We are developing a novel m onochromatic, a berration-corrected d ual- beam l ow e nergy e lectron m icroscope (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 Results Variety 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 Microscopy Has 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 Microscopy Advantages • 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/genome Challenges • 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 Analysis M. Mankos a , K. Shadman a , A. T. N’Diaye b , A. K. Schmid b , H.H.J. Persson c , and R.W. Davis c a Electron Optica, 1000 Elwell Court #110, Palo Alto, CA 94303, b NCEM, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA c Stanford Genome Technology Center, Stanford University School of Medicine, 855 California Avenue, Palo Alto, CA 94304 LECTRON OPTICA Acknowledgments References References Optics Simulations Plan Phase 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 1 st , 3 rd and 5 th order analysis to understand resolution limit prior to aberration correction • 5 th 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 5 th 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 5 th order geom. and 3 rd & 4 th rank chrom. aberrations - Monochromator needed to make 3 rd & 4 th rank chrom. aberrations negligible • Further improvement requires pentode MAC to cancel 5 th 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 1 st , 3 rd & 5 th order analysis for objective lens and tetrode MAC • Tetrode MAC improves resolution to ~ 1nm @ 200eV • Design of pentode MAC for C s5 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 MAC and monochromator 10eV diffraction 3 rd order spherical 2 nd rank chromatic total 5 th order spherical 3 rd order coma 3 rd rank chromatic 4 th rank chromatic 5 th order coma diffraction total 5 th order spherical 3 rd rank chromatic 4 th rank chromatic 5 th order coma total 4 th rank chromatic 5 th order coma diffraction 3 rd order spherical 2 nd rank chromatic total 5 th order spherical 3 rd order coma 4 th rank chromatic 3 rd order field curvature 5 th order coma 3 rd order astigmatism diffraction total 5 th order spherical 4 th rank chromatic 5 th order coma 3 rd order field curvature 3 rd order astigmatism diffraction total 4 th rank chromatic 5 th order coma 3 rd order field curvature 3 rd order astigmatism Standard LEEM 100eV LEEM with Tetrode MAC LEEM with Pentode MAC and monochromator Electron Energy Electron Reflectivity C G 1 e - e - MAC Lens Cs Cc Sequencing Approach . . CG TA TA GC CG AT CG . . . . AT GC CG GC TA GC CG . . TA GC ⤴ ⤴ MAD-LEEM ⤴ ⤴ Stretch out DNA on flat surface Convert nucleotide- specific electron reflectivity to grey levels Process image and store sequence Magnetic objective Electrostatic objective -21832V -16148V -8262V 0V Tetrode MAC MAC Principle DNA Helix Stretching Ladder 2.2nm 0.34nm 0.5nm 0.7nm 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 Electrostatic objective Combined magnetic objective Tetrode mirror corrector Magnification 10.02 9.49 1.00 C s3 [m] 28,337 14,279 -14,286 C c [m] 81.27 52.59 -52.65 C s5 [m] -81,728,764,353 -36,379,741,952 -71,746,471 C sc [m] 863,307,148 429,392,488 124,694 C cc [m] -487,553 -275,505 200.2 MAD-LEEM Key Features Monochromator • 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 Objective lens Projection optics Electron source (Imaging beam) Illumination optics Electron source (Charging beam) Illumination optics Beam splitter Symmetry mirror Aberration corrector 0-500eV Monochromator