LMB Cambridge - Helsinki Institute of Physics

hip.fi

LMB Cambridge - Helsinki Institute of Physics

Impact of silicon pixel detectors on

structural biology

Wasi Faruqi

MRC Laboratory of Molecular Biology,

Hills Road,

Cambridge CB2 0QH,

UK

IWORID 10 Helsinki, 1 st July, 2008

6/26/2008

LMB, Cambridge


Kings College Chapel, Cambridge

6/26/2008

LMB, Cambridge


MRC Laboratory of Molecular Biology,

Cambridge

6/26/2008

LMB, Cambridge


Direct Detection in Silicon Pixel

• Hybrid Pixel Detectors

Medipix2

Detectors

http://medipix.web.cern.ch/MEDIPIX/

• CMOS Detectors

Monolithic Active Pixel Sensors (MAPS) designed at the

STFC Rutherford Lab.

Pixellated silicon, readout built into each pixel.

http://mi3.shef.ac.uk/

6/26/2008

LMB, Cambridge


Acknowledgements

LMB Cambridge

•Richard Henderson

•Greg McMullan

•Shaoxia Chen

CERN (Medipix2)

•Lukas Tlustos,

•Xavi Llopart,

•M.Campbell

•RAL-STFC (MAPS)

•R.Turchetta

•M. Prydderch,

•MI3 Collaboration (RCUK)

6/26/2008

LMB, Cambridge


Main techniques used in structure

determination

X-ray (Protein) Crystallography

Electron Cryo-Microscopy

Nuclear Magnetic Resonance

(along with other supporting technologies)

6/26/2008

LMB, Cambridge


Main scientific aims

•Structural analysis of proteins and

macromolecular complexes to atomic or near

atomic resolution

•Electron Cryo-Microscopy frequently used

as a complementary technique to X-ray

Crystallography

•Example: Hepatitis B virus solved by both

techniques – Example in later slide

6/26/2008

LMB, Cambridge


Electron Detectors & X-ray Detectors

Electron Detection: What are the main differences

between X-ray Detectors and Electron Detectors?

Electrons 100 – 300 keV

X-rays 10 – 20 keV

Electrons very easily scattered and stopped by matter,

So, need to:

(a) install detector in a vacuum chamber, and

(b) entrance window not possible on the detector

Cannot use gas detectors

6/26/2008

LMB, Cambridge


Schematic of Microscope

LMB-Cambridge

6/26/2008

LMB, Cambridge


Scientific Background to Electron Cryo-Microscopy

Three Main types of Analysis (and the resolution attained

in the analysed structures):

1. Single Particle (molecule) Analysis 3.8 -10 Å

2. Electron Crystallography of ordered specimen, i.e. 2-D

crystals ~3Å … near-atomic resolution

3. Electron Tomography 20-100 Å … cell biology

Electron energy preferred: 300 keV (less lens

aberrations, less multiple scattering in sample, less

absorption)

6/26/2008

LMB, Cambridge


Electron Cryo-Microscopy

•Image molecules in native aqueous

environment in vitreous ice (prevent de-hydration)

•Trap important conformations in intermediate states of a

kinetic cycle by rapid freezing : equivalent to time-resolved

measurements

•Images have low contrast : need sophisticated software and

lots of averaging

•Radiation damage to specimen a severe limitation – hence

need very good detectors!

6/26/2008

LMB, Cambridge


Single Particle Analysis

No crystals required .. Makes it possible, e.g. to analyse

structure of membrane proteins, which are very difficult to

crystalise,

Can be applied to large macromolecular complexes

• Powerful technique when used in conjunction with atomic

structures obtained with x-ray crystallography

•Applications: Virus particles, ribosomes, etc

•‘Best’ Resolution with this technique :Rotavirus capsid 3.8 Ǻ

•6.5 Million copies of the capsid protein used for the map

•From 8400 virus particles (excellent symmetry)

6/26/2008

LMB, Cambridge


Rotavirus Epidemiology

Deaths due

to rotavirus

(diarrhea)

6/26/2008

Causes ~600,000 deaths annually

Infection occurs from feces. Only 10 to 100 infectious particles suffice.

