기본 푹신, 크리스탈 바이올렛, 말라카이트 그린, 메틸렌 블루, 사프라닌과 같은 기본 염료는 일반적으로 양성 염료 역할을 합니다. 반면, 산성 염료의 음전하 발색단은 음전하를 띤 세포벽에 의해 반발되어 음성 얼룩이 됩니다. 일반적으로 사용되는 산성 염료로는 산성 푹신, 에오신, 로즈 벵갈 등이 있습니다.

일부 염색 기술에는 표본에 하나의 염료만 적용하는 것이 포함됩니다. 다른 것들은 하나 이상의 염료가 필요합니다. 단순 염색에서는 표본의 특정 구조를 강조하기 위해 단일 염료가 사용됩니다. 단순 염색은 표본에 두 가지 이상의 유기체가 포함되어 있더라도 일반적으로 표본의 모든 유기체가 동일한 색상으로 나타나게 합니다. 대조적으로, 차별 염색은 여러 염색과의 상호 작용을 기반으로 유기체를 구별합니다. 즉, 서로 다르게 염색된 표본에 있는 두 유기체가 서로 다른 색상으로 나타날 수 있습니다. 임상 환경에서 일반적으로 사용되는 차별 염색 기술에는 그람 염색, 항산성 염색, 내생포자 염색, 편모 염색 및 캡슐 염색이 포함됩니다.

이 텍스트는 다음에서 수정되었습니다 <a href="https://openstax.org/books/microbiology/pages/2-4-staining-microscopic-specimens">Openstax, Microbiology 2e, Section 2.4: Staining microscopic specimens</a>

광학 현미경으로 표본을 시각화하기 위해 두 가지 기본 준비 유형, 즉 습식 마운트와 고정 표본이 사용됩니다.
가장 간단한 유형의 준비 ...

Education

교육

JoVE Core

Cell Biology

세포생물학

Unit 1

제1단원

Visualizing Cells, Tissues, and Molecules

세포, 조직 및 분자의 시각화

KEY TERMS AND CONCEPTS

주요 용어 및 개념

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

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 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

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 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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