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

I campioni biologici devono essere preparati per la microscopia elettronica per proteggere il campione dai danni causati dal potente fascio di elettroni e dalla bassa pressione creata dal vuoto.
La preparazione del campione inizia con la fissazione chimica e la completa disidratazione. I fissativi chimici come la glutaraldeide o la formaldeide reticolano le proteine, mentre il tetrossido di osmio viene spesso utilizzato per fissare i lipidi di membrana.
Successivamente, il campione viene disidratato gradualmente con concentrazioni crescenti di etanolo o acetone per evitare il restringimento del campione.
Per la TEM, i campioni sono incorporati in una resina epossidica dura. Un ultramicrotomo taglia quindi il campione in sezioni d'urto ultrasottili attraverso le quali gli elettroni possono passare.
Queste sezioni sono colorate con metalli pesanti, come l'acetato di uranile. Le molecole che legano i metalli appaiono più scure e forniscono contrasto all'immagine.
Per il SEM, i campioni vengono ulteriormente essiccati utilizzando anidride carbonica liquida in un processo chiamato essiccazione del punto critico. I campioni vengono quindi rivestiti con polverizzazione catodica con un sottile strato di metallo conduttivo, come l'oro, prevenendo danni termici al campione e fornendo una migliore qualità dell'immagine.

preparazione dei campioni per la microscopia elettronica

Preparation of Samples for Electron Microscopy

Per essere visualizzati da un microscopio elettronico, sia a trasmissione che a scansione, i campioni biologici devono essere fissati (stabilizzati) in modo che il fascio di elettroni non li distrugga ed essiccati accuratamente (essiccati/disidratati) in modo che il vuoto non li intacchi. La fissazione deve essere eseguita il più rapidamente possibile poiché le proprietà del campione inizieranno a cambiare non appena verrà rimosso dal suo ambiente naturale. Ad esempio, in un campione di tessuto, i livelli di ossigeno iniziano a diminuire, causando un aspetto alterato dei mitocondri.

Progressi nella preparazione dei campioni TEM

Criofissazione

In questa tecnica, le cellule e gli organelli più piccoli possono essere congelati mediante congelamento a immersione, mentre le cellule e i tessuti più grandi possono essere congelati utilizzando l'alta pressione. Il congelamento a immersione si ottiene posizionando una piccola quantità di campione su una griglia EM fredda e quindi immergendolo rapidamente nell'etano liquido riempito in un serbatoio e circondato da azoto liquido. Il congelamento rapido garantisce che l'acqua non cristallizzi. Nel congelamento ad alta pressione, l'azoto liquido viene utilizzato per congelare rapidamente il campione e viene sottoposto a una pressione di circa 2000 bar per rallentare la formazione dei cristalli di ghiaccio. I campioni criofissati vengono sezionati in un crio-ultramicrotomo costituito da un coltello diamantato congelato.

Proiezione dell'ombra

La piattaforma rotante in una camera a vuoto contiene il campione, che viene poi rivestito con metallo pesante. La rotazione del campione garantisce un rivestimento metallico sul lato anteriore e un effetto ombra sul retro. La tecnica è particolarmente utile quando si studiano i dettagli ultrastrutturali di batteri o virus, DNA, RNA o proteine isolate.

Colorazione negativa

Piccoli campioni, come ribosomi, molecole di enzimi, virus, ecc., possono essere sottoposti a imaging direttamente senza sezionamento. I campioni vengono miscelati con molibdato di ammonio, acetato di uranile o acido fosfotungstico, che sono opachi agli elettroni. Sulla griglia si forma uno strato metallico, tranne dove si trova il campione, creando un'immagine negativa del campione. Il contrasto si ottiene come risultato del modello di distribuzione del metallo.

Preparazione di campioni essiccati e non essiccati per SEM

Il SEM può visualizzare tre tipi di campioni, vale a dire (i) essiccati e conduttivi, (ii) essiccati e non conduttivi e (c) non essiccati (bagnati). I campioni naturalmente conduttivi essiccati possono essere facilmente montati e sottoposti a imaging. Tuttavia, i campioni essiccati e non conduttivi devono essere rivestiti con uno strato conduttivo per proteggere il campione dal surriscaldamento e migliorare la qualità dell'immagine. Questi campioni vengono resi conduttivi rivestendo il campione con un sottile strato d'oro utilizzando un rivestimento a spruzzo. Utilizza ioni di argon per staccare gli atomi d'oro da una piastra d'oro e rivestire la superficie del campione con oro, rendendo la superficie conduttiva agli elettroni.

I campioni bagnati possono produrre gas nell'ambiente sotto vuoto del microscopio.

Il SEM umido, chiamato anche SEM ambientale o ESEM, deve controllare la sublimazione dell'acqua nel campione durante l'imaging. Si ottiene rivestendo innanzitutto il campione con uno strato sottile di un polimero membranoso trasparente ai fasci di elettroni e separando il campione bagnato dal vuoto. Quindi la temperatura diminuisce, il che diminuisce la pressione del vapore saturo. Queste condizioni consentono il funzionamento dello strumento a un vuoto più elevato che preserva le caratteristiche ultrastrutturali del campione e riduce il rumore. Un Deben Coolstage può anche essere utilizzato per visualizzare campioni bagnati congelandoli a -25°C, preservando la struttura del campione e prevenendo il degassamento mentre gli elettroni lo bombardano.

Preparazione dei campioni per la microscopia elettronica

Per essere visualizzati da un microscopio elettronico, sia a trasmissione che a scansione, i campioni biologici devono essere fissati (stabilizzati) in modo che il fascio di elettroni ...

preparation-of-samples-for-electron-microscopy

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Cell Biology

Biologia cellulare

Unit 1

Unità 1

Visualizing Cells, Tissues, and Molecules

Visualizzazione di cellule, tessuti e molecole

KEY TERMS AND CONCEPTS

TERMINI E CONCETTI CHIAVE

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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Surface-Only Imaging with TIRF Microscopy

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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...

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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

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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