Name: electron microscopy sample prep methods
Uploaded: 2023-04-30T12:01:16.000Z
Duration: 1 min 20 s
Channel: JoVE

يجب تحضير العينات البيولوجية للفحص المجهري الإلكتروني لحماية العينة من التلف الناتج عن شعاع الإلكترون القوي والضغط المنخفض الناتج عن الفراغ.
يبدأ تحضير العينة بالتثبيت الكيميائي والجفاف الكامل. تربط المثبتات الكيميائية مثل الجلوتارالديهايد أو الفورمالديهايد البروتينات ، بينما غالبا ما يستخدم رابع أكسيد الأوزميوم لإصلاح دهون الغشاء.
بعد ذلك ، يتم تجفيف العينة تدريجيا مع زيادة تركيزات الإيثانول أو الأسيتون لتجنب انكماش العينة.
بالنسبة إلى TEM ، يتم تضمين العينات في راتنجات الايبوكسي الصلبة. ثم يقوم الميكروتوم الفائق بتقطيع العينة إلى مقاطع عرضية رفيعة للغاية يمكن أن تمر بها الإلكترونات.
هذه الأقسام ملطخة بالمعادن الثقيلة ، مثل أسيتات اليورانيل. تبدو الجزيئات التي تربط المعادن أغمق وتوفر تباينا مع الصورة.
بالنسبة إلى SEM ، يتم تجفيف العينات باستخدام ثاني أكسيد الكربون السائل في عملية تسمى تجفيف النقطة الحرجة. ثم يتم طلاء العينات بطبقة رقيقة من المعدن الموصل ، مثل الذهب ، مما يمنع التلف الحراري للعينة ويوفر جودة صورة أفضل.

تحضير العينات للمجهر الإلكتروني

Preparation of Samples for Electron Microscopy

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لكي يتم تصويرها بواسطة المجهر الإلكتروني، سواء بالنقل أو المسح، يجب تحضير العينات البيولوجية (تثبيتها) حتى لا يدمرها شعاع الإلكترون و تجفيفها بالكامل حتى لا يؤثر الفراغ عليها. يجب أن يتم التثبيت في أسرع وقت ممكن لأن خصائص العينة ستبدأ في التغير بمجرد إزالتها من بيئتها الطبيعية. على سبيل المثال، في عينة الأنسجة، تبدأ مستويات الأكسجين في التناقص، مما يتسبب في تغيير مظهر الميتوكوندريا.

التقدم في إعداد عينات TEM

التثبيت بالتبريد

في هذه التقنية، يمكن تجميد الخلايا والعضيات الصغيرة عن طريق التجميد الغاطس، بينما يمكن تجميد الخلايا والأنسجة الأكبر حجمًا باستخدام الضغط العالي. يتم تحقيق التجميد الغاطس عن طريق وضع كمية صغيرة من العينة على شبكة EM باردة ثم غمرها بسرعة في الإيثان السائل المملوء في خزان ومحاط بالنيتروجين السائل. يضمن التجميد السريع عدم تبلور الماء. في التجميد عالي الضغط، يتم استخدام النيتروجين السائل لتجميد العينة بسرعة، ويتعرض لضغط ~ 2000 بار لإبطاء تكوين بلورات الجليد. يتم تقسيم العينات المثبتة بالتبريد إلى مشراح فائق البرودة يتكون من سكين الماس المجمد.

اسقاط الظل

تحتوي المنصة الدوارة في غرفة مفرغة على العينة، والتي يتم بعد ذلك تغليفها بمعدن ثقيل. يضمن دوران العينة طلاءًا معدنيًا على الجانب الأمامي وتأثير الظل على الجانب الخلفي. تعتبر هذه التقنية مفيدة بشكل خاص عند دراسة تفاصيل البنية التحتية للبكتيريا أو الفيروسات أو الحمض النووي (DNA) أو الحمض النووي الريبوزي (RNA) أو البروتينات المعزولة.

تلطيخ سلبي

يمكن تصوير العينات الصغيرة، مثل الريبوسومات، وجزيئات الإنزيم، والفيروسات، وما إلى ذلك، مباشرة دون تقطيع. يتم خلط العينات مع موليبدات الأمونيوم، أو أسيتات اليورانيل، أو حمض الفوسفوتونجستيك، وهي معتمة للإلكترون. يتم تشكيل طبقة معدنية على الشبكة، باستثناء المكان الذي تقع فيه العينة، مما يؤدي إلى تكوين صورة سلبية للعينة. يتم تحقيق التباين نتيجة لنمط توزيع المعادن.

