Sample Drift Research Articles

3D-Aligner: advanced computational tool for correcting image distortion in expansion microscopy

Expansion Microscopy (ExM) is an innovative and cost-effective super-resolution microscopy technique that has become popular in cell biology research. It achieves super-resolution by physically expanding specimens. Since its introduction, ExM has undergone continuous methodological developments to enhance its resolution and labeling capabilities. However, ExM imaging often encounters sample drift during image acquisition due to the physical movement of the expanded hydrogel, posing a significant challenge for accurate image reconstruction. Despite many proposed experimental solutions to mitigate sample drift, a universal solution has yet to be established. In response to this challenge, we developed 3D-Aligner, an advanced and user-friendly image analysis tool designed to computationally correct drift in ExM images for precise three-dimensional image reconstruction and downstream quantification. We demonstrate that 3D-Aligner effectively determines and corrects drift in ExM images with different expansion rates and various fluorescently labeled biological targets, showcasing its capabilities and robustness in drift correction. Additionally, we validate the precision of 3D-Aligner by comparing drift values across different labeled targets and highlight the importance of drift correction in quantification of biological structures.

Application of a novel local and automatic PCA algorithm for diffraction pattern denoising in TEM-ASTAR analysis in microelectronics

This paper introduces a novel denoising method for TEM-ASTAR™ Diffraction Pattern (DP) datasets, termed LAT–PCA (Local Automatic Thresholding – Principal Component Analysis). This approach enhances the established PCA algorithm by partitioning the 4D dataset (a 2D map of 2D DPs) into localized windows. Within these windows, PCA identifies a basis where the physical signal predominantly resides in the higher-order principal components. By thresholding lower-order components, the method effectively reduces noise while retaining the essential features of the DPs. The locality of the approach, focusing on small windows, enhances computational efficiency and aligns with the localized nature of the crystallographic grain signals in ASTAR. The automatic aspect of the method employs a theoretical pure noise distribution, i.e. a Marchenko-Pastur Distribution, to set a threshold, beyond which the components are mostly noise.The LAT–PCA method offers significant reductions in acquisition and post-processing times. With denoised data, selecting the correct parameters for accurate phase maps and grain orientations becomes more straightforward, facilitating robust quantitative grain analysis. Experiments performed on a silicon-germanium-carbon sample validate the method's efficacy. The sample was analyzed with varying acquisition times to produce a high signal-to-noise ratio reference dataset and a lower ratio test dataset. The LAT–PCA algorithm's denoising results on the test dataset were benchmarked against the reference, demonstrating considerable improvements and reliability.In summary, LAT–PCA is an effective, automatic solution for denoising TEM DP datasets. Its adaptability to different noise levels and local processing capability makes it a valuable tool for enhancing dataset quality and reducing the time required for data acquisition and analysis. This method can minimize acquisition time, conserve microscope usage, and reduce sample drift and deterioration, leading to more accurate characterizations with fewer deformation artifacts.

3D-Aligner: An advanced computational tool designed to correct image distortion in expansion microscopy for precise 3D reconstitution and quantitative analysis.

Expansion Microscopy (ExM) is an innovative and cost-effective super-resolution microscopy technique that has become popular in cell biology research. It achieves super-resolution by physically expanding specimens. Since its introduction, ExM has undergone continuous methodological developments to enhance its resolution and labeling capabilities. However, ExM imaging often encounters sample drift during image acquisition due to the physical movement of the expanded hydrogel, posing a significant challenge for accurate image reconstruction. Despite many proposed experimental solutions to mitigate sample drift, a universal solution has yet to be established. In response to this challenge, we developed 3D-Aligner, an advanced and user-friendly image analysis tool designed to computationally correct drift in ExM images for precise three-dimensional image reconstruction and downstream quantification. We demonstrate that 3D-Aligner effectively determines and corrects drift in ExM images with different expansion rates and various fluorescently labeled biological targets, showcasing its capabilities and robustness in drift correction. Additionally, we validate the precision of 3D-Aligner by comparing drift values across different labeled targets and highlight the importance of drift correction in quantification of biological structures.

