Sample Drift Research Articles

Picometer-scale atom position analysis in annular bright-field STEM imaging

We study the effects of specimen mistilt on the picometer-scale measurement of local structure by combing experiment and simulation in annular bright-field scanning transmission electron microscopy (ABF-STEM). A relative distance measurement method is proposed to separate the tilt effects from the scan noise and sample drift induced image distortion. We find that under a typical experimental condition a small specimen tilt (∼6 mrad) in 25 nm thick SrTiO3 along [001] causes 11.9 pm artificial displacement between O and Sr/TiO columns in ABF image, which is more than 3 times of scan noise and sample drift induced image distortion ∼3.2 pm, suggesting the tilt effect could be dominant for the quantitative analysis of ABF images. The artifact depends on the crystal mistilt angle, specimen thickness, defocus, convergence angle and uncorrected aberration. Our study provides useful insights into detecting and correcting tilt effects during both experiment operation and data analysis to extract the real structure information and avoid mis-interpretations of atomic structure as well as the properties such as oxygen octahedral distortion/shift.

Fast Projection Matching for X-ray Tomography

X-ray 3D tomographic techniques are powerful tools for investigating the morphology and internal structures of specimens. A common strategy for obtaining 3D tomography is to capture a series of 2D projections from different X-ray illumination angles of specimens mounted on a finely calibrated rotational stage. However, the reconstruction quality of 3D tomography relies on the precision and stability of the rotational stage, i.e. the accurate alignment of the 2D projections in the correct three-dimensional positions. This is a crucial problem for nano-tomographic techniques due to the non-negligible mechanical imperfection of the rotational stages at the nanometer level which significantly degrades the spatial resolution of reconstructed 3-D tomography. Even when using an X-ray micro-CT with a highly stabilized rotational stage, thermal effects caused by the CT system are not negligible and may cause sample drift. Here, we propose a markerless image auto-alignment algorithm based on an iterative method. This algorithm reduces the traditional projection matching method into two simplified matching problems and it is much faster and more reliable than traditional methods. This algorithm can greatly decrease hardware requirements for both nano-tomography and data processing and can be easily applied to other tomographic techniques, such as X-ray micro-CT and electron tomography.

Calibrated sub-micron temperature mapping of an operating plasmonic HAMR device by thermoreflectance imaging

Thermal characterization of nano-featured devices is a critical challenge for the development of high performance devices. Although far-field thermoreflectance imaging is limited in spatial resolution by the optical diffraction limit, it is more amenable to absolute temperature calibration of plasmonic devices than existing near-field scanning probe tip methods. We have built an advanced thermoreflectance microscope capable of 50 ns time-resolved, diffraction-limited temperature imaging that can account and correct for thermal expansion, sample drift, numerical aperture, and polarization induced variations in the apparent thermoreflectance coefficient of nanoscale structures. We developed a per-pixel transient calibration technique using this microscope to measure the absolute temperature of an operating heat-assisted magnetic recording (HAMR) head, including features as narrow as 200 nm. The resulting temperature information can be used to experimentally validate numerical models in the design process of such plasmonic devices.

Determining oxygen relaxations at an interface: A comparative study between transmission electron microscopy techniques

Nowadays, aberration corrected transmission electron microscopy (TEM) is a popular method to characterise nanomaterials at the atomic scale. Here, atomically resolved images of nanomaterials are acquired, where the contrast depends on the illumination, imaging and detector conditions of the microscope. Visualization of light elements is possible when using low angle annular dark field (LAADF) STEM, annular bright field (ABF) STEM, integrated differential phase contrast (iDPC) STEM, negative spherical aberration imaging (NCSI) and imaging STEM (ISTEM). In this work, images of a NdGaO3-La0.67Sr0.33MnO3 (NGO-LSMO) interface are quantitatively evaluated by using statistical parameter estimation theory. For imaging light elements, all techniques are providing reliable results, while the techniques based on interference contrast, NCSI and ISTEM, are less robust in terms of accuracy for extracting heavy column locations. In term of precision, sample drift and scan distortions mainly limits the STEM based techniques as compared to NCSI. Post processing techniques can, however, partially compensate for this. In order to provide an outlook to the future, simulated images of NGO, in which the unavoidable presence of Poisson noise is taken into account, are used to determine the ultimate precision. In this future counting noise limited scenario, NCSI and ISTEM imaging will provide more precise values as compared to the other techniques, which can be related to the mechanisms behind the image recording.

