Phase Plates for Single Particles Radostin Danev Max Planck - - PowerPoint PPT Presentation

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Phase Plates for Single Particles Radostin Danev Max Planck - - PowerPoint PPT Presentation

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Max Planck Institute of Biochemistry Martinsried, Germany MAX PLANCK SOCIETY Phase Plates for Single Particles Radostin Danev Max Planck Institute of Biochemistry, Martinsried, 82152, Germany NRAMM workshop, November 2014, San Diego, USA

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Max Planck Institute

  • f Biochemistry

Martinsried, Germany

MAX PLANCK SOCIETY Max Planck Gesellschaft Max-Planck-Gesellschaft

Phase Plates for Single Particles

NRAMM workshop, November 2014, San Diego, USA

Radostin Danev

Max Planck Institute of Biochemistry, Martinsried, 82152, Germany

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MPI für Biochemie Max Planck Gesellschaft MPI für Biochemie Max-Planck-Gesellschaft

What is a phase plate?

Zernike phase plate

ZPC-TEM

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MPI für Biochemie Max Planck Gesellschaft MPI für Biochemie Max-Planck-Gesellschaft

Phase plate types

central beam OL aperture central beam central beam carbon film carbon film hole

Conventional TEM CTEM Zernike Phase Plate ZPP Volta Phase Plate VPP

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MPI für Biochemie Max Planck Gesellschaft MPI für Biochemie Max-Planck-Gesellschaft

ZPP vs. VPP, in-focus CTF example

VPP

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MPI für Biochemie Max Planck Gesellschaft MPI für Biochemie Max-Planck-Gesellschaft

VPP evolution

Niquist frequency ~ 4.3A

VPP image series FFT

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MPI für Biochemie Max Planck Gesellschaft MPI für Biochemie Max-Planck-Gesellschaft

The Volta potential hypothesis

carbon film central beam

  • The effect is not due to beam-induced

contamination or etching – confirmed experimentally.

  • The effective “charge” of the irradiated

area is negative (it produces less phase shift than the surrounding film)!

  • The properties of the area recover to

normal in ~ 1 day (depends on the temperature).

  • > The central beam changes the electronic

properties of the irradiated carbon film area.

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MPI für Biochemie Max Planck Gesellschaft MPI für Biochemie Max-Planck-Gesellschaft

beam

Volta potential - surface chemistry hypothesis

  • The carbon surface is in a chemical equilibrium with the residual gases in the

vacuum.

  • Electron irradiation breaks the bonds and causes local de-termination of the surface.
  • Unsaturated dangling bonds cause excess of electrons on the surface.

carbon film carbon film

  • - - - - - - - -
  • - - - - - - - -

+ + + + + + + + + + + + + + + + + +

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MPI für Biochemie Max Planck Gesellschaft MPI für Biochemie Max-Planck-Gesellschaft

A constant potential disk on a surface

  • 1 V potential
  • 1 um diameter disk
  • 215 V.nm -> /2

phase shift @ 200 kV.

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MPI für Biochemie Max Planck Gesellschaft MPI für Biochemie Max-Planck-Gesellschaft

Beam-induced phase shift series

  • 12 nm thick carbon film; beam current: 1.0 nA; beam diameter: 1 mm
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MPI für Biochemie Max Planck Gesellschaft MPI für Biochemie Max-Planck-Gesellschaft

VPP – phase shift vs. beam current

  • The phase shift depends on the total dose and not on the dose rate.
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MPI für Biochemie Max Planck Gesellschaft MPI für Biochemie Max-Planck-Gesellschaft

VPP beam spots

day 1 day 1? day 3 ZPP hole ZPP hole day 1 day 3, day 1 spot has disappeared!

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MPI für Biochemie Max Planck Gesellschaft MPI für Biochemie Max-Planck-Gesellschaft

VPP beam spot recovery

  • The recovery speed of beam spots does not depend on the temperature.
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MPI für Biochemie Max Planck Gesellschaft MPI für Biochemie Max-Planck-Gesellschaft

New PP holder and phase plate

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MPI für Biochemie Max Planck Gesellschaft MPI für Biochemie Max-Planck-Gesellschaft

FEI tools for phase plate navigation

  • 6 slots x 125 positions  750 fresh areas
  • Single area for ~1 hr operation

Phase Plate Slot

On Next button Used area of the Phase Plate

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MPI für Biochemie Max Planck Gesellschaft MPI für Biochemie Max-Planck-Gesellschaft

FEI tools for phase plate alignments

Alignments routine use

  • n-plane

condition Beam shift pivot point

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MPI für Biochemie Max Planck Gesellschaft MPI für Biochemie Max-Planck-Gesellschaft

Phase plate lifetime tests

  • The first phase plate of the new type installed in the Tecnai F20 was

used for > 6 ½ months with no observable deterioration in performance.