Remaining useful life prediction across operating conditions based on deep subdomain adaptation network considering the weighted multi-source domain

As one of the core contents of transfer learning, domain adaptation (DA) has been widely used to predict of the remaining useful life (RUL) of rolling bearings across operating conditions. However, existing RUL prediction methods often adopt single-source DA and do not consider the sample's drift phenomenon and migration degree in the source domain. At the same time, the distribution of bearing samples in different subdomains (degradation stages) differs, forcing global adaptation to inevitably lead to negative migration. Therefore, a deep subdomain adaptation network considering weighted multi-source domain (DSAN-WM) is proposed to improve the prediction accuracy of RUL across operating conditions. Firstly, a migration metric is developed to measure the migration degree of samples in the source domain, and a high-quality multi-source domain sample set is constructed. Secondly, a fine-grained feature distribution alignment strategy for subdomains based on adaptive degradation stage identification is proposed to reduce the difference in sample distribution in the same subdomain. Thirdly, for each decision boundary obtained by the multi-source domain model, consistent alignment and degradation constraints based on physical knowledge are established to improve the consistency and prediction accuracy of each regressor output. Lastly, the effectiveness of the proposed method is verified using eight migration scenarios of two bearing data sets, and the superiority of the method is proven through a comparison with advanced deep learning and transfer learning approaches.

Automated Sample Drift Correction for Low Intensity Electron Counted TEM Images

Automated Sample Drift Correction for Low Intensity Electron Counted TEM Images

An end-to-end approach for single-cell infrared absorption spectroscopy of bacterial inclusion bodies: from AFM-IR measurement to data interpretation of large sample sets

BackgroundInclusion bodies (IBs) are well-known subcellular structures in bacteria where protein aggregates are collected. Various methods have probed their structure, but single-cell spectroscopy remains challenging. Atomic Force Microscopy-based Infrared Spectroscopy (AFM-IR) is a novel technology with high potential for the characterisation of biomaterials such as IBs.ResultsWe present a detailed investigation using AFM-IR, revealing the substructure of IBs and their variation at the single-cell level, including a rigorous optimisation of data collection parameters and addressing issues such as laser power, pulse frequency, and sample drift. An analysis pipeline was developed tailored to AFM-IR image data, allowing high-throughput, label-free imaging of more than 3500 IBs in 12,000 bacterial cells. We examined IBs generated in Escherichia coli under different stress conditions. Dimensionality reduction analysis of the resulting spectra suggested distinct clustering of stress conditions, aligning with the nature and severity of the applied stresses. Correlation analyses revealed intricate relationships between the physical and morphological properties of IBs.ConclusionsOur study highlights the power and limitations of AFM-IR, revealing structural heterogeneity within and between IBs. We show that it is possible to perform quantitative analyses of AFM-IR maps over a large collection of different samples and determine how to control for various technical artefacts.

A novel STM for quality atomic resolution with piezoelectric motor of high compactness and simplicity

Scanning tunneling microscope (STM) is a renowned scientific tool for obtaining high-resolution atomic images of materials. Herein, we present an innovative design of the scanning unit with a compact yet powerful inertial piezoelectric motor inspired by the Spider Drive motor principle. The scanning unit mainly consists of a small 9 mm long piezoelectric tube scanner (PTS), one end of which is coaxially connected to the main sapphire body of the STM. Of particular emphasis in this design is the piezoelectric shaft (PS), constructed from piezoelectric material instead of conventional metallic or zirconium materials. The PS is a rectangular piezoelectric stack composed of two piezoelectric plates, which are elastically clamped on the inner wall of the PTS via a spring strip. The PTS and PS expand and contract independently with each other to improve the inertial force and reduce the threshold voltage. To ensure the stability of the PS and balance the stepping performance of the inertial motor, a counterweight, and a matching conical spring are fixed at the tail of the PS. This innovative design allows for the assessment of scanning unit performance by applying a driving signal, threshold voltage is 50 V at room temperature. Step sizes vary from 0.1 to 1 µm by changing the driving signal at room temperature. Furthermore, we successfully obtained atomic-resolution images of a highly oriented pyrolytic graphite (HOPG) sample and low drift rates of 23.4 pm/min and 34.6 pm/min in X-Y plane and Z direction, respectively, under ambient conditions. This small, compact STM unit has the potential for the development of a rotatable STM for use in cryogen-free magnets, and superconducting magnets.

Localization of fixed dipoles at high precision by accounting for sample drift during illumination

Single molecule localization microscopy relies on the precise quantification of the position of single dye emitters in a sample. This precision is improved by the number of photons that can be detected from each molecule. Particularly recording at cryogenic temperatures dramatically reduces photobleaching and would, hence, in principle, allow the user to massively increase the illumination time to several seconds. The downside of long illuminations, however, would be image blur due to inevitable jitter or drift occurring during the illuminations, which deteriorates the localization precision. In this paper, we theoretically demonstrate that a parallel recording of the fiducial marker beads together with a fitting approach accounting for the full drift trajectory allows for largely eliminating drift effects for drift magnitudes of several hundred nanometers per frame. We showcase the method for linear and diffusional drift as well as oscillations, assuming fixed dipole orientations during each illumination.

