We are developing a novel monochromatic, aberration-corrected
dual-beam low energy electron microscope (MAD-LEEM) aimed at
imaging DNA sequences as well as macromolecules, nanoparticles and
surfaces. The key advantages of this approach compared to current
sequencing techniques are long reads, no need for DNA labeling, and
imaging with low energy electrons. Longer reads reduce the
complexity needed to assemble the sequence and minimize errors. The
absence of heavy-atom labels, e.g. needed for proposed TEM-based
sequencing techniques, simplifies sample preparation and improves
accuracy. The use of low energy electrons ensures that radiation
damage is minimized, i.e. high doses needed to achieve high
throughput and low cost can be used. This novel design promises to
significantly improve the performance of a LEEM and extend its
applications to a variety of samples that may benefit from high
resolution imaging at low landing energies without charging
effects, including biological samples, oxides, and catalysts.
The goal of the experimental work is to establish that electron
reflectivity is sensitive enough to distinguish individual
nucleotides or pairs.Reflectivity Contrast
Substrate Selection• Smooth and conductive substrates optimal
for imaging molecules in LEEM• Selected mica-peeled gold on glass
substrates manufactured by Nanoink - Sub-nm roughness is ideal for
imaging molecules attached on surfaces - Good correlation between
AFM and LEEM imaging obtained
DNA Imaging ResultsVariety of samples with oligomers supplied by
Integrated DNA Technologies was characterized using AFM and LEEM:•
Single-stranded (ss) DNA - Long single-base ultramers (T-200mer,
A-100mer, C-100mer) - Short single-base oligomers (G-20mer,
T-20mer) - Dithiol-modified oligomers (5ʼ-/5DTPA/A-20mer,
5ʼ-/5DTPA/C-20mer)• Double-stranded (ds) DNA - Hybridized short
oligomer (G-20mer) with dithiol-m. oligomer (5ʼ-/5DTPA/C-20mer)
Electron MicroscopyHas potential to significantly extend
individual read length and accuracy• Transmission electron
microscopy (TEM) techniques utilize the contrast from DNA bases
labeled with heavy atoms (Halcyon Molecular, ZS Genetics)• Main
drawbacks - Need for labeling leads to read errors and complex
sequence assembly - Radiation damage (>80keV) limits the total
electron dose and throughput
Low Energy Electron MicroscopyAdvantages • Imaging nucleotide
sequences of unlimited length, labels not needed• High contrast for
biological specimens at low energies, staining not needed•
Minimized radiation damage due to very low landing energy•
Monolayer sensitivity (small inelastic path, Å-resolution in z)•
High throughput (projection, high dose) = affordable sequencing
cost/genomeChallenges• Lateral spatial resolution limited in
todayʼs LEEMs by aberrations to ~ 5nm• Charging on insulating
layers• Nucleotide contrast
Abstract
Experiments
Conclusions
Progress Towards An Aberration-Corrected Low Energy Electron
Microscope for DNA Sequencing and Surface AnalysisM. Mankosa, K.
Shadmana, A. T. N’Diayeb, A. K. Schmidb, H.H.J. Perssonc, and R.W.
Davisca Electron Optica, 1000 Elwell Court #110, Palo Alto, CA
94303, b NCEM, Lawrence Berkeley National Laboratory, Berkeley, CA
94720, USAc Stanford Genome Technology Center, Stanford University
School of Medicine, 855 California Avenue, Palo Alto, CA 94304
This work has been performed in cooperation with H.H.J. Persson
and R.W. Davis at the Stanford Genome Technology Center, Stanford
University in Palo Alto, CA and A.T. NʼDiaye and A.K. Schmid at the
LBNL National Center for Electron Microscopy in Berkeley, CA.
