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

Próbki biologiczne muszą być przygotowane do mikroskopii elektronowej, aby chronić próbkę przed uszkodzeniem przez silną wiązkę elektronów i niskie ciśnienie wytwarzane przez próżnię.
Przygotowanie próbki rozpoczyna się od chemicznego utrwalenia i całkowitego odwodnienia. Utrwalacze chemiczne, takie jak aldehyd glutarowy lub formaldehyd, sieciują białka, podczas gdy tetratlenek osmu jest często używany do wiązania lipidów błonowych.
Następnie próbka jest stopniowo odwadniana przy rosnących stężeniach etanolu lub acetonu, aby uniknąć kurczenia się próbki.
W przypadku TEM próbki są zatopione w twardej żywicy epoksydowej. Następnie ultramikrotom kroi próbkę na ultracienkie przekroje, przez które mogą przechodzić elektrony.
Sekcje te są zabarwione metalami ciężkimi, takimi jak octan uranylu. Cząsteczki wiążące metale wydają się ciemniejsze i zapewniają kontrast z obrazem.
W przypadku SEM próbki są dalej suszone przy użyciu ciekłego dwutlenku węgla w procesie zwanym suszeniem w punkcie krytycznym. Próbki są następnie napylane cienką warstwą przewodzącego metalu, takiego jak złoto, co zapobiega uszkodzeniom termicznym próbki i zapewnia lepszą jakość obrazu.

przygotowanie próbek do mikroskopii elektronowej

Preparation of Samples for Electron Microscopy

Aby próbki biologiczne można było uwidocznić za pomocą mikroskopu elektronowego, transmisyjnego lub skaningowego, należy je utrwalić (ustabilizować), aby wiązka elektronów ich nie zniszczyła, i dokładnie wysuszyć (wysuszyć/odwodnić), aby próżnia nie miała na nie wpływu. Utrwalanie należy przeprowadzić tak szybko, jak to możliwe, ponieważ właściwości próbki zaczną się zmieniać, gdy tylko zostanie ona usunięta ze swojego naturalnego środowiska. Na przykład w próbce tkanki poziom tlenu zaczyna spadać, powodując zmianę wyglądu mitochondriów. 

 Postępy w przygotowaniu próbek TEM 

 Kriofiksacja 

 W tej technice małe komórki i organelle można zamrażać metodą zanurzeniową, natomiast większe komórki i tkanki można zamrażać pod wysokim ciśnieniem. Zamrażanie wgłębne osiąga się poprzez umieszczenie małej ilości próbki na zimnej siatce EM, a następnie szybkie zanurzenie w ciekłym etanie wypełnionym zbiornikiem i otoczonym ciekłym azotem. Szybkie zamrażanie gwarantuje, że woda nie krystalizuje. W przypadku zamrażania wysokociśnieniowego do szybkiego zamrożenia próbki stosuje się ciekły azot, który następnie poddaje się ciśnieniu ~2000 barów, aby spowolnić tworzenie się kryształków lodu. Próbki krioutrwalone są krojone w krioultramikrotomie składającym się z zamrożonego noża diamentowego. 
 Rzucanie cienia 

 Platforma obrotowa w komorze próżniowej utrzymuje próbkę, która następnie jest powlekana metalem ciężkim. Obrót próbki zapewnia metaliczną powłokę na przedniej stronie i efekt cienia na spodniej stronie. Technika ta jest szczególnie przydatna podczas badania szczegółów ultrastrukturalnych bakterii lub wirusów, DNA, RNA lub izolowanych białek. Barwienie negatywne 

 Małe próbki, takie jak rybosomy, cząsteczki enzymów, wirusy itp., można obrazować bezpośrednio, bez dzielenia na kawałki. Próbki miesza się z molibdenianem amonu, octanem uranylu lub kwasem fosforowolframowym, które są nieprzezroczyste dla elektronów. Na siatce, z wyjątkiem miejsca, w którym znajduje się próbka, tworzy się warstwa metalu, tworząc negatywowy obraz próbki. Kontrast uzyskuje się w wyniku wzoru rozmieszczenia metalu. 

 Przygotowanie wysuszonych i niewysuszonych próbek do SEM 

 SEM może obrazować trzy typy próbek, a mianowicie (i) wysuszone i przewodzące, (ii) wysuszone i nieprzewodzące oraz (c) niewysuszone (mokre). Naturalnie przewodzące próbki, które zostały wysuszone, można łatwo zamontować i zobrazować. Jednakże próbki wysuszone i nieprzewodzące muszą być pokryte warstwą przewodzącą, aby zabezpieczyć próbkę przed przegrzaniem i poprawić jakość obrazu. Próbkom tym nadaje się przewodność poprzez pokrycie próbki cienką warstwą złota za pomocą powlekarki napylającej. Wykorzystuje jony argonu do wybijania atomów złota ze złotej płytki i pokrywania powierzchni próbki złotem, dzięki czemu powierzchnia przewodzi elektrony. 

 Mokre próbki mogą odgazować w próżniowym środowisku mikroskopu. 

 Mokry SEM, zwany także środowiskowym SEM lub ESEM, musi kontrolować sublimację wody w próbce podczas obrazowania. Osiąga się to poprzez pokrycie próbki cienką warstwą membranowego polimeru, który jest przezroczysty dla wiązek elektronów i oddziela mokrą próbkę od próżni. Następnie obniża się temperaturę, co powoduje zmniejszenie prężności pary nasyconej. Warunki te pozwalają na pracę instrumentu przy wyższej próżni, która pozwala zachować cechy ultrastrukturalne próbki i redukuje hałas. Urządzenie Deben Coolstage można również wykorzystać do obrazowania mokrych próbek poprzez zamrożenie ich do temperatury -25°C, co pozwala zachować strukturę próbki i zapobiega odgazowaniu podczas bombardowania jej przez elektrony.

Przygotowanie próbek do mikroskopii elektronowej

Aby można je było uwidocznić za pomocą mikroskopu elektronowego, zarówno transmisyjnego, jak i skanującego, próbki biologiczne muszą być utrwalone (ustabilizowane), aby wiązka elektronów nie ...

preparation-of-samples-for-electron-microscopy

Education

JoVE Core

Cell Biology

Biologia komórki

Unit 1

Jednostka 1

Visualizing Cells, Tissues, and Molecules

Wizualizacja komórek, tkanek i cząsteczek

KEY TERMS AND CONCEPTS

Kluczowe terminy i pojęcia

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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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...

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Immunogold Labeling in Electron Microscopy

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Cryo-EM for Native Biomolecule Imaging

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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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