•Why is low dose necessary?...TransCure Single Particle Cryo-EM Course | August 2018 | Ricardo Guerrero-Ferreira Beam deflection coils Tilt:beam bents off-axis, then towards the

TransCure Single Particle Cryo-EM Course | August 2018 | Ricardo Guerrero-Ferreira

• Why is low dose necessary?

Low dose imaging

Adapted from: Guerrero-Ferreira and Wright, 2014. JSB

TransCure Single Particle Cryo-EM Course | August 2018 | Ricardo Guerrero-Ferreira

Acquisition conditions• Record (exposure)• Focus/Tracking• Trial • View (search)

Low dose imaging

TransCure Single Particle Cryo-EM Course | August 2018 | Ricardo Guerrero-Ferreira

Search Mode Exposure Mode

Low dose imaging

TransCure Single Particle Cryo-EM Course | August 2018 | Ricardo Guerrero-Ferreira

Exposure area

• Minimize the dose on the area where your sample is

sample

objective lens

objective aperture

SEARCH

Low dose imaging

TransCure Single Particle Cryo-EM Course | August 2018 | Ricardo Guerrero-Ferreira

Exposure area

• Minimize the dose on the area where your sample is

FOCUS/TRACKING

Low dose imaging

sample

objective lens

objective aperture

TransCure Single Particle Cryo-EM Course | August 2018 | Ricardo Guerrero-Ferreira

Exposure area

• Minimize the dose on the area where your sample is

EXPOSURE

Low dose imaging

sample

objective lens

objective aperture

TransCure Single Particle Cryo-EM Course | August 2018 | Ricardo Guerrero-Ferreira

• Find an area

• Check condenser lens astigmatism

• Set eucentric height (z-height, z-shift, or z-adjustment)– Tilting of the specimen leads to

minimal movement of the image

Alignments: Eucentric height

TransCure Single Particle Cryo-EM Course | August 2018 | Ricardo Guerrero-Ferreira

Beam deflection

coils

Shift: beam is displaced but travels parallel to the optical axis Specimen

Alignments: Pivot points• Done to align the deflection coils

– Beam shift– Beam tilt

• Shift and tilt should be completely independent

TransCure Single Particle Cryo-EM Course | August 2018 | Ricardo Guerrero-Ferreira

Beam deflection

coils

Tilt: beam bents off-axis, then towards the same point on the specimen but at an angle Specimen

Alignments: Pivot points• Done to align the deflection coils

– Beam shift– Beam tilt

• Shift and tilt should be completely independent– No beam shift results when a beam tilt is applied

TransCure Single Particle Cryo-EM Course | August 2018 | Ricardo Guerrero-Ferreira

Beam deflection

coils

Specimen displaced in Z

Specimen

Alignments: Pivot points• Done to align the deflection coils

– Beam shift– Beam tilt

• Shift and tilt should be completely independent– No beam shift results when a beam tilt is applied– If wrong: incorrect defocus will be

calculated

TransCure Single Particle Cryo-EM Course | August 2018 | Ricardo Guerrero-Ferreira

• Wobble the objective lens

• Adjust the beam until rotation occurs around the center of the screen

• The image should not move when the focus is changed

Alignments: Rotation center

TransCure Single Particle Cryo-EM Course | August 2018 | Ricardo Guerrero-Ferreira

• It is done properly on a carbon film and running live FFT at the same time

• Use the objective stigmator and adjust

Alignments: Astigmatism correction

TransCure Single Particle Cryo-EM Course | August 2018 | Ricardo Guerrero-Ferreira

• It is done properly on a carbon film and running live FFT at the same time

• Contrast and FFT should not change when the beam is tilted.

Alignments: Coma-free

TransCure Single Particle Cryo-EM Course | August 2018 | Ricardo Guerrero-Ferreira

SpecimenFront focal planeBack focal plane

ObjectiveObjective

Mini condenser

C3

C2 aperture

C2The last condenser crossover is at the front focal plane of the objective lens

The diffraction pattern is focused at the back focal plane (where the objective lens crossover is)

Alignments: Parallel illumination (3 condenser system)

Adapted from: Matthijn Vos, 2014

TransCure Single Particle Cryo-EM Course | August 2018 | Ricardo Guerrero-Ferreira

TEM Modes

Nanoprobe Microprobe