Transcript Slide 1
Detectors for imaging macro-molecules at atomic resolution JP Abrahams, Biophysical Structural Chemistry, Leiden University With thanks to: • Jules Hendrix, MAR Research, Hamburg • Christian Broennimann, PSI, Villingen • Diederik Ellerbroek, Bruker-Nonius, Delft Biophysical Structural Chemistry Central question: how do molecules interact to create life? Genome Proteome information gene substance molecule Cell organism Research in life science proteomics bio-informatics Other techniques for identifying genes, proteins and/or ligands genetics array techniques Genes, proteins metabolites Transfection: In vivo studies Extraction: in vitro studies ATOMS MOLECULES CELL ORGANISM structural biology biochemistry / biophysics cell biology physiology Theory development X-ray crystallography 1. Grow crystals 3. Solve phases and refine structure 2. Measure diffraction Cryo-EM EM images Identify particles 300 nm (Re-)align particles 3D model Generate a 3D reconstruction Courtesy: R Koning, J Plaisier, HK Koerten 20 nm Optics of diffraction and imaging detector lens detector object object diffraction diffraction image focus Diffraction pattern Detector requirements in structural biology Characteristic Required for: diffract image Large size Low background ++ +/- Low point-spread Low background + ++ High DQE1 Reduction of sample damage ++ ++ High dynamic range Linear sampling of all intensities ++ +/- Fast readout Efficient data collection ++ ++ Low cost There’s more to life than + detectors + Detective Quantum Efficiency : DQE = (Signalout/Noiseout)2/(Signalin/Noisein)2 Overview of detectors used in X-ray diffraction & electron microscopy X-ray Electron Electron diffraction diffraction imaging Chemical detection: obsolete photography obsolete state- of the art Multi-wire obsolete never used irrelevant Television camera: FAST obsolete never used irrelevant Image plate state of the used not used art sometimes CCD state of the state of the up and art art coming Pixel detectors near future ????? ????? Image plate detectors Based on a system (storage phosphor) for medical applications Advantages: • practically no intrinsic noise; • large size • high spatial resolution • large dynamic range Disadvantages: • long read-out time Manufacturers: MAR Research, Rigaku Technology is tried & trusted, no major future developments are foreseen MAR image plate detector CCD detectors Photon detection of an X-ray phosphor by a CCD. Advantages: • fast readout; • low noise; • reasonable spatial resolution Disadvantages: • limited dynamic range; • small size requires de-magnifying optics; • reasonable spatial resolution; • expensive Manufacturers: Bruker-Nonius, MAR Research, ADSC Technology is recent and still developing Courtesy Bruker-Nonius CCD detectors – new developments: Lens-based de-magnification Courtesy Bruker-Nonius MAR-research CCD detectors – new developments: Next generation CCD’s JFET hybrid pre-amps: 2x faster, 2x lower noise Normal (buried channel) mode: 4x higher dynamic range Back illuminated CCD: 2-3X higher quantum efficiency Fairchild CCD486 Courtesy Bruker-Nonius Pixel detectors Courtesy MAR Research Direct detection of electrons by pixel electrodes. Advantages: • fast readout; • low noise; • high spatial resolution; • high dynamic range Disadvantages: • Very recent technology; first commercial products are anticipated for Autumn 2002 Manufacturers: MAR Research X-rays Courtesy Christian Broennimann, PSI Al Technology is recent and still developing p+ n+ n++ Edrift - V bias + MAR Research Solid State Direct Conversion detector MAR Research Solid State Direct Conversion detector Dimensions: 430mm x 358mm Pixelsize: 140mm x 140 mm Number of pixels: 7.8 Mpixels Readout time: less that 1 s MAR Research Solid State Direct Conversion detector Pixel Detectors: Principle X-rays Si pn-junction Al 3.6 eV to create 1 eh-pair p+ - V bias + Edrift n+ n++ Detector 0.2 mm X-rays Pixel Sensor 0.3 mm 0.2 mm Sensor Chip Pixel Read-out Chip Pixel electronics Bump Bonds Ext/Comp Clock Treshold correction Radiation hard Bump Pad Global Tresh CS Amp 1.7fF Cal Comp + Enable/ Disable Analog Block F12 Clock Gen Ext Clock 15 bit SR counter Reset Digital Block RBI RBO Paul Scherrer Institut PILATUS Detector with 3 Modules Bank Data • Active Area: 238.7 x 35.3 mm2 • 157 x 1098 = 172386 pixels • 48 chips (radiation hard) • 2.38 mm gap between modules • Readout-time: 6 ms • Energy Range: Eg >4 keV • XY-addressing of each pixel • Threshold adjust of each pixel • Analog signal of each pixel Ch. Brönnimann Paul Scherrer Institut Diffraction pattern recorded with PILATUS Detector at Beamline 6s at the SLS Data Taking • Lysozyme crystal • 1 deg. Rotation (of a 45 deg data set) • 2s exposure, E=12 keV • Data taken at 7 detector positions • Flatfield correction for each detector position Ch. Brönnimann Future developments: digital holography? Single molecule diffraction Computational phase retrieval Continuous or over-sampled diffraction pattern Courtesy Miao, Hodgson & Sayre, PNAS 98, p 6642 Summary & conclusions The ideal detector in structural biology has the following characteristics: For X-ray diffraction: • Large, high-resolution, high dynamic range detectors with a fast readout. Detectors coming close to these specifications are available (CCD-based detectors) and more promising ones are around the corner (solid state direct conversion) For electron diffraction: • Similar requirements as for X-ray diffraction. CCD detectors are already very good, but may be overtaken in future by solid state direct conversion detectors. For electron imaging: • even higher resolution is required, but a high dynamic range is not as essential. It is not certain if direct conversion detectors will achieve a resolution that is sufficient; using large detectors will help, but this may require a re-think of the engineering of electron microscopes.