Microscopy Setup Research Articles

Simultaneous single particle tracking, phase retrieval and PSF reconstruction.

3D particle tracking and localization provide direct means to monitor biomolecular processes within nano-scale environments. However, optical aberrations due to inhomogeneous refractive indices are a major shortcoming in probing these processes in situ. In particular, point spread functions (PSF) may be distorted resulting in poor localization and linking across frames. This issue is particularly important when using pre-calibrated PSFs that do not take into account sample induced aberrations. The sample induced aberrations are often removed using experimental techniques such as adaptive optics (AO) by introducing new optical components to microscope setups. Here, we propose a computational method, leveraging a Bayesian framework, as an alternative to the AO techniques. Our Bayesian method is capable of simultaneous particle tracking, phase retrieval and PSF reconstruction directly from a given data set, without adding any new hardware to the optical setup. Moreover, our method is data efficient by rigorously propagating uncertainty from all existing sources in the problem, such as the uncertainty in the shape of PSF, often ignored. We benchmark our method using a wide range of synthetic and experimental data.

High-speed imaging of giant unilamellar vesicle production in cDICE.

Giant unilamellar vesicles (GUVs) are cell-sized containers that are commonly used as three-dimensional model membranes in biophysics, as in vitro model systems in synthetic biology, and even as cargo carriers in various other research fields. Despite their ubiquitous use, there is still no one-size-fits-all GUV production method. Over the years, numerous methods have been developed, attempting to meet the demanding requirements of robustness, reliability, and high yield while simultaneously achieving robust encapsulation. Double emulsion-based methods are often praised for their apparent simplicity and good yields; hence, methods like continuous droplet interface crossing encapsulation (cDICE) that make use of this principle, have gained popularity in recent years. In cDICE, aqueous droplets that originate from a capillary orifice are continuously forced through an oil-water interface by centrifugal force, thereby forming a lipid bilayer and thus GUVs. Although cDICE and related methods are frequently used in the field, the complexity of the underlying principles and fluid dynamics has not been considered previously, and how exactly the GUVs are being formed remains unknown. To elucidate the process of GUV formation in cDICE, we have developed a high-speed microscopy setup that allows us to visualize GUV formation in real time. We focused on the capillary orifice, where initial droplet formation occurs, and on the oil-water interface, where droplets are converted into GUVs. Our experiments reveal a complex droplet formation process at the capillary orifice and suboptimal droplet transfer through the water-oil interface, which we explain using fluid dynamics and theoretical modeling. Our results are a first step towards explaining the widely observed variation in encapsulation efficiency and size polydispersity in cDICE. Ultimately, these results will contribute to a better understanding of GUV formation processes in cDICE and in extension, in double emulsion-based methods in general.

Near-Surface Nanomechanics of Medical-Grade PEEK Measured by Atomic Force Microscopy.

Detecting subtle changes of surface stiffness at spatial scales and forces relevant to biological processes is crucial for the characterization of biopolymer systems in view of chemical and/or physical surface modification aimed at improving bioactivity and/or mechanical strength. Here, a standard atomic force microscopy setup is operated in nanoindentation mode to quantitatively mapping the near-surface elasticity of semicrystalline polyether ether ketone (PEEK) at room temperature. Remarkably, two localized distributions of moduli at about 0.6 and 0.9 GPa are observed below the plastic threshold of the polymer, at indentation loads in the range of 120-450 nN. This finding is ascribed to the localization of the amorphous and crystalline phases on the free surface of the polymer, detected at an unprecedented level of detail. Our study provides insights to quantitatively characterize complex biopolymer systems on the nanoscale and to guide the optimal design of micro- and nanostructures for advanced biomedical applications.