LECTRONOPTICA
Acknowledgments
ReferencesReferences
Optics Simulations
PlanPhase I (2011-2013)• Electron Optics: Detailed column design
and modeling of spatial resolution with aberration corrector and
monochromator• Experiments: Measurement and analysis of
reflective/emissive properties of individual DNA bases at low
energies (0 to 1000 eV); Investigation of contrast
Phase II (2013 - )Realization of MAD-LEEM prototype with
resolution needed to distinguish individual nucleotides and capable
of achieving throughput equivalent to reading one genome (3Gbp) in
< 8 hours with error rates < 10-6.
Motivation
Objective Lens Analysis• Investigated electrostatic and combined
magnetic immersion objective lenses optimized for LEEM• Completed
1st, 3rd and 5th order analysis to understand resolution limit
prior to aberration correction• 5th order aberrations are key to
get sub-nm resolution
Mirror Aberration Corrector (MAC) Analysis• Completed analysis
of tetrode MAC as proposed by Rose & Preikszas up to 5th order
by the differential algebra method (Mirror-DA software by MEBS,
Ltd.)• MAC focuses electrons with larger aperture angles and lower
energies less than electrons with smaller angles and larger
energies, i.e. opposite to a con- ventional lens
• Extended analysis to higher landing energies to improve
resolution - Fine-tuned tetrode MAC spherical and chromatic
aberration coefficients to cancel aberrations of used objective
lens for a range of electron energies
- Resolution limited by 5th order geom. and 3rd & 4th rank
chrom. aberrations - Monochromator needed to make 3rd & 4th
rank chrom. aberrations negligible• Further improvement requires
pentode MAC to cancel 5th order spherical aberration (design in
progress)
Electron Reflectivity Results
2.5eV 2.9eV 3.0eV 3.1eV 3.2eV 3.5eV
Field of view : 8μm
Optics Summary
Landing energy 3.3eV 5.0eV 10.0eV
Field of view : 8μm
AFM LEEM
2.3eV 2.9eV 3.5eV
Field of view : 8μm
AFM LEEM
Standard LEEM+ Tetrode MAC
Tetrode MAC + Monochromator
Pentode MAC + Monochromator
Pentode MAC + Monochromator +coma correction
• Completed 1st, 3rd & 5th order analysis for objective lens
and tetrode MAC• Tetrode MAC improves resolution to ~ 1nm @ 200eV•
Design of pentode MAC for Cs5 correction in progress - Extends
resolution into the sub-nm regime
4.3eV 3.1eV
2.9eV
Reduced 5’-/5DTPA/C-20mer
Reduced 5’-/5DTPA/A-20mer
Au substrate 5’-/5DTPA/C-20mer 5’-/5DTPA/A-20mer
Au substrate
dithiol-C20 islands
Au substrate
dithiol-A20 islands
5’-/5DTPA/C-20mer
5’-/5DTPA/A-20mer
• Detailed electron-optical analysis of key MAD-LEEM components
completed - Tetrode MAC has widely tunable negative aberration
coefficients - Compensates the aberrations of the objective lens
down to ~1nm resolution - Monochromator is critical for further
resolution improvement - Pentode MAC with monochromator has
potential for sub-nm resolution• LEEM imaging and electron
reflectivity spectra at low electron energies indicate that high
contrast is achievable for DNA structures on a Au surface•
Monochromatic illumination, aberration correction and charge
control opens a new opportunity for nm scale imaging in biology,
nanotechnology, semicon- ductors, ceramics, ferroelectrics,
dielectrics, polymers ...
Specimen
Specimen
This project was supported by Grant Number R43HG006303 from the
National Human Genome Research Institute (NHGRI). The content is
solely the responsibility of the authors and does not necessarily
represent the official views of the NHGRI or the National
Institutes of Health.LEEM imaging was performed at the National
Center for Electron Microscopy, Lawrence Berkeley National
Laboratory, and was supported by the Office of Science, Office of
Basic Energy Sciences, Scientific User Facilities Division, of the
U.S. Department of Energy under Contract No. DE-AC02—05CH11231. ATN
acknowledges support from the Alexander von Humboldt
Foundation.
