Comparison of x-ray and electron beams for structural, chemical and elemental analysis R.F. Egerton Physics Department, University of Alberta, Canada

Structural analysis by x-rays and electrons Hard x-ray diffraction and diffractive imaging  structure X-ray absorption fine structure (EXAFS, NEXAFS)  structure Soft x-ray absorption in water window  elemental or chemical map Electron diffraction and diffractive imaging (100 – 300 keV)  structure TEM scattering-contrast imaging (amplitude contrast)  structure TEM phase-contrast imaging (obj. defocus or phase plate)  structure STEM annular dark-field (ADF) imaging  structure, Z-contrast image Electron energy-loss imaging  elemental map etc. Electron energy-loss spectroscopy  composition, structure

I0I0 Plasmon Co M 23 Nucleus core levels valence band conduction band e-e- e-e- Echenique et al, PRB 20 (1979), p potential charge dielectric Single e - e-e- b Electron Energy-Loss Spectrum

Some practical considerations X-ray synchrotronsTEM + EELS, EDX spectroscopy < 10 sites in USAseveral major centers + many routine instruments Zone–plate focusing toFocusing to < 0.1 nm 20nm with ~ 5% efficiencywith 100% efficiency Detectable concentrationDetectable mass < g < 1 ppm by fluorescence Micron-thick specimensSpecimens < 500 nm thick (but overlap of structure)(spec. prep. time-consuming) Environmental cells easyEnvironmental cells difficult but feasible with MEMS Recording time ~ hoursRecording time ~ secs, mins

X-rays and electrons are ionizing radiation: X-ray absorption  photoelectrons  radiolysis Electron inelastic scattering  secondaries  radiolysis (PMMA: > 80% of radiolysisis due to secondaries) How electrons differ from x-rays: They have charge  efficient focusing by magnetic lenses but Coulomb repulsion  limitation on incident flux Also, electrostatic charging of insulating specimens (rupture)  deflection of incident and imaging beam (microlensing) Electrons have rest mass and appreciable momentum  knock-on displacement damage Energy transfer few eV or tens of eV for high-angle scattering But this is rare, so knock-on damage is mainly observed in conducting specimens, where radiolysis is absent.

Effects of knock-on damage (conducting specimen): Atom displacement in the bulk (E d ~ tens of eV) Atom displacement at grain boundaries (E d ~ few eV) Atom displacement from a surface (e-beam sputtering) Atom displacement along a surface ( radiation-enhanced diffusion ) Decreasing displacement energy E d and decreasing incident-energy threshold. For 180° scattering, E 0 th = (511 keV)[1+AE d /561eV] 1/2 - 1]

Graphite irradiated by 200keV electrons for 10 minutes at 600  C (Dose ~ 500 C/cm 2 ) Egerton, Phil. Mag. 35 (1977) materialE d ( eV )E th ( keV ) diamond graphite aluminum copper gold M gO ,460 Bulk (volume) displacement  atomic clusters Simulation of neutron damage in nuclear fuel rods etc.

Electron-induced sputtering incident electron entrance surface exit surface high-angle ‘elastic’ collisions with a single atom incident electron Inside specimen, can create interstitials and vacancies

Calculated cross sections for e-sputtering No effect below threshold energy. Thinning rate ( monolayer/s ) =  (J/e) ~ 10 for  = 100 barn and J =10 4 A/cm 2 (10pA in 1nm 2 ) J >10 6 A/cm 2 for CFEG & C s -corr.

