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

معظم عينات الأنسجة البيولوجية سميكة وغير شفافة ويجب معالجتها قبل ملاحظتها تحت المجهر.
تتم معالجة الأنسجة أولا باستخدام محلول تثبيت يقتل الخلايا ويشل حركة المكونات الخلوية للحفاظ على بنيتها.
يتم تمرير العينات الثابتة من خلال تدرج متزايد من الكحول ، مما يؤدي إلى تجفيف العينة.
لمنع تلف العينات الهشة ، يتم دمجها في مادة طاردة للماء مثل شمع البارافين وتقطيعها إلى أقسام رفيعة باستخدام ميكروتوم.
يتم نقل الأقسام الرقيقة إلى شرائح ، حيث تضاف بضع قطرات من المذيبات العضوية لإزالة الشمع الملتصق.
بعد ذلك ، يتم تلطيخ الأقسام ، ويضاف وسيط تثبيت ، مع غطاء في الأعلى. يجب أن يكون لوسيط التثبيت نفس معامل الانكسار مثل الغطاء والشريحة للحصول على أفضل دقة للصورة.
العينة جاهزة الآن للتصور تحت المجهر.

التثبيت والتجزئة

Fixation and Sectioning

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يتم استخدام نوعين أساسيين من التحضير لتصوير العينات باستخدام المجهر الضوئي: العينات الرطبة والعينات الثابتة.

أبسط نوع من التحضير هو التركيب الرطب، حيث يتم وضع العينة في قطرة من السائل على الشريحة. يمكن إيداع العينة السائلة مباشرة على الشريحة باستخدام قطَّارة. يمكن وضع العينات الصلبة، مثل كشط الجلد، على الشريحة قبل إضافة قطرة من السائل لتحضير القاعدة الرطبة. في بعض الأحيان يكون السائل مجرد ماء، ولكن غالبًا ما يتم إضافة البقع لتعزيز التباين. بمجرد إضافة السائل إلى الشريحة، يتم وضع ساترة في الأعلى، وتكون العينة جاهزة للفحص تحت المجهر.

الطريقة الثانية لتحضير العينات للفحص المجهري الضوئي هي التثبيت. يشير "تثبيت" العينة إلى عملية ربط الخلايا بالشريحة. غالبًا ما يتم التثبيت عن طريق التسخين (التثبيت الحراري) أو معالجة العينة كيميائيًا. بالإضافة إلى ربط العينة بالشريحة، يؤدي التثبيت أيضًا إلى قتل الكائنات الحية الدقيقة في العينة، وإيقاف حركتها واستقلابها مع الحفاظ على سلامة مكوناتها الخلوية للمراقبة.

لتثبيت العينة بالحرارة، يتم نشر طبقة رقيقة من العينة على الشريحة (تسمى اللطاخة)، ثم يتم تسخين الشريحة لفترة وجيزة فوق مصدر الحرارة. غالبًا ما تكون المثبتات الكيميائية مفضلة لتسخين عينات الأنسجة. يمكن للعوامل الكيميائية مثل حمض الأسيتيك، و الإيثانول، والميثانول، والفورمالديهايد (الفورمالين)، والجلوتارالدهيد أن تفسد البروتينات، وتوقف التفاعلات الكيميائية الحيوية، وتثبت هياكل الخلايا في عينات الأنسجة.

بالإضافة إلى التثبيت، يتم تطبيق الصبغ بشكل شبه دائم لتلوين ميزات معينة للعينة قبل فحصها تحت المجهر الضوئي. تحتوي البقع أو الأصباغ على أملاح تتكون من أيون موجب وأيون سالب. اعتمادًا على نوع الصبغة، قد يكون الأيون الموجب أو السالب هو حامل اللون (الأيون الملون)؛ والأيون الآخر غير الملون يسمى الأيون المضاد. إذا كان الكروموفور هو أيون موجب الشحنة، فسيتم تصنيف الصبغة على أنها صبغة أساسية؛ إذا كان الأيون السالب هو حامل اللون، تعتبر الصبغة صبغة حمضية.

يتم اختيار الأصباغ للتلوين بناءً على الخواص الكيميائية للصبغة والعينة التي يتم ملاحظتها، والتي تحدد كيفية تفاعل الصبغة مع العينة. في معظم الحالات، يفضل استخدام صبغة إيجابية، وهي صبغة تمتصها الخلايا أو الكائنات الحية التي يتم ملاحظتها، مما يضيف لونًا إلى الأشياء محل الاهتمام لجعلها تبرز على الخلفية. ومع ذلك، هناك سيناريوهات يكون من المفيد فيها استخدام صبغة سلبية تمتصها الخلفية ولكن ليس بواسطة الخلايا أو الكائنات الحية الموجودة في العينة. ينتج عن التلوين السلبي مخططًا تفصيليًا أو صورة ظلية للكائنات الحية على خلفية ملونة.

نظرًا لأن الخلايا عادةً ما تحتوي على جدران خلوية سالبة الشحنة، فإن الكروموفورات الموجبة في الأصباغ الأساسية تميل إلى الالتصاق بجدران الخلايا، مما يجعلها بقع موجبة. وبالتالي، فإن الأصباغ الأساسية مثل الفوشين الأساسي، والبنفسجي البلوري، والأخضر الملكيت، وأزرق الميثيلين، والسافرانين عادة ما تكون بمثابة بقع إيجابية. من ناحية أخرى، يتم صد الكروموفورات سالبة الشحنة في الأصباغ الحمضية بواسطة جدران الخلايا سالبة الشحنة، مما يجعلها بقع سلبية. تشمل الأصباغ الحمضية شائعة الاستخدام حمض الفوشين والأيوسين ووردية البنغال.

تتضمن بعض تقنيات الصبغ تطبيق صبغة واحدة فقط على العينة؛ البعض الآخر يتطلب أكثر من صبغة واحدة. في التلوين البسيط، يتم استخدام صبغة واحدة للتأكيد على هياكل معينة في العينة. إن الصبغة البسيطة ستؤدي عمومًا إلى ظهور جميع الكائنات الحية في العينة بنفس اللون، حتى لو كانت العينة تحتوي على أكثر من نوع واحد من الكائنات الحية. في المقابل، يميز التلوين التفريقي الكائنات الحية بناءً على تفاعلاتها مع البقع المتعددة. وبعبارة أخرى، قد يظهر كائنان حيان في عينة ملطخة بشكل مختلف بألوان مختلفة. تشمل تقنيات التلوين التفريقي المستخدمة بشكل شائع في البيئات السريرية تلطيخ الجرام، والتلطيخ المقاوم للأحماض، وتلطيخ الجراثيم الداخلية، وتلطيخ الأسواط، وتلطيخ الكبسولة.

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يتم استخدام نوعين أساسيين من التحضير لتصور العينات بالمجهر الضوئي: الحوامل الرطبة والعينات الثابتة.
أبسط نوع من التحضير ...

Education

JoVE Core

Cell Biology

بيولوجيا الخلية

Unit 1

الوحدة الأولى

Visualizing Cells, Tissues, and Molecules

تصوير الخلايا والأنسجة والجزيئات

KEY TERMS AND CONCEPTS

المصطلحات والمفاهيم الأساسية

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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