LMB, Cambridge


Symmetry:

60 x 13

=780 VP6 molecules

6/26/2008

LMB, Cambridge

Zhang et al. (2008)


Side Chains

3.8 Å

X-ray crystallography (2F o

–F c

)

Cryo-EM

6/26/2008

Settembre et al.

LMB, Cambridge

Xing Zhang et al. (2008)


Electron Crystallography

• Averaging done in crystal… many identical scattering

particles

• Main application: membrane proteins (but not

exclusively)

• Near-atomic resolution (~2.5 Å) achieved.

6/26/2008

LMB, Cambridge


Bacteriorhodopsin

•Membrane protein family

•Light driven proton pump; photocycle

•Structural changes at atomic resolution studied

by trapping intermediates in reaction cycle

•Diffraction data collected with LMB CCD

camera

6/26/2008

LMB, Cambridge


Electron Diffraction Studies on Bacteriorhodopsin

(with R.Henderson and S. Subramaniam)

6/26/2008

LMB, Cambridge

A 7-second diffraction pattern from bacteriorhodopsin with spots visible to 2Å -1


BR Model

6/26/2008

LMB, Cambridge Henderson, et al JMB, 213, 899-929 (1990)


BR

6/26/2008

LMB, Cambridge

Courtesy R.Henderson


Electron Tomography

1. View specimen from large number of angles,

but limits due to radiation damage

2. Combine views into 3-D reconstruction

3. Image needs re-focusing and re-aligning after

each step due to imperfections in stage construction

4. Automation and electronic detectors essential!

6/26/2008

LMB, Cambridge


Reconstruction

Tomography - Reconstruction of a 3D Model

Based on a Series of Projections

Projections

Reconstruction

6/26/2008

LMB, Cambridge

A. Koster et al. 1997


Investigation into Mitotic Proteins using

Electron Tomography

John Kilmartin and Sam Li (LMB)

Identify proteins common to yeast and

mammalian cells using mass spectrometry.

Explore function with biochemical techniques and

study structure with electron tomography.

6/26/2008

LMB, Cambridge


6/26/2008

LMB, Cambridge


Recent structures obtained using

Cryo-EM on next slide

a. 70S E.coli ribosome complexed with mRNA and fMettRNA

(11.5 Ǻ)

b. Hepatitis B virus (7.4 Ǻ)

c. Actin filaments decorated with myosin heads (30-35

Ǻ)

d. 2D crystal Light Harvesting Complex II (3.4 Ǻ)

Baker & Henderson‘Electron cryomicroscopy’

International Tables for Crystallography, 451-479,(2002)

6/26/2008

LMB, Cambridge


Molecular structures obtained by Electron Cryo-Microscopy Magnification

Micrographs:170K, Models: 1.2 Million

Montage

70S Ribosome

6/26/2008

Hepatitis B Virus Actin-Myosin (Muscle) Light Harvesting Comple

LMB, Cambridge


Hepatitis B Structure

Cryo-EM X-ray Crystallography

Andrew Leslie LMB

6/26/2008

LMB, Cambridge


Scientific Background to Electron Cryo-Microscopy

Three Main types of Analysis (and the resolution attained

in the analysed structures):

1. Single Particle (molecule) Analysis 4 -10 Å

2. Electron Crystallography of ordered specimen, i.e. 2-D

crystals ~3Å … near-atomic resolution

3. Electron Tomography 20-100 Å … cell biology

Electron energy preferred: 300 keV (less lens aberrations,

less multiple scattering in sample, less absorption)

6/26/2008

LMB, Cambridge


High Resolution Imaging Detector

Requirements for Cryo-EM

1. Electronic detector with computer control.. eliminate film!

2. Number of independent pixels : 4000 by 4000

3. Pixel Size 10 – 50 µm (has to fit in commercial microscopes)

4. High sensitivity with no noise – ability to add multiple frames

5. Radiation damage; should be able to withstand

at least 1 MRad this would be ~ a one year dose for cryo-EM

6. Readout time preferably short

6/26/2008

LMB, Cambridge


Medipx2(Quad) in 300 kV Microscope

Mounting

6/26/2008

LMB, Cambridge


300 kV EM with detector installed

6/26/2008

LMB, Cambridge

Medipix Quad


Detectors: Quality Factors

Sensitivity: Detective Quantum Efficiency (DQE),

(S/N) 2 output /(S/N)2 input (=1 for perfect detector)