Pseudoheterodyne interferometry for multicolor near-field imaging.

We report the development and characterization of a detection technique for scattering-type scanning near-field optical microscopy (s-SNOM) that enables near-field amplitude and phase imaging at two or more wavelengths simultaneously. To this end, we introduce multispectral pseudoheterodyne (PSH) interferometry, where infrared lasers are combined to form a beam with a discrete spectrum of laser lines and a time-multiplexing scheme is employed to allow for the use of a single infrared detector. We first describe and validate the implementation of multispectral PSH into a commercial s-SNOM instrument. We then demonstrate its application for the real-time correction of the negative phase contrast (NPC), which provides reliable imaging of weak IR absorption at the nanoscale. We anticipate that multispectral PSH could improve data throughput, reduce effects of sample and interferometer drift, and help to establish multicolor s-SNOM imaging as a regular imaging modality, which could be particularly interesting as new infrared light sources become available.

RegiSTORM: channel registration for multi-color stochastic optical reconstruction microscopy

BackgroundStochastic optical reconstruction microscopy (STORM), a super-resolution microscopy technique based on single-molecule localizations, has become popular to characterize sub-diffraction limit targets. However, due to lengthy image acquisition, STORM recordings are prone to sample drift. Existing cross-correlation or fiducial marker-based algorithms allow correcting the drift within each channel, but misalignment between channels remains due to interchannel drift accumulating during sequential channel acquisition. This is a major drawback in multi-color STORM, a technique of utmost importance for the characterization of various biological interactions.ResultsWe developed RegiSTORM, a software for reducing channel misalignment by accurately registering STORM channels utilizing fiducial markers in the sample. RegiSTORM identifies fiducials from the STORM localization data based on their non-blinking nature and uses them as landmarks for channel registration. We first demonstrated accurate registration on recordings of fiducials only, as evidenced by significantly reduced target registration error with all the tested channel combinations. Next, we validated the performance in a more practically relevant setup on cells multi-stained for tubulin. Finally, we showed that RegiSTORM successfully registers two-color STORM recordings of cargo-loaded lipid nanoparticles without fiducials, demonstrating the broader applicability of this software.ConclusionsThe developed RegiSTORM software was demonstrated to be able to accurately register multiple STORM channels and is freely available as open-source (MIT license) at https://github.com/oystein676/RegiSTORM.git and https://doi.org/10.5281/zenodo.5509861 (archived), and runs as a standalone executable (Windows) or via Python (Mac OS, Linux).

Automated piezoresponse force microscopy domain tracking during fast thermally stimulated phase transition in CuInP2S6 * *Notice: This manuscript has been authored by UT-Battelle, LLC, under contract DE-AC05-00OR22725 with the US Department of Energy (DOE). The United States Government retains and the publisher, by accepting the article for publication, acknowledges that the United States Government retains a nonexclusive, paid-up, irrevocable, worldwide

Real-time tracking of dynamic nanoscale processes such as phase transitions by scanning probe microscopy is a challenging task, typically requiring extensive and laborious human supervision. Smart strategies to track specific regions of interest (ROI) in the system during such transformations in a fast and automated manner are necessary to study the evolution of the microscopic changes in such dynamic systems. In this work, we realize automated ROI tracking in piezoresponse force microscopy during a fast (≈0.8 °C s−1) thermally stimulated ferroelectric-to-paraelectric phase transition in CuInP2S6. We use a combination of fast (1 frame per second) sparse scanning with compressed sensing image reconstruction and real-time offset correction via phase cross correlation. The applied methodology enables in situ fast and automated functional nanoscale characterization of a certain ROI during external stimulation that generates sample drift and changes local functionality.

Serial intravital 2-photon microscopy and analysis of the kidney using upright microscopes.

