CRYO-Scanning Electron Microscopy

Problems for the observation of Bio-samples

Observing hydrated Bio-samples in a conventional SEM

Water evaporates too rapidly sample shrinks and distorts.

contaminates the SEM chamber.

Standard procedures for observation of biological material in an SEM

Living cells and tissues

Chemically fix (kill and preserve)

Dehydrate Embed and section

All water, and water- and solvent- soluble matter removed by dehydration, resulting in:

1) Shrinkage and distortion of hydrated material.

2) Loss of distinction between liquid and gas-filled spaces and loss of waxes and resins.

3) Swelling and distortion of inherently desiccated tissues.

4) Long preparation procedure.

Coat sample with conductive coating

SEM observation

Post-staining

Why use CRYO-SEM?

CRYO – SEM

Immobilisation of water and soluble materials by freezing them. Vulnerable biological structures are then well preserved.

then observing them in a CRYO SEM.

CRYO-SEM secondary electron image: mucus on the sample surface is visible, representing the inherent surface morphology of the rat tongue (image 2).

Morphology of rat tongue

Image 2: Cryo-fixed sample

Image 1: Critical point dried sample

What is CRYO-SEM?Conventional SEM

+a CRYO stage in the sample chamber, anda CRYO preparation chamber with a coating system.

• Sample preparation

• Etching

• Coating

• Observation

Operational procedures

Cryo SEM workflow

1. Stick specimen to stub.

2. Fit stub to holder.

3. Fit holder to vacuum transfer device.

4. Plunge into slush.

5. Transfer into preparation chamber.

6. Fracture specimen if needed.

7. Sublimation of ice on the sample surface in an SEM (etching).

8. Sputter coat in preparation chamber.

9. Observation.

CRYO-specimen holders and adhesives

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Conductive adhesives: mixture of Tissue-Tek and Colloidal graphite (ratio 1:1)

Sample preparation

Live material

CRYO

preparation chamber

CRYO-planing using

CRYO-microtome

Direct freeze in preparation chamber

CRYO-fixation:Plunge in N2 Slush

CRYO-pliers

High pressure freezer

CRYO preparation chamber

Imaging

Example of sample damage when focusing at high magnification

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Things to consider:• Signal to noise ratio.

• Resolution of the image.

• Depth of field.

• Avoid charging.

• Avoid scanning in a small area.

After etching

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Purpose of Etching: sublimating any frost that is accumulated on the frozen

sample surface during its handling.

Method: Raise temperature of the CRYO stage from LN2 temperature to -90oCwhile the sample is observed in SEM. When all ice on the sample surface is sublimated, cool down the stage to LN2 temperature.

Before etching

Etching

Purpose of Coating: making sample conductive on the surface to avoid charging.

Normally, bio-samples are not conductive. It causes charging under the electron beam.

Charging results in:- deflection of the beam and secondary electrons- periodic bursts of secondary electrons

After etching, the sample is transferred to the CRYO preparation chamber and coated with a conductive coating at near LN2 temperature.

Au or Al coating makes the sample surface conductive to avoid charging. Also, the secondary electron yield of the metal coating is much higher than the bio-sample.

Coating

ObservationCRYO-SEM parameters (accelerating voltage and beam current) need to be carefully set up in order to avoid sample damage by electron beam. The prepared sample is observed on the CRYO stage below -140oC to avoid further dehydration of the sample.

What can be observed with Cryo-SEM?

Biological samples

• External morphology and internal anatomy of biological samples

• Soils and root/soil interfaces

• Gels and mucilages

Other samples

• Food products including emulsions

• Medicine, especially emulsions and lotions

• Ice, snowflakes

• Fluid inclusions in rocks, including oil and brine in sandstone

• Resins, paints, polymers

Example 1 – Plunge freezing method

with permission from Dr. Ryan McQuinn, ANU

Images of Arabidopsis flower with pollenPollen absorbed on the anthers and pistils would easily be lost in the chemical fixation process. With the plunge freezing method, pollen

was fixed in the original position on anthers and pistils.

Pollen absorbed on the anthers Arabidopsis pistil with some pollen attached

Example 2 – CRYO planing method

CRYO-planed protoxylem of Azorellamacquariensis and Colobanthusmuscoides

CRYO-SEM not only visualises the ultra-structure of the CRYO-planed hydrated samples, but also allows the water level in hydrated samples to be controlled by etching techniques. It is critical for understanding the embolism repair in relation to xylem structure.

Easy Come, Easy Go: Capillary Forces Enable Rapid Refilling of Embolized Primary Xylem Vessels

Vivien Rolland, Dana M. Bergstrom, Thomas Lenné, Gary Bryant, Hua Chen, Joe Wolfe, N. Michele Holbrook, Daniel E. Stanton, Marilyn C. BallPlant Physiology: Volume.168. pages 1636-1647, 2015Protoxylem elements: Scale bars

10 µm (A–F) and 2 µm (A′, A′′, and C′).

Example 3 – CRYO fracturing method

CRYO-fractured flower petal

Summary

CRYO-SEM- introduces less artefacts for a hydrated biological sample in

comparison with the conventional chemical fixation technique.

- can be used for observation of both the morphology and the anatomy of biological samples.

- provides a quick, reliable and effective way for viewing the inherent state of hydrated samples.