Integrated financial data stream mining based on memory curve

Integrated financial data stream mining is an important method to study financial data stream with the sample drift. In view of the defects of the traditional integrated financial data stream mining, human memory curve is introduced into the financial data stream mining, and the memorizing based financial data stream mining (MFSM) is proposed. The model regards the base classifier as the knowledge gained by the system, through the “Memory” mechanism, it not only makes the base classifier useful in history be kept in the “Memory” attributing to the strong memory capacity, and improve the stability of the prediction, but also select the base classifier with good classification effect from the “Memory bank” to participate in the integrated prediction, so as to improve the adaptability to the sample variation. Based on the MFSM model, a memorizing based integrated financial data stream mining (MISM) is put forward, which makes use of the Ebbinghaus memory curve to design the memory curve of the system, and utilizes the selective integration to simulate human’s “Memory” mechanism. After compared with 4 typical financial data stream mining algorithms, the results show that: The MISM algorithm has high classification accuracy and strong overall adaptability to the sample drift, especially for the sample drift that ocurrs repeated and the complex sample drift existing in the practical application. It not only can adapt to the new sample variations quickly, but also can effectively resist the impact of the random sample fluctuations on the system performance.

Novel in –Situ heating transmission electron microscope holder for atomic resolution

This study presents novel design of “in-situ” TEM heating holder. In-situ experimentations are important because hot deformation, dynamic recovery and dynamic recrystallization are important processes which are not well understood because it is very difficult to assess the nature microstructural processes solely from the traditional post mortem study of samples. We have designed an optimum heating holder that is dynamically stable for accuracy and image processes, capable of elevating the sample temperature up to 2000 K, with minimum controllable drift of the sample position. Sample drift is calculated. Thermal stressed induces in the sample are calculated. The in-depth finite element simulations provide detailed information about thermal and dynamic behavior of the heating holder. Finite element results show that the proposed design outperform the current heating technology. Based on the finite element results, a revised design is proposed that has lower temperature gradient among the sample, lower temperature difference between the sample and the TEM grids, lower thermal stresses in the sample, lower heat losses to the microscope parts. Furthermore, the present analysis provides the basis for the design of optimal heating holder for accurate measurements.DOI: http://dx.doi.org/10.5755/j01.mech.23.2.13544

A protocol for registration and correction of multicolour STED superresolution images.

Multicolour fluorescence imaging by STimulated Emission Depletion (STED) superresolution microscopy with doughnut-shaped STED laser beams based on different wavelengths for each colour channel requires precise image registration. This is especially important when STED imaging is used for co-localisation studies of two or more native proteins in biological specimens to analyse nanometric subcellular spatial arrangements. We developed a robust postprocessing image registration protocol, with the aim to verify and ultimately optimise multicolour STED image quality. Importantly, this protocol will support any subsequent quantitative localisation analysis at nanometric scales. Henceforth, using an approach that registers each colour channel present during STED imaging individually, this protocol reliably corrects for optical aberrations and inadvertent sample drift. To achieve the latter goal, the protocol combines the experimental sample information, from corresponding STED and confocal images using the same optical beam path and setup, with that of an independent calibration sample. As a result, image registration is based on a strategy that maximises the cross-correlation between sequentially acquired images of the experimental sample, which are strategically combined by the protocol. We demonstrate the general applicability of the image registration protocol by co-staining of the ryanodine receptor calcium release channel in primary mouse cardiomyocytes. To validate this new approach, we identify user-friendly criteria, which - if fulfilled - support optimal image registration. In summary, we introduce a new method for image registration and rationally based postprocessing steps through a highly standardised protocol for multicolour STED imaging, which directly supports the reproducibility of protein co-localisation analyses. Although the reference protocol is discussed exemplarily for two-colour STED imaging, it can be readily expanded to three or more colours and STED channels.

Development of a real-time wave field reconstruction TEM system (I): incorporation of an auto focus tracking system.