DQE(0) zero spatial frequency

DQE(spatial frequency)

Resolution: Modulation Transfer Function (MTF)

Framing Speeds… inverse of readout time

Radiation Hardness … useful lifetime

Dynamic Range … ability to record very weak and very

strong parts of an image simultaneously (diffraction only)

Defects …… Faults in fabrication, etc

6/26/2008

LMB, Cambridge


Monte Carlo simulation of electron trajectories in silicon.

Detector thickness = 300 microns, pixel=55 microns

Extension of simulations to include energy deposition

6/26/2008

LMB, Cambridge

McMullan, et al Ultramicroscopy,

107,(2007), 401-413


MTF at Nyquist Frequency

6/26/2008

LMB, Cambridge

McMullan, et al

Ultramicroscopy, 107, (2007),

401-413


Single Electron Clusters; 240 incident electrons at 120 keV

250

Counts/electron vs Ext Vthl

High Threshold

Low Threshold

200

No counts

1 count

Counts/electron

150

100

2 counts

50

0

1190 1240 1290 1340 1390 1440 1490

Ext Vthl (1190 __ 120 keV)

3

4

counts

6/26/2008

LMB, Cambridge


Increased probability of several pixels counting an electron at

lower thresholds. Seed pixel in centre of array. E=120 keV

(a) (b) (c) (d) (e)

(f) (g) (h) (i) (j)

6/26/2008

(k) (l) (m) (n) (o)

LMB, Cambridge


DQE(0) and DQE(Nyquist)

DQE

6/26/2008

LMB, Cambridge

Threshold(keV)


Some examples of Single Particle

Imaging using the Medipix2_Quad

Detector

All images recorded at 120 keV

McMullan & Faruqi

Nucl. Instr. and Meth. A 591 (2008) 129–133

6/26/2008

LMB, Cambridge


Rotavirus with 1.6(left) and 160(right) electrons/pixel

0.04 electron/Å 2 (at specimen) 4 electrons/Å 2

Rotavirus imaged with Medipix2 Quad

6/26/2008

LMB, Cambridge


Single Lambda Phage on MPX2 Quad

6/26/2008

LMB, Cambridge


TMV: Results of image alignment

Negatively stained TMV

protein stacked disks:

(a) first image in a series of

65,

(b) simple sum of the 65

images (blurred),

(c) aligned sum of the

images (sharp),

(d) sample movement in Å.

The scale bar in (b)

indicates 500 Å

6/26/2008

LMB, Cambridge


Tobacco Mosaic Virus (from 65 images)

Magn. =180K

6/26/2008

LMB, Cambridge


MAPS CMOS Detector

- charged particles

• no bias voltages

• charge diffusion

• 100% fill factor

Epilayer

Substrate

6/26/2008

Turchetta et al

NIM A458 (2001) 677-689

LMB, Cambridge


Monolithic Active Pixel Sensor (MAPS)

General Background

Monolithic Active Pixel Sensor (MAPS ) –designed at

RAL*

Size: 525 by 525, 25 μm square pixels

Non-Radhard, standard 0.5 μm CMOS technology

Each pixel contains four diodes

Electrons drift to one of the four diodes in pixel

Charge summed from all diodes and converted to a voltage

One ADC per column; all pixels in a row read out in

parallel

*Prydderch, et al Nucl.Instr. & Meth. A512,(2003),358

6/26/2008

LMB, Cambridge


Monte Carlo simulation of electron trajectories in silicon.