  • The first phase plate installed in the Titan Krios has been there for > 4

months with no performance degradation.

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MPI für Biochemie Max Planck Gesellschaft MPI für Biochemie Max-Planck-Gesellschaft

Phase plate lifetime – grid 121, Tecnai F20

0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 5 10 15 20 25 30

phase shift / pi week

Phase shift after 100 nC exposure on-plane

extrapolated from 25 nC total exposure measurement

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MPI für Biochemie Max Planck Gesellschaft MPI für Biochemie Max-Planck-Gesellschaft

Phase plate lifetime – PP1, Titan Krios

0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 5 10 15 20 25 30

phase shift / pi week

Phase shift after 100 nC exposure on-plane

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MPI für Biochemie Max Planck Gesellschaft MPI für Biochemie Max-Planck-Gesellschaft

VPP CTF performance

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MPI für Biochemie Max Planck Gesellschaft MPI für Biochemie Max-Planck-Gesellschaft

VPP CTF performance

  • 500 nm defocus; ~ 18% signal loss, expected ~ 12%
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MPI für Biochemie Max Planck Gesellschaft MPI für Biochemie Max-Planck-Gesellschaft

ZPP vs VPP images

VPP-TEM de-fringed ZPP-TEM ZPP-TEM

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MPI für Biochemie Max Planck Gesellschaft MPI für Biochemie Max-Planck-Gesellschaft

Cryo-EM, ZPP vs VPP

  • Sample & data collection by Fuku; primary neuron culture.

ZPP VPP

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MPI für Biochemie Max Planck Gesellschaft MPI für Biochemie Max-Planck-Gesellschaft

VPP vs. ZPP

VPP ZPP

long life constant phase shift less information loss?!? no fringes! user friendly easy to automate

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MPI für Biochemie Max Planck Gesellschaft MPI für Biochemie Max-Planck-Gesellschaft

Important practical points for VPP use

  • Beam shift pivot points - the beam must pass through the same spot on the

phase plate in both Focus and Exposure low-dose modes. This allows the phase plate to be “renewed” at any time by moving to another place on the PP film followed by “conditioning” (continuous irradiation for ~ 30 sec) in Focus mode.

  • On-plane condition – best contrast (lowest cuton); ~ 0.25  phase shift; no PP

film grain projected on the image (a la Ronchigram).

  • Defocus – for tomography work VPP requires some defocus for best transfer of

the high spatial frequencies. The defocus is set so that the conventional (no phase shift) CTF has its first maximum at ~ 2/3 Nyquist. Typical defocus value: ~ 500 nm underfocus.

  • Heating – to ~ 300 oC is essential for maintaining the phase plate in “Volta mode”.

It prevents contamination and accelerates the “healing” (~ 1 – 2 days) of the used spots on the PP.

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MPI für Biochemie Max Planck Gesellschaft MPI für Biochemie Max-Planck-Gesellschaft

Beam shift pivot points

phase plate back focal plane specimen specimen plane

Exposure Focus Exposure Focus

Correct pivot point setting Incorrect pivot point setting

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MPI für Biochemie Max Planck Gesellschaft MPI für Biochemie Max-Planck-Gesellschaft

PP on-plane & off-plane

central beam phase plate

Volta Phase Plate

  • n-plane

(parallel illumination)

phase plate

Volta Phase Plate

  • ff-plane

(non-parallel illumination)

back focal plane

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MPI für Biochemie Max Planck Gesellschaft MPI für Biochemie Max-Planck-Gesellschaft

VPP – on-plane vs. off-plane

  • beam current: 1.0 nA;
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MPI für Biochemie Max Planck Gesellschaft MPI für Biochemie Max-Planck-Gesellschaft

VPP – on-plane vs. off-plane

  • CTFs on-plane & off-plane; 50 nm cut-on periodicity on-plane.
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MPI für Biochemie Max Planck Gesellschaft MPI für Biochemie Max-Planck-Gesellschaft

VPP – heating is essential

phase plate at 60oC: Contamination & charging phase plate at 225oC: Volta regime

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MPI für Biochemie Max Planck Gesellschaft MPI für Biochemie Max-Planck-Gesellschaft