Low energy electron scattering directly related to the sampleʼs
electronic structure:• Different nucleotides have different
electronic structure => electron scattering coefficients are
nucleotide dependent => results in observable contrast
diffraction
Standard LEEM LEEM with Tetrode MAC LEEM with Pentode MACand
monochromator10eV
diffraction
3rd orderspherical
2nd rankchromatictotal
5th orderspherical
3rd ordercoma
3rd rankchromatic
4th rankchromatic
5th ordercoma
diffraction
total
5th orderspherical
3rd rankchromatic
4th rankchromatic
5th ordercoma
total
4th rankchromatic
5th ordercoma
diffraction
3rd orderspherical
2nd rankchromatic
total
5th orderspherical
3rd ordercoma
4th rankchromatic
3rd orderfield curvature
5th ordercoma
3rd orderastigmatism
diffraction
total
5th orderspherical
4th rankchromatic
5th ordercoma
3rd orderfield curvature
3rd orderastigmatism
diffraction
total 4th rankchromatic
5th ordercoma
3rd orderfield curvature 3
rd orderastigmatism
Standard LEEM100eV LEEM with Tetrode MAC LEEM with Pentode
MACand monochromator
Electron Energy
Elec
tron
Refl
ectiv
ity
C
G
1
e-
e-
MAC
Lens
Cs Cc
Sequencing Approach..
CGTATAGCCGATCG..
.
.ATGCCGGCTAGCCG..
TAGC
⤴⤴MAD-LEEM⤴⤴
Stretch out DNA on flat surface
Convert nucleotide-specific electron
reflectivity to grey levels
Process image and store sequence
Magneticobjective
Electrostaticobjective
-21832V -16148V -8262V 0V
Tetrode MACMAC Principle
DNA Helix Stretching Ladder
2.2nm
0.34
nm
0.5n
m
0.7n
m
LEEM results• Reduced dithiol-modified oligomers show most
promising results - Formed small islands on Au surface - Suitable
for spectroscopy measurements• Un-reduced dithiol-modified
oligomers - Formed fractal-like structures - Not suitable for
spectroscopy measurements• Long ss ultramers and hybridized ds
oligomers - Charged up severely in the LEEM - Likely due to the
missing salt rinse step
• Electron reflectivity spectra from Au substrates with and
without immobilized DNA were acquired in a LEEM over a range of
landing energies from 0-20eV• Deposited DNA samples are easily
visible over a range of landing energies - Small change in landing
energy has strong impact on achievable contrast
• Electron reflectivity spectra at low electron energies
demonstrate the high contrast achievable for bulk DNA structures on
a Au surface• Early results indicate that immobilized islands with
different bases (5ʼ-/5DTPA/C-20mer vs. 5ʼ-/5DTPA/A-20mer) produce
different ʻsignaturesʼ when compared to the underlying Au layer
Unreduced 5’-/5DTPA/C-20mer
1eV electron energy
Electrostaticobjective
Combined magnetic objective
Tetrode mirror corrector
Magnification 10.02 9.49 1.00
Cs3 [m] 28,337 14,279 -14,286
Cc [m] 81.27 52.59 -52.65
Cs5 [m] -81,728,764,353 -36,379,741,952 -71,746,471
Csc [m] 863,307,148 429,392,488 124,694
Ccc [m] -487,553 -275,505 200.2
MAD-LEEMKey FeaturesMonochromator• Energy spread reduced to <
50meV (from 0.5-2eV)
Aberration corrector• Resolution improved to < 1nm @ 100eV
(from 5nm)
Dual beam illumination• 2 coaxial flood beams eliminate
charging, high pressure (e.g. ESEM) is not needed
Screen(CCD)
Specimen/Stage
Objectivelens
Projection optics
Electron source
(Imaging beam)
Illumination optics
Electron source(Chargingbeam)
Illumination optics
Beamsplitter
Symmetrymirror
Aberration corrector
0-500eV
Monochromator