Detector thickness = 300 microns, pixel=55 microns

Extension of simulations to include energy deposition

6/26/2008

LMB, Cambridge

McMullan, et al Ultramicroscopy,

107,(2007), 401-413


MAPS in 300 keV Mounting

6/26/2008

LMB, Cambridge


MI-3 Consortium Members

Professor NM Allinson, University of Sheffield

Professor PP Allport, University of Liverpool

Dr RL Bates, University of Glasgow

Professor AR Cossins, University of Liverpool

Professor MM El Gomati, University of York

Dr AR Faruqi, MRC Laboratory of Molecular Biology

Dr MJ French, CCLRC

Professor A Holland, Open University

Professor RJ Ott, Institute of Cancer Research

Dr V O'Shea, University of Glasgow

Professor RD Speller, University College London

Dr R Turchetta, CCLRC

Dr K Wells, University of Surrey

6/26/2008

LMB, Cambridge


Imaging of 100 mesh grid in MAPS

6/26/2008

LMB, Cambridge


ADC Response for Single Electrons

at 40 keV and 120 keV

6/26/2008

LMB, Cambridge


Sensitivity:

Noise:

MAPS Summary at 120 keV

~50 ADC Units/electron

~2 ADC Units

Signal/Noise: 20-25

Radiation Hardness:

Active area

10-15 kRad . Needs improvement!

525 x 525 pixels need larger areas

Faruqi, Henderson, Turchetta et al Nucl. Instr.& Meth 546,

170-175, (2005)

6/26/2008

LMB, Cambridge


Radhard

Technology

512 x 512, 25 µm

Radiation Damage to STAR250

(FillFactory/Cypress Corp.) at 300 keV

C

Radiation Dose:

A: 200kRad

(annealed for 4

weeks)

B: 200 kRad

C: 1000 kRad

Contrast values

labelled in bottom

left image

B

B

C

A

A

6/26/2008

LMB, Cambridge


Startracker & Film response to electrons 10 - 300 keV

50

Response - Film values normalised

40

30

20

10

0

0 50 100 150 200 250 300

Electron energy (keV)

Startracker

Film

6/26/2008

LMB, Cambridge


Large Area Sensor : Specifications

Pixel Size 40µm

Array Size 1.4K by 1.4K

Array Dimensions 5.6cm x 5.6cm

Epi Thickness 15µm

Frame Rate

10 f/s max

Noise floor

28e

Radiation tolerance ~


Large Area Sensor Wafer

Courtesy of

Andy Clark &

Renato Turchetta

MI3

Collaboration

6/26/2008

LMB, Cambridge


Large Area Sensor - Mounted

Courtesy of

Andy Clark &

Renato Turchetta

MI3

Collaboration

6/26/2008

LMB, Cambridge


6/26/2008

Detectors for Protein Crystallography : Main

Requirements

High Efficiency, low noise (high DQE)

Large active area; can get better S/N by increasing distance

Excellent spatial resolution; need to resolve ~500 orders (3K x3K)

Very large dynamic range (strong & weak spots)

High rate capability (no dead time, shutterless operation)

No spatial distortions or non-uniformity of response; any corrections should

be stable over long periods

Ability to operate at a wide range of wavelengths for MAD, 0.6Ǻ –2.5Ǻ

Low cost, reliable (low maintenance)

Continuous readout to eliminate beam shutter (closed during readout)

PILATUS 1M and 6M (PSI, Villigen) - talks during conference

Medipix3: Does it offer special advantages for Crystallography?

LMB, Cambridge


Medipix3

Silicon sensor, 300 or 500 µm for very high efficiency

1 or 2 counters per pixel – continuous R/W with no dead time

Pixel Size : 55 µm square, very well suited for microcrystallography.