  • 2.00
  • 1.00

0.00 1.00 2.00 0.1 0.2 0.3 0.4 0.5 0.6 0.7 CTEM, def. 4 um VPP-TEM, def. 500 nm, ps 0.3pi VPP-TEM, def. 500 nm, ps 0.1pi VPP-TEM, def. 0, ps 0.3pi VPP-TEM, def. 0, ps 0.1pi

VPP for tomography - CTFs

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MPI für Biochemie Max Planck Gesellschaft MPI für Biochemie Max-Planck-Gesellschaft

Phase plates for Single Particle Analysis – ideal world

  • Automatically collect images in-focus with the phase plate.
  • Easy automatic picking of the particles.
  • No CTF fitting or correction.
  • Reconstruct the data and get 3 A resolution with 10,000 particles.
  • Press the “Publish” button and order a Mai Tai on Waikiki beach …
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MPI für Biochemie Max Planck Gesellschaft MPI für Biochemie Max-Planck-Gesellschaft

The VPP for Single Particle Analysis

+ Works well with the FEI EPU software. + A couple of additional steps before starting the automated acquisition – insert a phase plate, wait ~ 5 min for the thermal drift of the PP to settle and adjust the on-plane condition.

  • For best results requires a switch of the phase plate every ~ 100-200
  • images. This will be automated in next versions of EPU.
  • At present, CTF fitting and sign-flipping is necessary due to low

focusing accuracy.

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MPI für Biochemie Max Planck Gesellschaft MPI für Biochemie Max-Planck-Gesellschaft

SPA of GroEL with the VPP, Tecnai F20

  • Sample: GroEL; provided by Thomas; ~ 800 kDa; manual data collection.
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MPI für Biochemie Max Planck Gesellschaft MPI für Biochemie Max-Planck-Gesellschaft

Reconstructions by EMAN and RELION

  • SPA reconstruction: 50 micrographs; 3081 particles; D7 symmetry; resolution ~ 8.3 A.
  • No CTF correction
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MPI für Biochemie Max Planck Gesellschaft MPI für Biochemie Max-Planck-Gesellschaft

SPA of β-galactosidase with the VPP, Tecnai F20

  • Sample: purchased from Sigma; ~ 450 kDa
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MPI für Biochemie Max Planck Gesellschaft MPI für Biochemie Max-Planck-Gesellschaft

SPA of β-galactosidase with the VPP, Tecnai F20

  • Sample: purchased from Sigma; ~ 450 kDa
  • SPA reconstruction: Eagle CCD camera, EPU data collection, 484 micrographs; 6221 good particles;

D2 symmetry; resolution ~ 12A

  • CTF corrected
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MPI für Biochemie Max Planck Gesellschaft MPI für Biochemie Max-Planck-Gesellschaft

D3 virus, Tecnai F20

  • Sample: D3 virus capsid; provided by James Conway through Matthijn Vos
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MPI für Biochemie Max Planck Gesellschaft MPI für Biochemie Max-Planck-Gesellschaft

D3 virus, Tecnai F20

  • Sample: D3 virus capsid; provided by James Conway through Matthijn Vos
  • SPA reconstruction: 626 micrographs; 2205 particles; I1 symmetry; resolution ~ 8.1 A
  • CTF corrected
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MPI für Biochemie Max Planck Gesellschaft MPI für Biochemie Max-Planck-Gesellschaft

D3 virus – CTF corrected vs not corrected

  • Sample: D3 virus capsid; provided by James Conway through Matthijn Vos

CTF corrected – 8.1 A CTF uncorrected – 14.4 A

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MPI für Biochemie Max Planck Gesellschaft MPI für Biochemie Max-Planck-Gesellschaft

D3 virus, Titan Krios

  • Sample: D3 virus capsid; provided by James Conway through Matthijn Vos
  • SPA reconstruction: K2 camera, 155 manually acquired micrographs; 936 particles; I1 symmetry;

resolution ~ 8 A

  • CTF uncorrected
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MPI für Biochemie Max Planck Gesellschaft MPI für Biochemie Max-Planck-Gesellschaft

PP focus accuracy requirement

  • 2.00
  • 1.00

0.00 1.00 2.00 0.5 1 1.5 2 2.5 3 CTEM def. -1 um PP def. -70 nm PP def. +40 nm

CTF k, 1/nm

7Å

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MPI für Biochemie Max Planck Gesellschaft MPI für Biochemie Max-Planck-Gesellschaft

Strategies for SPA with the phase plate

  • Close to focus approach – ideal approach with a PP

+ No CTF zeros. + No CTF fitting & correction steps.