تحضير العينات المجففة وغير المجففة لـ SEM

يمكن لـ SEM تصوير ثلاثة أنواع من العينات، وهي (1) المجففة والموصلة، (2) المجففة وغير الموصلة، و(ج) غير المجففة (الرطبة). يمكن بسهولة تركيب وتصوير العينات الموصلة بشكل طبيعي والمجففة. ومع ذلك، يجب أن تكون العينات المجففة وغير الموصلة مغلفة بطبقة موصلة لحماية العينة من الحرارة الزائدة وتحسين جودة الصورة. يتم جعل هذه العينات موصلة عن طريق طلاء العينة بطبقة رقيقة من الذهب باستخدام طبقة الطلاء بالرش. ويستخدم أيونات الأرجون لطرد ذرات الذهب من لوحة ذهبية وطلاء سطح العينة بالذهب، مما يجعل السطح موصلاً للإلكترونات.

يمكن للعينات الرطبة أن تنطلق الغازات في البيئة المحيطة الفراغية للمجهر.

يجب أن يتحكم Wet SEM، المعروف أيضًا باسم Environmental SEM أو ESEM، في تسامي الماء في العينة أثناء التصوير. ويتم ذلك عن طريق طلاء العينة أولاً بطبقة رقيقة من البوليمر الغشائي الذي يكون شفافًا لحزم الإلكترون ويفصل العينة الرطبة عن الفراغ. ثم تنخفض درجة الحرارة، مما يقلل من ضغط البخار المشبع. تسمح هذه الظروف بتشغيل الأداة عند فراغ أعلى يحافظ على ميزات البنية التحتية للعينة ويقلل الضوضاء. يمكن أيضًا استخدام دبن كولستيج لتصوير العينات الرطبة عن طريق تجميدها إلى -25 درجة مئوية، مما يحافظ على بنية العينة ويمنع إطلاق الغازات أثناء قصفها بالإلكترونات.
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ليتم تصورها بواسطة المجهر الإلكتروني ، سواء كان الإرسال أو المسح الضوئي ، يجب تثبيت العينات البيولوجية (استقرارها) بحيث يتم تثبيت شعاع الإلكترون ...

preparation-of-samples-for-electron-microscopy

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

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 is why early stabilization matters.

TEM sample prep can use cryofixation, shadow casting, or negative staining. In cryofixation, small cells and organelles are plunge frozen on a cold EM grid in liquid ethane surrounded by liquid nitrogen. Larger cells and tissues can be frozen by high-pressure freezing with liquid nitrogen and about 2000 bar of pressure. This slows ice crystal formation. Cryofixed samples are then cut in a cryo-ultramicrotome with a frozen diamond knife.

Shadow casting places the sample on a rotating platform in a vacuum chamber and coats it with heavy metal. The rotation helps cover the front side and creates a shadow effect on the back side. This method is useful for ultrastructural details in bacteria, viruses, DNA, RNA, and isolated proteins. Negative staining is used for small samples such as ribosomes, enzyme molecules, and viruses. The sample is mixed with an electron-opaque stain such as ammonium molybdate, uranyl acetate, or phosphotungstic acid, which leaves a metal layer around the sample and forms a negative image.

SEM sample prep depends on whether the sample is desiccated, conductive, non-conductive, or wet. Naturally conductive dry samples can be mounted and imaged directly. Dry but non-conductive samples need a thin gold coating from a sputter coater. Argon ions knock gold atoms off a gold plate and deposit them on the sample surface. This makes the surface conductive and helps prevent overheating while improving image quality.

Wet samples can outgas in the vacuum of the microscope. For these specimens, wet SEM, also called Environmental SEM or ESEM, controls water sublimation during imaging. One approach uses a thin membranous polymer that is transparent to electron beams and separates the wet specimen from the vacuum. The temperature is then lowered, which reduces saturated vapor pressure. A Deben Coolstage can also image wet samples by freezing them to -25°C, which preserves structure and limits outgassing while electrons strike the specimen.

Electron Microscopy Sample Prep Methods

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 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...

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

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 biological samples are fixed, dried, coated, or frozen for TEM and SEM, including cryofixation, negative staining, and wet sample imaging.

Electron Microscopy Sample Prep Methods | 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.

Electron Microscopy Sample Prep Methods

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