Microscope Basics

Midlands Cryo-EM Workshop

2021
Christos Savva

1931
2021
 Electron Microscopy

Electromagnetic
Glass Lenses
Lenses

MicrobiologyInfo.com
 Transmission Electron Microscopy
- The Transmission electron microscope is required for high-
resolution structural studies
- Requires ultra-thin specimens <0.2 μm
- 100 kV electrons λ=0.037 Å
- Lens aberrations limit resolution to ~1 Å (0.5Å in some cases)
Electron Micrograph: Picture taken with an EM

Gold particles on carbon

film. Lattice spacing of
2.04 Å

Image:
Electron Microscopy Sciences
 Transmission Electron Microscopy

Electrons attracted to nucleus of atom.

The higher the atomic number (Z) the
higher the scattering.
Figures 4 and 5 Frank Krumeich
 A very Basic TEM
(that probably wont work)

Gun/Filament/Cathode: Source of electrons

-300,000 V
Anode: Attracts the electrons

240 V Condenser: Focuses the electrons

Aperture
Specimen holder/loader
High Tension Generator Aperture
Objective: Creates the first image of the sample

Projector: Magnifies the first image further

~1x10-8 mbar

Camera
Vacuum Pump
The Transmission Electron Microscope
 Wave Coherence

• Spatial Coherence: Are the electrons coming from the same direction and in
phase?
• Temporal coherence: Do the electrons have the same energy/speed
(wavelength)?

Coherent Waves

Incoherent Waves
 TEM Gun types
Thermionic: W or LaB6 Field Emission Gun (Schottky)

100nm

Temporal 1.0-2.0 Energy Spread (eV) 0.2-0.3

>104 Source Size (nm) <30
Spatial
105 Brightness (A/cm2xsr) 108
10-3 Vacuum required (Pa) 10-8
100-300 Life Span (hrs) ~8000
 Electron Beam Coherence

Thermionic gun Field Emission Gun

 Gun Dependent Resolution

30 Å

Thermionic Gun

3.9 Å

Field Emission Gun

(Alice Clark, 2014)

 Lenses

Image Plane

F
Object

Lens

F=Focal point
 Lenses

Image:
myscope.training
 Lens aberrations

Lenses are not perfect and suffer from aberrations

Perfect Lens

Spherical Aberration (Cs)

Requires expensive hardware to correct

 Chromatic aberration (CC)
• Energy spread of electrons due to gun temporal coherence
• Thick samples also result in many electrons with lost energy
• Cs correctors available but expensive
• Energy filters can remove energy loss electrons
• More important for tomography of thick specimens

Chromatic Aberration of the objective lens

 FFT of sample

Lens aberrations
Objective Lens Astigmatism

Astigmatic
• Lenses are not perfect and suffer from aberrations

Perfect Lens

Spherical Aberration
Astigmatism is easy to correct on modern microscopes

Corrected
 Condenser Lens Astigmatism

Condenser lens: produces an image of your electron source

Check for a circular beam

Beam shape

Astigmatic Non Astigmatic

Beam on detector

www.rodenburg.org
 Electron beam path through the lenses
Microscopes with 2 condenser lenses

Spot Size
Beam diameter

Diffraction plane

Image plane

Image: myscope.training
 Choice of Accelerating Voltage

What kV should I use? What to consider:

- Resolution: Wavelength at 300 kV vs smaller that at 100 kV
- Aberrations: higher effect of lens aberrations higher at lower
kV
- Radiation damage: 1.5 x less dam age at 300 kV vs 100 kV
- Useful information: 25% higher elastic/inelastic ratio at 100 kV
- Detector performance: DDD perform worst at lower kV.
Detectors for lower kV scopes have reduced field of view
- Cost: 100 kV much cheaper than 200 or 300 kV
- Sample thickness: Can image thicker samples at higher kV

Peet et al. 2019 and Naydenova et al. 2020

A 100 kV FEG TEM

Tundra Cryo-TEM
 Specimen Holders: Side entry
Samples kept at ~-175℃

Dubochet MKI Gatan 626: 1 grid

Gatan 910: 3 grids at once Simple Origin 200: Takes 2 autogrids

 Specimen Holders: Side entry
Advantages
- Cheap (£40-80K…yes that’s cheap!!)
- Can be used on almost any side-entry TEM (Anti-contaminator required)
- Was the only option until recent years

Disadvantages
- Fiddley to use
- Fragile/easy to break
- Require pumping/heating for optimal performance (Pumping station and Heater)
- Low throughput (Sample exchange takes ~45-60min)
- Each load cycle introduces moisture to the column
- Stability not great: Drift, vibration prone
- Require manual LN2 top-up
 Autoloaders