Serial intravital 2-photon microscopy of the kidney and other abdominal organs is a powerful technique to assess tissue function and structure simultaneously and over time. Thus, serial intravital microscopy can capture dynamic tissue changes during health and disease and holds great potential to characterize (patho-) physiological processes with subcellular resolution. However, successful image acquisition and analysis require significant expertise and impose multiple potential challenges. Abdominal organs are rhythmically displaced by breathing movements which hamper high-resolution imaging. Traditionally, kidney intravital imaging is performed on inverted microscopes where breathing movements are partly compensated by the weight of the animal pressing down. Here, we present a custom and easy-to-implement setup for intravital imaging of the kidney and other abdominal organs on upright microscopes. Furthermore, we provide image processing protocols and a new plugin for the free image analysis software FIJI to process multichannel fluorescence microscopy data. The proposed image processing pipelines cover multiple image denoising algorithms, sample drift correction using 2D registration, and alignment of serial imaging data collected over several weeks using landmark-based 3D registration. The provided tools aim to lower the barrier of entry to intravital microscopy of the kidney and are readily applicable by biomedical practitioners.

Fast4DReg - fast registration of 4D microscopy datasets.

Unwanted sample drift is a common issue that plagues microscopy experiments, preventing accurate temporal visualization and quantification of biological processes. Although multiple methods and tools exist to correct images post acquisition, performing drift correction of three-dimensional (3D) videos using open-source solutions remains challenging and time consuming. Here, we present a new tool developed for ImageJ or Fiji called Fast4DReg that can quickly correct axial and lateral drift in 3D video-microscopy datasets. Fast4DReg works by creating intensity projections along multiple axes and estimating the drift between frames using two-dimensional cross-correlations. Using synthetic and acquired datasets, we demonstrate that Fast4DReg can perform better than other state-of-the-art open-source drift-correction tools and significantly outperforms them in speed. We also demonstrate that Fast4DReg can be used to register misaligned channels in 3D using either calibration slides or misaligned images directly. Altogether, Fast4DReg provides a quick and easy-to-use method to correct 3D imaging data before further visualization and analysis.

Measuring the fluidity of the membrane of Bacillus subtilis using TIR-FCS.

Bacteria need to constantly maintain the fluidity of their membrane in response to external stimuli such as changes in temperature, growth medium, etc. So far, bacterial membrane remodeling could only be assessed indirectly via biochemical methods like fatty acid analysis or in live bacteria using environment-sensitive dyes. Fluorescence correlation spectroscopy (FCS), a gold-standard technique in membrane dynamics investigations, was however barely used to study bacterial membranes. This is likely due to an increased technical complexity, caused by the small size of bacteria and mounting difficulties, which complicates FCS measurements on confocal microscopes as they are usually performed. Here, we show that imaging FCS on a TIRF microscope (TIR-FCS) can remediate this thanks to an increased axial resolution with respect to confocal and ability to correct for sample drift using image registration. To facilitate the analysis of the large amount of data generated by TIR-FCS, we developed a simple method to automatically exclude artefactual FCS curves. As a result, we could perform high-throughput measurements of the diffusion coefficients of lipophilic dyes like Nile Red or TopFluor-sphingomyelin in the membrane of B. subtilis. Using this assay, we observed the effects of membrane remodeling in response to known stimuli like a cold shock. We also looked at the effects of the widely used membrane-perturbing drug benzyl alcohol, revealing an unexpectedly temperature-dependent fluidization effect. Finally, we used TIR-FCS to explore membrane fluidity adaptation during the sporulation process. We believe that this method has significant potential in microbiology and can easily be applied in the future to different problems, to study membrane proteins diffusion or to understand the effects of selected drugs on membrane viscosity.

Development of temporal series 4D-STEM and application to relaxation time measurement.

Diffraction patterns contain useful information about the materials. Recent developments in four-dimensional scanning transmission electron microscopy and the acquisition of the spatial distribution of diffraction patterns have produced significant results. The acquisition of a temporal series of diffractions is achieved for a stationary beam. However, the acquisition of spatiotemporal distribution of diffraction patterns has only been established under limited conditions. In this study, we developed a simple method that enables the recording of the spatiotemporal distribution of diffraction patterns and applied it to the relaxation time measurement that is robust to sample drift.

SEM3De: image restoration for FIB-SEM.

FIB-SEM (Focused Ion Beam-Scanning Electron Microscopy) is a technique to generate 3D images of samples up to several microns in depth. The principle is based on the alternate use of SEM to image the surface of the sample (a few nanometers thickness) and of FIB to mill the surface of the sample a few nanometers at the time. In this way, huge stacks of images can thus be acquired.Although this technique has proven useful in imaging biological systems, the presence of some visual artifacts (stripes due to sample milling, detector saturation, charge effects, focus or sample drift, etc.) still raises some challenges for image interpretation and analyses. With the aim of meeting these challenges, we developed a freeware (SEM3De) that either corrects artifacts with state-of-the-art approaches or, when artifacts are impossible to correct, enables the replacement of artifactual slices by an in-painted image created from adjacent non-artifactual slices. Thus, SEM3De improves the overall usability of FIB-SEM acquisitions. SEM3De can be downloaded from https://sourceforge.net/projects/sem3de/ as a plugin for ImageJ.