Spatial resolution of imaging and spectroscopy Electrons have small deBroglie wavelength ( 15 keV) and can be focused efficiently by electromagnetic lenses  High spatial resolution in imaging, diffraction and spectroscopy, as in the (S)TEM. Electron lenses have high spherical and chromatic aberration but these aberrations can now be corrected. Instrumental resolution ~ 0.05 nm for E 0 ~ 200 keV. This is the practical resolution for conducting (e.g. metal) specimens where knock-on displacement (inefficient) is the only damage mechanism

Ionization damage versus knock-on displacement in organic samples Microscopy Research & Technique 75 (2012) 1550

Non-conducting (e.g. organic) specimens Resolution is limited by ionization damage (radiolysis) Dose-limited resolution  (SNR) C -1 (DQE. F.D c /e) -1/2 SNR ~ 3 to 5 (Rose criterion) C = contrast between resolution elements DQE = performance of recording system F = specimen/detector attenuation (e.g. TEM objective aperture) D c /e = critical dose in electrons/area

Calculated contrast C and dose-limited resolution  for a boundary in polymer (projected structure, 10% density change) TEM bright-field scattering contrast Resolution improves with increasing thickness until F becomes small ( most electrons absorbed by objective aperture ) Low kV is better for a very thin specimen (  ~ C -1 D c -1/2 ) but worse for thicker one.

Calculated contrast C and dose-limited resolution  for 10% density change in a polymer (e.g. PMMA) Phase contrast Contrast and resolution both improve with increasing thickness, until the phase shift exceeds 3  /4. For thin specimens,  ~ C ph -1 D c -1/2 ~ E 0 1/2 E 0 -1/2 i.e. independent of kV, but higher kV allows thicker specimen -> smaller . Assumes an ideal phase plate (future possibility) overlap problems

Dark-field imaging in scanning mode (ADF-STEM) Pennycook, Condensed Matter Physics (2005)

ADF-STEM imaging of a polymer (10% density change) Resolution versus inner detector angleResolution versus incident energy

Three-dimensional imaging with x-rays or electrons via tomography or diffractive imaging Required dose less for electrons due to stronger elastic scatter (Henderson etc.) Damage dose (in Gray) same for electrons and x-rays (ionization damage) Figure modified from Howells et al. JESRP 170 (2009) Damage data from DP fading for calalase, protein purple membrane, bacteriorhodopsin, ribosomes etc. (Glaeser et al., Howells et al.)

TEM cryo-microscopy of organics: Repeated structure (e.g. crystal) lowers the required dose  atomic resolution in phase-contrast images except for mechanical distortion and electrostatic charging of the specimen Brilot et al. JSB (2012)  5nm  direct-e camera 5 frames/sec Li et al. Nat.Meth 10 (2013) 584

X-ray direct imaging: resolution restricted to ~ 20nm (zone plate) Diffractive imaging capable of atomic resolution but DLR is limited by radiation damage ( e.g. 10nm ) unless damage can be outrun (<100fs pulses) Pulsed-laser-activated photoemission electron source  Short electron pulses, down to single electrons (Zewail) Used to study Solid-state phase transitions Metal-insulator transition Nucleation and crystallization dynamics Nanomechanical systems Surface-charging effects Plasmonics in nanostructures Dynamics of chemical reactions Free-electron laser gives femtosecond x-ray pulses

Short-pulse x-ray diffraction: H. Chapman et al., Nat. Phys. 2 (2006) fs pulse containing photons (2.9keV, 0.32nm) gives a diffraction pattern of a patterned Si 3 N 4 membrane before vaporizing it at 60,000 K. Chapman et al. Nature 470 (2011) 73 10fs, 70fs and 200fs pulses of 1.8keV (0.7nm) x-rays focused to 7 microns (900 J/cm 2, dose = 700MGy/pulse) give DPs of a membrane-protein complex (size ~ 10nm) and demonstrate no damage below 70 fs (see below)  30MGy damages cooled protein

Chapman et al. (2011) Liquid-jet injector  and pnCCD detectors (30Hz) Photosystem-1 protein image  reconstructed from from 15,000 DPs by coherent diffractive imaging DP’s from detectors 

Conditions for damage-free diffractive imaging 1.Flux high enough to generate sufficient signal before the object is destroyed. 2. Many objects can be used, improving the signal (as in a crystallized object) but for randomly-oriented objects the statistics in each DP must be adequate (e.g diffracted photons, maybe less with sophisticated software). 3. Photoelectrons may escape from a small isolated object, making damage less than in an extended crystal. 4. Pulse length < 200 fs for efficiency. Nuclear motion (damage) occurs after about 30 fs, so the diffraction pattern gets blurred, then electrons arriving after destruction contribute nothing to the DP background. 5. For diffractive imaging, X-ray beam must be coherent over a diameter ~ particle size or over unit cell (for a crystalline object).

