cryo-scanning electron microscopymicroscopy.anu.edu.au/files/cryo-sem_presentation.pdf ·...
TRANSCRIPT
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
9
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
12
Things to consider:• Signal to noise ratio.
• Resolution of the image.
• Depth of field.
• Avoid charging.
• Avoid scanning in a small area.
After etching
13
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.