TFS Glacios 200 kV TFS Krios 300 kV JEOL Cryo-ARM 200 JEOL Cryo-ARM 300
 Microscope Stage
• The stage or goniometer supports the specimen holder
• On Autoloader systems the holder is always inserted
• Moves along X, Y and Z directions and tilts along ⍺ (and does the same to
your sample)
Philips CM200 Stage Krios Stage
⍺

X Z

Y
 Setting the sample to Eucentric Position
Image Plane

F=Focal point=Eucentric Focus

Objective Lens

F’
 Detectors for the TEM

Detective Quantum Efficiency (DQE) =SNR2o/SNR2i

A measure of the signal to noise ratio degradation
Perfect detector has DQE of 1

Detector Advantages Drawbacks

Film - Large area - Limited to 50 exposures
- Descent DQE - Needs developing-Scanning
- Adds moisture to microscope
CCD - Easy to use - Low DQE (0.1)
- Instant
Direct electron Detectors - High DQE - Expensive
- Fast frame rates-Movies

McMullan et. al.

Ultramicroscopy, 2014
 CCD vs DED

Complementary metal-oxide semiconductor (CMOS)

www.directelectron.com
 Integration vs Counting
Integration
• Short exposures
• High Dose-rates
applications
• Lower DQE
Electron enters Electron signal is Charge collects in
detector. scattered. each pixel.

Counting
• Very low dose rate
(0.5-15 e-/pixel/sec)
• Fast frame rates
• Long exposures
Events reduced to • Higher DQE
www.gatan.com highest charge pixels.
 DQE comparison of some detectors

Previous Generation DDD The GATAN K3 (Paul Mooney, GATAN)

McMullan et. al.

Ultramicroscopy, 2014 Nyquist: 2X the magnified pixel size
 Super-resolution
Counting Counting with Super-resolution

Events reduced to Events Events

highest charge pixels. localised to sub-pixel
accuracy.

• Super-resolution with Fourier cropping “binning” increases DQE

• Increases disk space requirement
• Even allows one to go beyond physical Nyquist:
- Recent example at our facility: Data at 81K (Pixel 1.09Å). Collected at SR bin1
- Resolution reached physical Nyquist (~2.2Å)
- Re-extracted SR movies during polishing step > Reached 1.9Å
 Movies instead of snapshots

• CCD and film limited to one exposure

• Fast frame rate of DED allows movie collection

Bai et. al, 2015, TBS

 Movies instead of snapshots

Single Frame Summed frames

(No alignment)

Power Spectra Summed frames

Unaligned /Aligned (With alignment)

McMullan, 2016
 Individual particle tracking

Particles can move in the ice

- Electrostatic attraction
- Release of stress in the ice

Scheres, 2014, Elife

 Radiation Damage

• Biological samples are radiation sensitive

• Bonds are broken and free radicals released
• Imaging performed using “Low Dose” methods
• Each micrograph receives a limited amount of
electrons to prevent structure deformation
• Typically we use 40-60 electrons/Å2 per micrograph
 Using movies to deal with radiation damage

“Old days” (before 2012)

Single image
1 second exposure at 40
e-/Å2/sec dose rate

Dose per image: 40 e-/Å2

Current date: New detectors
40 frames/sec, 1 second exposure at 40 e-/Å2/sec dose rate
Higher radiation damage:
Lower radiation damage: High frequencies are signal High frequencies are noise

Frame 1 2 3 4 ………………….. 40
Accumulated
Dose (e-/Å2) 1 2 3 4 ………………….. 40
 Radiation damage weighting

Scheres, 2014, eLife

 Microscope Apertures

• An aperture is a small hole in a strip of metal inserted in the

beam path
• Will only allow the central beam to go through

From Williams and Carter, 2009

 Condenser Aperture

C2 aperture

C2 aperture Produces a more coherent, parallel beam

 Objective Aperture

Specimen scattered electrons

e- e-

objective aperture

Increases Contrast
 Objective Aperture

Also acts as a low-pass filter removing high-resolution

information
Consider resolution cut-off. e.g on Krios:

30 μm > 4 Å
70 μm > 1.8 Å
100 μm > 1.4 Å
 Magnification and Pixel sizes
Nominal Magnification

Pixel size= Calibrated Magnification

Physical Pixel Size

Gold diffraction: 2.35Å Known atomic model fitting

Thanks and Questions