Can we do the same with electrons? 1.6-cell rf photocathode gun (BNl/SLAC/UCLA) 100fs electron pulses, with electrons/pulse. Instantaneous current = 1.6 – 160 Amp Problems: 1.Electron momentum (knock-on damage, negligible compared to ionization damage) 2. Electron charge: Coulomb repulsion effects (Kruit & Jansen, 1997): A. Space charge (effect on one electron of all others) compensate by refocusing B.Trajectory displacement (statistical, between electrons) unavoidable C. Energy broadening (Boersch-effect) increases chromatic aberration

Continuous beam (100keV electrons focused over 0.2m) Current density limited to ~ 2000 A/cm 2

Continuous beam (2.5MeV electrons focused over 0.2m) Maximum current density now ~ 65 MA/cm 2

Pulsed electron beam: if Coulomb repulsion same, 2.5MeV dose within 100fs = (1e-13)(65e6) = 6e-6 C/cm2 2.5MeV damage dose ~ 6e-2 C/cm2 So negligible damage in a single pulse, short pulses may offer no advantage. Kruit & Jensen equations include relativistic factor: V* = V(1+eV/m 0 c 2 ), but not magnetic attraction of parallel-trajectory electrons: F total = (e/2  0 )(  /  2 )r Also, Coulomb repulsion in a short pulse may be less. So the above dose estimates will be too low. Relativistic particle-bunch calculations are needed. Is it necessary to outrun primary damage (fs time scale) ? 1. Short x-ray pulses needed only to increase collection efficiency 2. Secondary damage has longer timescales.

Radiolysis time scale (Warkentin et al. 2012)

Secondary damage can be avoided if structural information is acquired on a nanosecond to millisecond time scale. This requires: Fast detectors Efficient signal collection Slow down secondary processes e.g. by cooling specimen The existence of these longer time scales implies a dose-rate dependence of the damage dose D c.

Dose-rate dependence of damage by x-rays Change in damage dose reflects free-radical secondary damage Warkentin et al. Acta Cryst.(2013)

Evidence for dose-rate effects in electron-irradiation of organic materials Wery & Mansot, Microsc. Microanal. Microstruct. 4 (1993) 87. Formation of f.c.c. lead (detected in diffraction pattern) in lead isooctanoate. Egerton and Rauf, Ultramicroscopy 80 (1999) 247. Loss of O,C and N from collodion at 90 K. + + _ computer simulation

Suggests that STEM can “outrun” mass loss (less damage in elemental map) if probe current not high enough to cause appreciable temperature rise Simulation for 1nm electron probe (as in STEM): dose D e for mass loss from organic polymer at 90 K

Conclusions Electrons and x-rays and electrons are both ionizing radiation Radiation damage higher for EXELFS than for EXAFS Damage may be less for elastic imaging by electrons because electrons are scattered more strongly  very thin specimens, sometimes difficult  image interpretation sometimes more complicated Electron beams can be focused down to 0.1 nm  very small analysis volume Energy resolution of EELS and XAS now comparable (0.01 – 1 eV) Femtosecond imaging/spectroscopy more difficult with electrons but cryo-TEM can now achieve atomic resolution from small organic crystals and large macromolecules.

