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Combined Tilt- and Focal-Series Tomography for HAADF-STEM


Tim Dahmen , 1 * Holger Kohr , 2 Andrew R. Lupini , 4 Jean-Pierre Baudoin , 5 Christian Kübel , 6


Patrick Trampert , 1 Philipp Slusallek , 1 and Niels de Jonge 3 1 German Research Center for Artifi cial Intelligence (DFKI) GmbH , 66123 Saarbrü cken , Germany 2 KTH Royal Institute of Technology , Lindstedtsvä gen 25 , Stockholm , SE 100 44 , Sweden 3 INM – Leibniz Institute for New Materials , 66123 Saarbrü cken , Germany 4 Oak Ridge National Laboratory , Materials Science and Technology Division , Oak Ridge , TN 37831-6071 5 Aix-Marseille Université , Faculté de Médecine, 27 Bd Jean Moulin , 13005 Marseille , France 6 Karlsruhe Institute of Technology (KIT) , Institute of Nanotechnology (INT) and Karlsruhe Nano Micro Facility (KNMF) , 76344


Eggenstein-Leopoldshafen , Germany * tim.dahmen@df i.de


Abstract: A new aid to tomography in the scanning transmission electron microscope (STEM) is called combined tilt- and focal-series (CTFS). This software controls the recording of a tilt series where for each specimen tilt an entire focal series is recorded. This approach is particularly useful for thick specimens where the tilt range may be limited. Use of CTFS leads to a signifi cant reduction of the missing wedge effect and a better representation of the 3D shapes of features in the specimen.


Introduction T e primary means to study cellular ultrastructure in three dimensions (3D) with nanoscale resolution is currently tilt-series transmission electron microscopy (TEM) [ 1 – 2 ]. In this imaging modality, images are acquired at diff erent projection angles realized by mechanically tilting the sample stage. However, high-quality TEM imaging strongly depends on the condition that the sample is thinner than a few hundreds of nanometers because undesired inelastic and multiple electron scattering can otherwise lead to blurring of the image [ 3 ]. For biological materials, sectioning is one way to prepare suffi ciently thin samples. It has the disadvantage of limiting the tomogram to a subsection of the sample. For thicker samples containing, for example, whole cells, both inelastic and multiple elastic scattering contribute substantially to the projection data. Scanning transmission electron microscope (STEM), where the sample is scanned by a focused electron beam, is capable of recording images with nanoscale resolution of thicker samples [ 4 – 6 ]. However, because of geometric blurring and scattering of the probe through the sample thickness, resolution decreases with increasing sample thickness. Especially for slab- like samples, the eff ective thickness of the specimen as seen by the electron beam is increasing for higher tilt angles ( Figure 1a ). For example, a specimen titled to 70° exhibits a three-fold larger effective thickness than at 0° tilt. Especially for samples thicker than a few hundred nanometers, this limits the achievable resolution. It thus would be advantageous if the maximum tilt could be limited to ~40°, where the


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thickness increase is only a factor of 1.3. However, tomographic reconstructions then suff er from severe missing wedge artifacts, visible as elongated objects (axial elongation).


Avoiding tilting altogether is also possible by recording


a stack of images with diff erent focal values. Making use of the limited depth of fi eld (DOF) of aberration-corrected STEM, a focal series can be used to obtain 3D STEM information [ 7 – 13 ]. Therein, axial information is retrieved from a stack of images with diff erent focal values. T e 3D data set can be deconvolved to improve the axial resolution and to remove out-of-focus information [ 14 ]. However, the focal series data suff ers from a vertically elongated point spread function, and the vertical resolution is further reduced by shadowing eff ects below strongly scattering objects [ 10 – 11 ].


In this article, we present a combined tilt- and focal-series (CTFS) scheme as a new recording method for 3D STEM imaging [ 15 – 16 ]. In this scheme, the specimen is rotated in relatively large tilt increments over a limited tilt range, and for every tilt position a focal series is recorded ( Figure 1b ).


Figure 1 : (a) The thickness of the sample as seen by the electron beam increases with 1/cos(β ) as the sample is tilted by an angle β . (b) Schematic overview of the CTFS recording scheme. The sample is tilted by relatively large angular increments, and for every tilt position a focal series is recorded.


doi: 10.1017/S1551929516000328 www.microscopy-today.com • 2016 May


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