We have developed a real-time wave field reconstruction transmission electron microscope system that enables auto focus tracking at a video rate. In the developed system, a high-speed image calculation technique using a graphical processing unit was incorporated along with two techniques facilitating high-speed focus control using high-voltage modulation and weighted image integration using exposure time control. By utilizing these techniques, the sample drift induced in the Z-axis direction can be measured and automatically corrected for every 1/30 of a second. The auto focus tracking system can be operated in the sample drift range of ±150 nm with a precision of ~0.4 nm. In addition to amorphous samples, the system can be used to examine thin crystalline samples, which indicates that atomic structure analysis can be performed in real time with high reliability even when heavy drift occurs in the Z-axis direction.

An in situ correlative STEM-EDS and HRTEM based nanoscale chemical characterization of solid-liquid interfaces in an aluminium alloy.

The chemistry and the structure of solid-liquid interface in an Al-Si based alloy during high temperature phase transformation were characterized at nanoscale using scanning Transmission Electron Microscopy-EDS and HRTEM. Such studies were until recently limited by large sample drift associated with conventional heating holders. This study was made possible thanks to the modern low-drift MEMS-chip based localized heating technology. The results reveal that (i) the structural interface between solid (111) oriented Si phase and the liquid phase (i.e. decay of crystalline order) coexisting at 600°C is 3.2 nm wide (ii) the STEM-EDS chemical maps show inhomogeneous distribution of the elements with the solid phase being rich in Si and the liquid phase rich in Al (iii) the HRTEM and the HAADF images display respectively dark and bright intensity bands along the interface which could be due to apparent enrichment of Cu at the interface region resulting in enhanced amplitude-contrast (darker band in HRTEM) and Z-contrast (bright band in HAADF) and (iv) intriguingly, the concentration profiles within (i.e. compositional width) and across the solid-liquid interface display element-specific complex and asymmetric variation in the chemical widths.

Division of Mitochondrial Nucleoids Visualized by Biplane FPALM/dSTORM

Mitochondrial DNA (mtDNA) is contained in nucleoids with accessory proteins and gene expression machinery proteins. To visualize them and screen sizes, 3D superresolution fluorescent photoactivable localization microscopy (FPALM) with Eos conjugated to the mitochondrial (mt) transcription factor-A (TFAM) [1], was employed in HepG2 cells. Also, 3D-immunocytochemistry of TFAM, mt-single-stranded-DNA-binding protein (mtSSB), or DNA, was used with the direct stochastic optical reconstruction microscopy (dSTORM). The localized points of FPALM/dSTORM data were remodeled using Delaunay tetrahedron modeling or by principal component analysis (PCA). Alternatively, PCA ellipsoid nucleoid images were normalized by Delaunay volume to obtain symmetrical ellipsoid (DVSE) models. For TFAM FPALM average DVSE of 80×80×159 nm dimensions were yielded, confirmed by TFAM dSTORM giving 78×78×170 nm; whereas mtDNA- and mtSSB-contoured ellipsoids were on average 65×65×152 nm and 44×44×133 nm, reflecting the nucleoid core and a space of unfolded mtDNA (or third-strand structures, such as D-loop), respectively. Due to the 2-fold lower axial vs. lateral resolution, only bulky DVSE models with a high aspect ratio and tilted towards the xy-plane were considered as two proximal nucleoids and suspicious to reflect a state immediately after the nucleoid division following mtDNA replication. Such nucleoid dividing pairs were visualized. The existence of twin nucleoids in mtDNA-dSTORM 3D images, representing snapshot of mtDNA “doubling”, i.e., predicted outcome of replication, supports our hypothetical assignments of similar nucleoids twins, when visualized via TFAM. The visualized nucleoid pairs (bulky, exhibiting high aspect ratios along the mt tubular axis) might represent direct observations of mt nucleoid division after mtDNA replication. Supported by grant 13-02033 (GACR).[1] M.J. Mlodzianoski et al. “Sample drift correction in 3D fluorescence photoactivation localization microscopy”. Opt. Express19, 15009-15019 (2011).

High Dynamic Range Pixel Array Detector for Scanning Transmission Electron Microscopy.

