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

Biyolojik doku örneklerinin çoğu kalın ve opaktır ve mikroskop altında gözlemlenmeden önce işlenmelidir.
Dokular ilk önce hücreleri öldüren ve yapılarını korumak için hücresel bileşenleri hareketsiz hale getiren bir sabitleme solüsyonu kullanılarak tedavi edilir.
Sabitlenen numuneler, numuneyi dehidre eden artan bir alkol gradyanından geçirilir.
Kırılgan numunelerin zarar görmesini önlemek için, parafin mumu gibi su itici bir malzemeye gömülür ve bir mikrotom ile ince bölümler halinde dilimlenir.
İnce kesitler, yapışan balmumunu çıkarmak için birkaç damla organik çözücünün eklendiği slaytlara aktarılır.
Daha sonra, bölümler boyanır ve üstüne bir lamel ile bir montaj ortamı eklenir. Montaj ortamı, en iyi görüntü çözünürlüğünü elde etmek için lamel ve sürgü ile aynı kırılma indisine sahip olmalıdır.
Numune artık mikroskop altında görselleştirmeye hazırdır.

sabitleme ve bölümleme

Fixation and Sectioning

Numuneleri ışık mikroskobu ile görselleştirmek için iki temel hazırlık türü kullanılır: ıslak montajlar ve sabit numuneler. En basit hazırlama türü, numunenin slayt üzerindeki bir damla sıvıya yerleştirildiği ıslak montajdır. Sıvı bir numune, bir damlalık kullanılarak doğrudan lam üzerine bırakılabilir. Deri kazıma gibi katı numuneler, ıslak montajı hazırlamak için bir damla sıvı eklenmeden önce slayt üzerine yerleştirilebilir. Bazen sıvı sadece sudur, ancak kontrastı arttırmak için sıklıkla lekeler eklenir. Sıvı slayta eklendikten sonra üstüne bir lamel yerleştirilir ve örnek mikroskop altında incelenmeye hazır hale gelir. Işık mikroskobu için numune hazırlamanın ikinci yöntemi sabitlemedir. Bir numunenin "sabitlenmesi", hücrelerin bir slayta eklenmesi işlemini ifade eder. Sabitleme genellikle numunenin ısıtılmasıyla (ısıyla sabitleme) veya kimyasal işlemle sağlanır. Numuneyi slayta tutturmanın yanı sıra, sabitleme aynı zamanda numunedeki mikroorganizmaları da öldürür, onların hareketlerini ve metabolizmalarını durdururken, gözlem için hücresel bileşenlerinin bütünlüğünü korur. Bir numuneyi ısıyla sabitlemek için numunenin ince bir tabakası lam üzerine yayılır (smear adı verilir) ve ardından lam bir ısı kaynağı üzerinde kısa süre ısıtılır. Kimyasal sabitleyiciler genellikle doku örneklerinin ısıtılması için tercih edilir. Asetik asit, etanol, metanol, formaldehit (formalin) ve glutaraldehit gibi kimyasal maddeler proteinleri denatüre edebilir, biyokimyasal reaksiyonları durdurabilir ve doku örneklerinde hücre yapılarını stabilize edebilir. Sabitlemenin yanı sıra, bir numunenin belirli özelliklerini ışık mikroskobu altında incelemeden önce renklendirmek için neredeyse her zaman boyama uygulanır. Lekeler veya boyalar pozitif iyon ve negatif iyondan oluşan tuzlar içerir. Boyanın türüne bağlı olarak pozitif veya negatif iyon, kromofor (renkli iyon) olabilir; ve diğer renksiz iyona karşı iyon denir. Kromofor pozitif yüklü iyon ise leke, bazik boya olarak sınıflandırılır; negatif iyon kromofor ise leke asidik bir boya olarak kabul edilir. Boyalar, boyanın ve gözlemlenen numunenin kimyasal özelliklerine göre boyama için seçilir; bu, boyanın numuneyle nasıl etkileşime gireceğini belirler. Çoğu durumda, pozitif bir lekenin (gözlenen hücreler veya organizmalar tarafından emilecek, ilgilenilen nesnelere arka planda öne çıkmalarını sağlayacak şekilde renk katacak bir boya) kullanılması tercih edilir. Bununla birlikte, örnekteki hücreler veya organizmalar tarafından değil, arka plan tarafından absorbe edilen negatif bir lekenin kullanılmasının avantajlı olduğu senaryolar vardır. Negatif boyama, renkli bir arka plan üzerinde organizmaların bir taslağını veya siluetini oluşturur. Hücreler tipik olarak negatif yüklü hücre duvarlarına sahip olduğundan, bazik boyalardaki pozitif kromoforlar hücre duvarlarına yapışma eğilimi gösterir ve onları pozitif lekeler haline getirir. Bu nedenle, bazik fuksin, kristal menekşe, malakit yeşili, metilen mavisi ve safranin gibi bazik boyalar tipik olarak pozitif lekeler olarak görev yapar. Öte yandan, asidik boyalardaki negatif yüklü kromoforlar, negatif yüklü hücre duvarları tarafından itilerek onları negatif lekeler haline getirir. Yaygın olarak kullanılan asidik boyalar arasında asit fuksin, eozin ve bengal gülü bulunur. Bazı boyama teknikleri numuneye yalnızca bir boyanın uygulanmasını içerir; diğerleri birden fazla boya gerektirir. Basit boyamada, numunedeki belirli yapıları vurgulamak için tek bir boya kullanılır. Basit bir leke, numune birden fazla organizma türü içerse bile genellikle numunedeki tüm organizmaların aynı renkte görünmesine neden olur. Buna karşılık, diferansiyel boyama, organizmaları çoklu lekelerle etkileşimlerine göre ayırt eder. Başka bir deyişle, diferansiyel olarak boyanmış bir örnekteki iki organizma farklı renklerde görünebilir. Klinik ortamlarda yaygın olarak kullanılan farklı boyama teknikleri arasında Gram boyama, aside dirençli boyama, endospor boyama, flagella boyama ve kapsül boyama yer alır.

Sabitleme ve Bölümleme

Numuneleri ışık mikroskobu ile görselleştirmek için iki temel hazırlama türü kullanılır: ıslak montajlar ve sabit numuneler.
En basit hazırlık türü ...

Education

JoVE Core

Cell Biology

Hücre Biyolojisi

Unit 1

Ünite 1

Visualizing Cells, Tissues, and Molecules

Hücrelerin, Dokuların ve Moleküllerin Görülebilir Kılınması

KEY TERMS AND CONCEPTS

TEMEL TERİMLER VE KAVRAMLAR

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

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

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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...

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 Hücre Biyolojisi, anlaşılması kolay ancak kapsamlı animasyonlu video derslerde karmaşık hücresel süreçleri gösterir. Bu koleksiyon, organeller ve zarlar, hücresel taşıma ve hücre enerjileri dahil olmak üzere hücrelerin temel bileşenlerine ve işlevlerine odaklanır. Gerçek dünya araştırma deneylerinde hücre biyolojisinin uygulanmasını gösteren bilim insanı eylem videoları ile tamamlanmaktadır.

Microscope Slide Prep and Staining

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