  • Requires precise focusing (+40 nm to -70 nm for 7A resolution) !!!
  • No CTF zeros -> cannot fit & correct the CTF, cannot measure SNR, cannot evaluate

image quality.

  • Hybrid approach – approach 1

+ Collect focus pairs: close to focus + defocused image. + Use the defocused image to fit the CTF and correct the close to focus image.

  • CTF fitting and correction steps.
  • Slower data acquisition + larger data volume.
  • Defocus approach – approach 2

+ Does not require precise focusing -> some defocus variation is actually necessary! + Can fit & correct the CTF -> precise defocus & phase shift determination, SNR measurement, image quality assessment.

  • CTF zeros -> information loss, half of the advantage of the PP is lost.
  • CTF fitting and correction steps.
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MPI für Biochemie Max Planck Gesellschaft MPI für Biochemie Max-Planck-Gesellschaft

SPA – ideal data collection approach

  • AF – autofocus position
  • D1, D2 – data images taken

close to focus AF D1 D2

AF?

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MPI für Biochemie Max Planck Gesellschaft MPI für Biochemie Max-Planck-Gesellschaft

SPA data collection approach 1 - free ice

  • AF – autofocus position
  • D1, D2 – data images taken

close to focus.

  • F1, F2 – focus & phase shift

measurement images taken at ~ 1 um defocus. AF F1 F1 D1 D2

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MPI für Biochemie Max Planck Gesellschaft MPI für Biochemie Max-Planck-Gesellschaft

SPA data collection approach 2 – thin carbon

  • AF – autofocus position
  • D1, D2 – data images taken at

~ 1 um defocus. The defocus in necessary for CTF fitting. AF D1 D2

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MPI für Biochemie Max Planck Gesellschaft MPI für Biochemie Max-Planck-Gesellschaft

PP CTF fitting

  • MATLAB code; fits astigmatism, defocus and phase shift.
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MPI für Biochemie Max Planck Gesellschaft MPI für Biochemie Max-Planck-Gesellschaft

EPU focusing accuracy, Tecnai F20

  • Tecnai F20, D3 dataset.

Asked defocus: 1500 nm

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MPI für Biochemie Max Planck Gesellschaft MPI für Biochemie Max-Planck-Gesellschaft

EPU data collection with the VPP – phase shift evolution

  • Tecnai F20, D3 virus dataset.
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MPI für Biochemie Max Planck Gesellschaft MPI für Biochemie Max-Planck-Gesellschaft

CTF deformation due to focus beam spots on the VPP

  • Titan Krios, EPU, Falcon camera

10 A

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MPI für Biochemie Max Planck Gesellschaft MPI für Biochemie Max-Planck-Gesellschaft

Usability of Phase plates for Single Particle Analysis

  • Quickly build a low-resolution (~ 10 A) initial model for new samples.
  • Evaluate sample quality – particle distribution, purity, state of

assembly, orientation etc.

  • A “native state” higher-resolution alternative to negative stain.
  • Tackle heterogeneous, small or flexible samples.
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MPI für Biochemie Max Planck Gesellschaft MPI für Biochemie Max-Planck-Gesellschaft

Main issues and limitations

  • Focusing accuracy:

– 3 image method. – combine beam tilt with CTF fitting for more accurate focusing? – OL hysteresis! – Ice thickness!

  • ~ 20% information loss – even thinner films for VPP (currently ~ 10 nm),

graphene?

  • CTF deformation due to focus beam tilt spots – increase beam tilt angle,

use CTF fitting for focusing.

  • Phase shift increase – switch phase plates automatically every N

exposures, will be in next versions of EPU.

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MPI für Biochemie Max Planck Gesellschaft MPI für Biochemie Max-Planck-Gesellschaft

Max Planck Institute

  • f Biochemistry

Martinsried, Germany

Acknowledgements

Wolfgang Baumeister Jürgen Plitzko Maryam Khoshouei Yoshiyuki Fukuda Alex Rigort Günter Pfeifer Inga Wolf Shoh Asano Yuxiang Chen Thomas Hoffmann Bart Buijsse Raymond Wagner Marc Storms Petr Herzig Kasim Sader Gijs van Duinen Matthijn Vos Bob IJtsma