Henderson, Quart. Rev. Biophys. 2 (1995) 171 Electronssoft x-rayhard x-ray Energy/inelastic event20 eV400 eV8 keV Energy/elastic event60 eV400 keV80 keV If the signal is elastic, X-rays are 10 4 to 10 5 times more damaging Protein/water contrast0.410 X-ray water-window contrast 25 times higher than in TEM-BF image This factor outweighs the noise advantage of BF-TEM: (400/60) 1/2 = 2.6 but TEM phase contrast ~ 40 times more contrast than BF (TMV in ice), giving (2.6)(40)/25 ~ x 4 advantage for electrons In practice, TEM resolution of biomolecules is often limited by beam-induced specimen movement and charging (micro-lensing).

Radiation units X-ray community (and most radiation specialists) measure radiation dose in terms of deposited energy, in units of Gray (= J/m 3 ) or MegaGray (MGy) Electron microscopists use “dose” = fluence = (beam current density)(time) = Coulomb/cm 2 or particles/area, usually e/nm 2 or e/Angstrom 2 1 C/cm 2  [10 4 / IMFP(nm)] [E av (eV) /  (g/cm 3 )] For 100keV electrons and typical organic material, IMFP ~ 100 nm, E av ~ 35 eV and  ~ 1.4 g/cm 3, giving 1 C/cm 2  2500 MGy or 1 electron/Angstrom 2  4 MGy Critical or characteristic dose D c : Amino acid (l-valine): C/cm 2, 5 MGy Chlorinated Cu-phalocyanine: 30 C/cm 2, 75 GGy

Usual assumption: damage proportional to accumulated dose (critical dose is independent of dose rate). This is the basis for using Gray or rad units Primary process leading to damage: < 1 fs absorption (x-rays) or inelastic scattering (electrons)  core- or valence-electron excitation (single-electron or plasmon)  bond breakage (may not be permanent, damage not 100% efficient)  creation of photelectrons or secondary electrons, Auger electrons Secondary processes: additional damage created by secondary electrons (~80% in PMMA) or photoelectrons (predominant damage process for hard x-rays) motion of atomic nuclei, leading to structural damage> 50 fs (thermal motion may contribute  temperature dependence of damage) Tertiary processes include:ns, ms, s, days... Loss of crystalline structure Diffusion from or into the irradiated area (composition change) Escape of material form the specimen (mass loss) Dielectric breakdown due to charge buildup Disruption of biological processes (e.g. cell death) These slower processes may nonlinear  dose-rate dependence of damage

Classification of dose-rate effects Diffusion leads to mass loss or precipitation, expect positive d-r effect Fast XFEL pulses allow diffract & destroy (Chapman et al., 2011) positive Diffusion allows recovery (Jiang & Spence, 2012) negative, threshold Beam heating causes mechanical motion (Downing, 1987) negative or faster radiolysis in polymer (Beamson; Egerton & Rauf) negative Electrostatic charging causes dielectric breakdown or Coulomb explosion  hole formation in oxides (Humphreys et al.) negative, threshold Implications: STEM, STXM give high dose rate for short dwell time. Scanning is beneficial if the dose-rate effect is positive. Diffusion effects continue after irradiation: better to scan once only [wet chromosomes, Williams et al. J. Microsc. 170 (1993) 155 ] Scanning is detrimental if the dose-rate effect is negative. Fixed-beam microscopy could then give less damage for the same information.

100keV electrons and 100fs pulses: Current density ~ 4e9 A/cm 2, dose ~ 4x10 -4 C/cm 2 per pulse

Electron energy30fs-dosedamage dose (1nm, protein) keV0.3 Mgy100 MGy 2.5 MeV 3 Mgy300 MGy 30fs-dose is below CW damage threshold for most organics So many pulses required for good spatial resolution  No advantage over CW irradiation unless short pulses fail to excite lattice motion. Calculations include relativistic factor: V* = V(1+eV/m 0 c 2 ), but not magnetic attraction of parallel-trajectory electrons: F total = (e/2  0 )(  /  2 )r So the above dose estimates are likely too low. In practice, other factors can limit the beam diameter: Spherical aberration, chromatic aberration, diffraction limit, geometric source size ( ~ 100  m in BNL apparatus, reduced by focusing the laser illumination or using a small emitter tip)