We describe a hybrid pixel array detector (electron microscope pixel array detector, or EMPAD) adapted for use in electron microscope applications, especially as a universal detector for scanning transmission electron microscopy. The 128×128 pixel detector consists of a 500 µm thick silicon diode array bump-bonded pixel-by-pixel to an application-specific integrated circuit. The in-pixel circuitry provides a 1,000,000:1 dynamic range within a single frame, allowing the direct electron beam to be imaged while still maintaining single electron sensitivity. A 1.1 kHz framing rate enables rapid data collection and minimizes sample drift distortions while scanning. By capturing the entire unsaturated diffraction pattern in scanning mode, one can simultaneously capture bright field, dark field, and phase contrast information, as well as being able to analyze the full scattering distribution, allowing true center of mass imaging. The scattering is recorded on an absolute scale, so that information such as local sample thickness can be directly determined. This paper describes the detector architecture, data acquisition system, and preliminary results from experiments with 80-200 keV electron beams.

Optimal Drift Correction for Superresolution Localization Microscopy with Bayesian Inference

Single-molecule-localization-based superresolution microscopy requires accurate sample drift correction to achieve good results. Common approaches for drift compensation include using fiducial markers and direct drift estimation by image correlation. The former increases the experimental complexity and the latter estimates drift at a reduced temporal resolution. Here, we present, to our knowledge, a new approach for drift correction based on the Bayesian statistical framework. The technique has the advantage of being able to calculate the drifts for every image frame of the data set directly from the single-molecule coordinates. We present the theoretical foundation of the algorithm and an implementation that achieves significantly higher accuracy than image-correlation-based estimations.

Drift correction for single-molecule imaging by molecular constraint field, a distance minimum metric.

BackgroundThe recent developments of far-field optical microscopy (single molecule imaging techniques) have overcome the diffraction barrier of light and improve image resolution by a factor of ten compared with conventional light microscopy. These techniques utilize the stochastic switching of probe molecules to overcome the diffraction limit and determine the precise localizations of molecules, which often requires a long image acquisition time. However, long acquisition times increase the risk of sample drift. In the case of high resolution microscopy, sample drift would decrease the image resolution.ResultsIn this paper, we propose a novel metric based on the distance between molecules to solve the drift correction. The proposed metric directly uses the position information of molecules to estimate the frame drift. We also designed an algorithm to implement the metric for the general application of drift correction. There are two advantages of our method: First, because our method does not require space binning of positions of molecules but directly operates on the positions, it is more natural for single molecule imaging techniques. Second, our method can estimate drift with a small number of positions in each temporal bin, which may extend its potential application.ConclusionsThe effectiveness of our method has been demonstrated by both simulated data and experiments on single molecular images.

Large area strain analysis using scanning transmission electron microscopy across multiple images

Here, we apply revolving scanning transmission electron microscopy to measure lattice strain across a sample using a single reference area. To do so, we remove image distortion introduced by sample drift, which usually restricts strain analysis to a single image. Overcoming this challenge, we show that it is possible to use strain reference areas elsewhere in the sample, thereby enabling reliable strain mapping across large areas. As a prototypical example, we determine the strain present within the microstructure of a Ni-based superalloy directly from atom column positions as well as geometric phase analysis. While maintaining atomic resolution, we quantify strain within nanoscale regions and demonstrate that large, unit-cell level strain fluctuations are present within the intermetallic phase.

Sub-nanometer drift correction for super-resolution imaging.

Spatial resolution of conventional far-field fluorescence microscopy is limited by diffraction of light. Single-molecule localization microscopy (SMLM), such as (direct) stochastic optical reconstruction microscopy (dSTORM/STORM), and (fluorescence) photoactivation localization microscopy (fPALM/PALM), can break this barrier by localizing single emitters and reconstructing super-resolution image with much higher precision. Nevertheless, a SMLM measurement needs to record a large number of image frames and takes considerable recording time. In this process, sample drift becomes a critical problem and cannot be neglected. In this Letter, we present a sub-nanometer precision, low-cost sample drift correction method based on minimizing normalized root-mean-square error (NRMSE) between bright field images. Two optical configurations are suggested for recording bright field and fluorescence images simultaneously or alternately. The method was demonstrated on simulated data, and better than 0.3 nm drift correction precision was achieved. It was also applied on dSTORM imaging of F-actins of 3T3 cell, and the quality of reconstructed super-resolution image was improved observably. This method does not require special hardware, extra labelling or markers, and no precision decline due to photobleaching. It can be applied as an add-on for SMLM setups and achieves sub-nanometer precision drift correction for post-measurement or real time drift compensation.

