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

Biological samples must be prepared for electron microscopy to protect the sample from damage by the powerful electron beam and the low pressure created by the vacuum.
Sample preparation starts with chemical fixation and complete dehydration. Chemical fixatives such as glutaraldehyde or formaldehyde cross-link the proteins, while osmium tetroxide is often used to fix the membrane lipids.
Next, the sample is dehydrated gradually with increasing concentrations of ethanol or acetone to avoid sample shrinkage.
For TEM, samples are embedded in a hard epoxy resin. An ultramicrotome then slices the sample into ultrathin cross-sections that electrons can pass through.
These sections are stained with heavy metals, such as uranyl acetate. Molecules that bind the metals appear darker and provide contrast to the image.
For SEM, samples are further dried using liquid carbon dioxide in a process called critical point drying. Samples are then sputter-coated with a thin layer of conductive metal, such as gold, preventing thermal damage to the sample and providing better image quality.

voorbereiding van monsters voor elektronenmicroscopie

Preparation of Samples for Electron Microscopy

Om te worden gevisualiseerd door een elektronenmicroscoop, hetzij door transmissie of door middel van scannen, moeten biologische monsters worden gefixeerd (gestabiliseerd) zodat de elektronenbundel ze niet vernietigt, en grondig worden gedroogd (uitgedroogd/gedehydrateerd), zodat het vacuüm ze niet beïnvloedt. Fixatie moet zo snel mogelijk worden uitgevoerd, omdat de eigenschappen van het monster zullen veranderen zodra het uit zijn natuurlijke omgeving wordt verwijderd. In een weefselmonster beginnen de zuurstofniveaus bijvoorbeeld te dalen, wat een veranderd uiterlijk van de mitochondriën veroorzaakt.

Vooruitgang in de voorbereiding van TEM-monsters

Cryofixatie

Bij deze techniek kunnen kleine cellen en organellen worden ingevroren door middel van invriezen, terwijl grotere cellen en weefsels kunnen worden bevroren door middel van hoge druk. Invriezen wordt bereikt door een kleine hoeveelheid monster op een koud EM-rooster te plaatsen en vervolgens snel in vloeibaar ethaan te dompelen, gevuld in een reservoir en omgeven door vloeibare stikstof. Het snelle bevriezen zorgt ervoor dat water niet kristalliseert. Bij invriezen onder hoge druk wordt vloeibare stikstof gebruikt om het monster snel te bevriezen, en het wordt onderworpen aan een druk van ~2000 bar om de vorming van ijskristallen te vertragen. Gecryofixeerde monsters worden in secties verdeeld in een cryo-ultramicrotoom dat bestaat uit een bevroren diamantmes.

Schaduwgieten

Het roterende platform in een vacuümkamer houdt het monster vast, dat vervolgens wordt bedekt met zwaar metaal. De rotatie van het monster zorgt voor een metalen coating aan de voorzijde en een schaduweffect aan de achterzijde. De techniek is vooral handig bij het bestuderen van ultrastructurele details van bacteriën of virussen, DNA, RNA of geïsoleerde eiwitten.

Negatieve kleuring

Kleine monsters, zoals ribosomen, enzymmoleculen, virussen, enz., kunnen direct in beeld worden gebracht zonder ze te snijden. Monsters worden gemengd met ammoniummolybdaat, uranylacetaat of fosfowolfraamzuur, die elektronenondoorzichtig zijn. Op het rooster wordt een metaallaag gevormd, behalve waar het monster ligt, waardoor een negatief beeld van het monster ontstaat. Contrast wordt bereikt als resultaat van het patroon van metaalverdeling.

Gedroogde en niet-gedroogde monsters voorbereiden voor SEM

SEM kan drie soorten monsters in beeld brengen, namelijk (i) uitgedroogd en geleidend, (ii) uitgedroogd en niet-geleidend, en (c) niet-uitgedroogd (nat). Natuurlijk geleidende monsters die zijn uitgedroogd, kunnen eenvoudig worden gemonteerd en afgebeeld. Uitgedroogde en niet-geleidende monsters moeten echter worden gecoat met een geleidende laag om het monster tegen oververhitting te beschermen en de beeldkwaliteit te verbeteren. Deze monsters worden geleidend gemaakt door het monster te bedekken met een dunne goudlaag met behulp van een sputtercoater. Het maakt gebruik van argonionen om goudatomen van een gouden plaat te slaan en het monsteroppervlak met goud te bedekken, waardoor het oppervlak geleidend wordt voor elektronen.

Natte monsters kunnen in de vacuümomgeving van de microscoop uitgassen.

Natte SEM, ook wel Environmental SEM of ESEM genoemd, moet tijdens de beeldvorming de watersublimatie in het monster controleren. Dit wordt bereikt door het monster eerst te bedekken met een dunne laag van een membraanachtig polymeer dat transparant is voor elektronenstralen en het natte monster scheidt van het vacuüm. Vervolgens wordt de temperatuur verlaagd, waardoor de verzadigde dampspanning afneemt. Deze omstandigheden maken het mogelijk dat het instrument bij een hoger vacuüm werkt, waardoor de ultrastructurele kenmerken van het monster behouden blijven en de ruis wordt verminderd. Een Deben Coolstage kan ook worden gebruikt om natte monsters in beeld te brengen door ze te bevriezen tot -25 ° C, waardoor de monsterstructuur behouden blijft en ontgassing wordt voorkomen terwijl elektronen deze bombarderen.

Voorbereiding van monsters voor elektronenmicroscopie

To be visualized by an electron microscope, either transmission or scanning, biological samples need to be fixed (stabilized) so the electron beam does ...

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 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

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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...

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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

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Electron Microscopy Sample Prep Methods

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