Can be re-arranged to be 110 µm for medical work

with higher energy x-rays

Pixel level intelligence – expect improved resolution (by reducing

effects of charge sharing between pixels)

Counting rates ~ 10 6 counts/second/pixel

Chips to be 4-side buttable (in future) for extended tiling

For more details, see Michael Campbell during IWORID10 or visit:

http://medipix.web.cern.ch/MEDIPIX/


Medipix3 – charge summing concept

The winner takes

all

• The incoming

quantum is assigned as

a single hit

• Charge processed is

summed in every 4

pixel cluster on an

event-by-event basis

55μ

Medipix

Collaboration

6/26/2008

LMB, Cambridge


55μ

4

5

6 7

DIGITAL CIRCUITRY

4. Control logic

(124)

5. 2x15bit counters

/ shift registers

(480)

6. Configuration

latches (152)

7. Arbitration

circuits (100)

Total digital 856

2

1 3

17 October 2006 Michael Campbell

55μ

ANALOG CIRCUITRY

1. Preamplifier (24)

2. Shaper (134)

3. Discriminators

and Threshold

Adjustment

Circuits (72)

Total analog 230


The Medipix3 Consortium

• University of Canterbury, Christchurch, New Zealand

• CEA, Paris, France

• CERN, Geneva, Switzerland,

• DESY-Hamburg, Germany

• Albert-Ludwigs

Ludwigs-Universität Freiburg, , Germany,

• University of Glasgow, Scotland, UK

• Leiden Univ., The Netherlands

• NIKHEF, Amsterdam, The Netherlands

• Laboratory of Molecular Biology, Cambridge, England, UK

• Mid Sweden University, Sundsvall, , Sweden

• Czech Technical University, Prague, Czech Republic

• ESRF, Grenoble, , France

• Universität Erlangen-Nurnberg

Nurnberg, Erlangen, , Germany

• University of California, Berkeley, USA

• VTT, Information Technology, Espoo, , Finland

• ISS, Forschungszentrum Karlsruhe, Germany

• Diamond Light Source, Oxfordshire, UK

6/26/2008

LMB, Cambridge


Microcrystallography – A new application?

Useful technique for very small crystals, ~20 µm 3

(large crystals more difficult to grow in many cases)

ESRF Microfocus Beam ID13, Focused beam size ~1 µm

Energy=13 keV,

Flux = 3x10 10 photons/sec/µm 2

XylanaseII structure determined to 1.5 Ǻ, diffraction pattern

next slide.

MW=21kDa, 40Ǻ x 39Ǻ X 57Ǻ

Detector needs to have small pixels, high DQE

Riekel, Schertler… Current Opinion Strc Biol (2005), 15, 556-562

6/26/2008

LMB, Cambridge


Micro-diffraction from Xylanase II

Data collected on

MarCCD165

Pixel size ~79 µm

square

Spot size limited by

detector resolution and

not by beam or crystal

6/26/2008

LMB, Cambridge


Summary: X-ray Detectors

Potential Advantages

Direct detection in Silicon – better resolution as no light scattering,

even better resolution with Medipix3

Fast readout – fast framing (few milliseconds) possible

Excellent S/N – noiseless readout, high dynamic range

Detector and electronics separate – choose detector material (Si,

GaAs, CdTe, …) for optimum efficiency

See also: PILATUS contribution to this conference

Downside

Large area detectors difficult and expensive to build

Individual detectors, ~2 cm 2 , tiled to obtain larger areas but gaps in

between chips/modules leads to some dead space

Technology not yet mature (?) – problems of ‘yield’

6/26/2008

LMB, Cambridge


Summary: Detectors for Electron

Microscopy

• Medipix2 is a superb detector up to 120 keV

• But, it may prove expensive to design 4K square arrays

without dead spaces

• Higher energies (300 keV) may be feasible but with higher

density compounds, e.g. Cd(Zn)Te

• CMOS detectors offer a good chance of a radiation hard,

4Kx4K square detector – but needs a lot more R&D effort

6/26/2008

LMB, Cambridge


Acknowledgements

LMB Cambridge

•Richard Henderson

•Greg McMullan

•David Cattermole

•Shaoxia Chen

CERN (Medipix2)

•Lukas Tlustos,

•Xavi Llopart,

•M.Campbell

http://medipix.web.cern.ch/MEDIPIX/

•RAL-STFC (MAPS)

•R.Turchetta, et al

•M. Prydderch, et al

•MI3 Collaboration (RCUK)

•http://mi3.shef.ac.uk/

6/26/2008

LMB, Cambridge

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