Imaging live cells at the nanometer-scale with single-molecule microscopy: obstacles and achievements in experiment optimization for microbiology.

Single-molecule fluorescence microscopy enables biological investigations inside living cells to achieve millisecond- and nanometer-scale resolution. Although single-molecule-based methods are becoming increasingly accessible to non-experts, optimizing new single-molecule experiments can be challenging, in particular when super-resolution imaging and tracking are applied to live cells. In this review, we summarize common obstacles to live-cell single-molecule microscopy and describe the methods we have developed and applied to overcome these challenges in live bacteria. We examine the choice of fluorophore and labeling scheme, approaches to achieving single-molecule levels of fluorescence, considerations for maintaining cell viability, and strategies for detecting single-molecule signals in the presence of noise and sample drift. We also discuss methods for analyzing single-molecule trajectories and the challenges presented by the finite size of a bacterial cell and the curvature of the bacterial membrane.

Accurate measurement of relative tilt and azimuth angles in electron tomography: a comparison of fiducial marker method with electron diffraction.

Electron tomography is a method whereby a three-dimensional reconstruction of a nanoscale object is obtained from a series of projected images measured in a transmission electron microscope. We developed an electron-diffraction method to measure the tilt and azimuth angles, with Kikuchi lines used to align a series of diffraction patterns obtained with each image of the tilt series. Since it is based on electron diffraction, the method is not affected by sample drift and is not sensitive to sample thickness, whereas tilt angle measurement and alignment using fiducial-marker methods are affected by both sample drift and thickness. The accuracy of the diffraction method benefits reconstructions with a large number of voxels, where both high spatial resolution and a large field of view are desired. The diffraction method allows both the tilt and azimuth angle to be measured, while fiducial marker methods typically treat the tilt and azimuth angle as an unknown parameter. The diffraction method can be also used to estimate the accuracy of the fiducial marker method, and the sample-stage accuracy. A nano-dot fiducial marker measurement differs from a diffraction measurement by no more than ±1°.

Putting a New Spin on Scanning Transmission Electron Microscopy

Local atomic scale ordering and structural distortions can significantly modify material properties. Aberration correction dramatically improves spatial resolution into the sub-Angstrom regime, unlocking information about material defects previously beyond reach. While STEM has proven essential to the atomic scale characterization of materials, for example at defects, interfaces, or even in perfect crystals, measurement of atomic displacements and distances has remained challenging due to the presence of sample drift. Distortion proportional to the rate of sample drift during image acquisition hinders the accurate measurement or even representation of atomic structure. Though modern STEM installations are optimized to reduce vibration, air flow/fields, and temperature fluctuations, sample drift generally remains.

Localization events-based sample drift correction for localization microscopy with redundant cross-correlation algorithm

Highly accurate sample drift correction is essential in super-resolution localization microscopy to guarantee a high spatial resolution, especially when the technique is used to visualize small cell organelle. Here we present a localization events-based drift correction method using a redundant cross-correlation algorithm originally developed to correct beam-induced motion in cryo-electron microscopy. With simulated, synthesized as well as experimental data, we have demonstrated its superior precision compared to previously published localization events-based drift correction methods. The major advantage of this method is the robustness when the number of localization events is low, either because a short correction time step is required or because the imaged structure is small and sparse. This method has allowed us to improve the effective resolution when imaging Golgi apparatus in mammalian cells.

Progress in quantitative single-molecule localization microscopy.

With the advent of single-molecule localization microscopy (SMLM) techniques, intracellular proteins can be imaged at unprecedented resolution with high specificity and contrast. These techniques can lead to a better understanding of cell functioning, as they allow, among other applications, counting the number of molecules of a protein specie in a single cell, studying the heterogeneity in protein spatial organization, and probing the spatial interactions between different protein species. However, the use of these techniques for accurate quantitative measurements requires corrections for multiple inherent sources of error, including: overcounting due to multiple localizations of a single fluorophore (i.e., photoblinking), undercounting caused by incomplete photoconversion, uncertainty in the localization of single molecules, sample drift during the long imaging time, and inaccurate image registration in the case of dual-color imaging. In this paper, we review recent efforts that address some of these sources of error in quantitative SMLM and give examples in the context of photoactivated localization microscopy (PALM).