NANOINDENTATION OF SOFT MATERIALS. ANALYSIS OF THE EXPERIMENTAL FACTORS IN CONSTRUCTING A MATHEMATICAL MODEL

The authors call for attention to the specifics of conducting experiments on nanoindentation of soft materials (elastomers, polymers), the features of the experimental setup, the material itself, the interaction of the material under study with the scanning elements of the setup, and environmental conditions. The paper shows which of them require to be taken into account in mathematical models, and which can be neglected, or can be almost completely compensated for by others. The following topics are considered: influence of cantilever bending and its inclination, humidity, plasticity, and viscosity, probe jump to the surface, determining the radius of the probe tip curvature, plastics, destruction of the sample during double indentation, size (scale) effect, sample drift, preservation of the probe shape before and after the experiment, time-varying surface properties, and surface energy during contact formation. This work is intended both to simplify further research and to focus efforts on solving acute problems.

Quantitative dSTORM superresolution microscopy

AbstractLocalization based superresolution technique provides the highest spatial resolution in optical microscopy. The final image is formed by the precise localization of individual fluorescent dyes, therefore the quantification of the collected data requires special protocols, algorithms and validation processes. The effects of labelling density and structured background on the final image quality were studied theoretically using the TestSTORM simulator. It was shown that system parameters affect the morphology of the final reconstructed image in different ways and the accuracy of the imaging can be determined. Although theoretical studies help in the optimization procedure, the quantification of experimental data raises additional issues, since the ground truth data is unknown. Localization precision, linker length, sample drift and labelling density are the major factors that make quantitative data analysis difficult. Two examples (geometrical evaluation of sarcomere structures and counting the γH2AX molecules in DNA damage induced repair foci) have been presented to demonstrate the efficiency of quantitative evaluation experimentally.

Quantifying the Structure and Chemistry of Semiconductor Devices with the Latest Advancements in Scanning Transmission Electron Microscopy

Electron microscopy has been revolutionized by the advent of the aberration corrector and advances in detector both for imaging and spectroscopy. Aberration correction dramatically improved spatial resolution into the sub-ångstrom regime, while new detectors have unlocked information about materials that was previously only imaginable.While these recent advances have proven essential to the atomic scale characterization of materials, many measurements have remained largely semi-quantitative. In particular, accuracy and precision for scanning transmission electron microscopy (STEM) was significantly hampered by the presence of sample drift and scan distortion. Until recently, this limitation has obscured the capabilities to characterize minute changes to the atomic structure that can ultimately define material properties.In this talk, we will introduce revolving scanning transmission electron microscopy (RevSTEM) for the characterization of semiconductors. The method uses a series of fast-acquisition STEM images, but with the scan coordinates rotated between successive frames. This scan rotation introduces a concomitant change in image distortion that is then used to analyze the sample drift rate and direction. Multiple case studies will be presented to demonstrate the power of this technique to characterize electronic materials. For example, we will show how picometer level precision and accuracy enabled the capability to directly quantify ferroelectric HfO2 thin films. We will also show how RevSTEM images can be used to accurately determine crystallographic parameters in real-space, and to determine the structural origins of polarization.When combined with modern spectroscopy tools, the technique can directly inform growth mechanisms in AlN based materials for fabrication of quantum well structures. Specifically, We will show the interface quality between various AlGaN/AlN quantum wells using a combination of high-angle annular dark-field (HAADF) imaging and energy dispersive X-ray spectroscopy (EDS). We will show that we can directly measure the lattice parameters for each layer and how they vary across each AlN/AlGaN interface. Using EDS, we correlate the lattice parameters and lattice parameter values to the chemical composition in each layer. We show that throughout multiple quantum well structures, there is an asymmetrical interface with a diffusion of Ga on one side of the AlGaN layer. Further, we will discuss fluctuation in the composition present throughout each layer and the role on the local strain distribution.

Performance evaluation of Hipee S2 point-of-care testing urine dipstick analyser: a cross-sectional study

ObjectiveWith advances in mobile technology, smartphone-based point-of-care testing (POCT) urinalysis hold great potential for disease screening and health management for clinicians and individual users. The purpose of this study is...