Sub-nanometer measurement of transient structural changes in dye-doped polystyrene microspheres

We present an interferometric spectral-domain optical coherence tomography microscopy setup to detect structural changes using interference of light reflected from different interfaces of the sample. We induce a reproducible nanometer-scale size change in dye-doped 10-µm polystyrene microspheres by the release of Stokes shift energy of dye molecules inside the microspheres, excited by a modulated 532-nm laser. The resulting optical path length difference was measured with a sensitivity of 0.4 p m / H z limited by photodetection noise, and reveals elastic as well as inelastic responses, which opens up possibilities for measuring the response of cell-sized biological objects.

Refractive axicon for X-ray microscopy applications: design, optimization, and experiment.

In a full-field transmission X-ray microscopy (TXM) setup, a condenser X-ray optical element is used to illuminate the sample by condensing the X-ray beam delivered by the synchrotron storage ring. On-going and future upgrades of synchrotron facilities to diffraction-limited storage rings will pose new challenges to these TXM setups, such as much smaller X-ray beams on the condenser. Here, we demonstrate that a refractive axicon can be used as an X-ray beam shaper to match the ring-shaped aperture of the condenser. Aiming at more efficient use of the incoming X-ray intensity, we explore several axicon designs both analytically and with numerical simulations. The axicons were produced by two-photon polymerization 3D printing on thin silicon nitride membrane substrates. The first characterization of the axicon was carried out at the TOMCAT beamline of the Swiss Light Source (Switzerland).

Electrical Actuation of DNA-Based Nanomechanical Systems.

DNA nanotechnology provides efficient methods for the sequence-programmable construction of mechanical devices with nanoscale dimensions. The resulting nanomachines could serve as tools for the manipulation of macromolecules with similar functionalities as mechanical tools and machinery in the macroscopic world. In order to drive and control these machines and to perform specific tasks, a fast, reliable, and repeatable actuation mechanism is required that can work against external loads. Here we describe a highly effective method for actuating DNA structures using externally applied electric fields. To this end, electric fields are generated with controllable direction and amplitude inside a miniature electrophoresis device integrated with an epifluorescence microscope. With this setup, DNA-based nanoelectromechanical devices can be precisely controlled. As an example, we demonstrate how a DNA-based nanorobotic system can be used to dynamically position molecules on a molecular platform with high speeds and accuracy. The microscopy setup also described here allows simultaneous monitoring of a large number of nanorobotic arms in real time and at the single nanomachine level.

SegPC-2021: A challenge & dataset on segmentation of Multiple Myeloma plasma cells from microscopic images

Multiple Myeloma (MM) is an emerging ailment of global concern. Its diagnosis at the early stages is critical for recovery. Therefore, efforts are underway to produce digital pathology tools with human-level intelligence that are efficient, scalable, accessible, and cost-effective. Following the trend, a medical imaging challenge on "Segmentation of Multiple Myeloma Plasma Cells in Microscopic Images (SegPC-2021)" was organized at the IEEE International Symposium on Biomedical Imaging (ISBI), 2021, France. The challenge addressed the problem of cell segmentation in microscopic images captured from the slides prepared from the bone marrow aspirate of patients diagnosed with Multiple Myeloma. The challenge released a total of 775 images with 690 and 85 images of sizes 2040×1536 and 1920×2560 pixels, respectively, captured from two different (microscope and camera) setups. The participants had to segment the plasma cells with a separate label on each cell's nucleus and cytoplasm. This problem comprises many challenges, including a reduced color contrast between the cytoplasm and the background, and the clustering of cells with a feeble boundary separation of individual cells. To our knowledge, the SegPC-2021 challenge dataset is the largest publicly available annotated data on plasma cell segmentation in MM so far. The challenge targets a semi-automated tool to ensure the supervision of medical experts. It was conducted for a span of five months, from November 2020 to April 2021. Initially, the data was shared with 696 people from 52 teams, of which 41 teams submitted the results of their models on the evaluation portal in the validation phase. Similarly, 20 teams qualified for the last round, of which 16 teams submitted the results in the final test phase. All the top-5 teams employed DL-based approaches, and the best mIoU obtained on the final test set of 277 microscopic images was 0.9389. All these five models have been analyzed and discussed in detail. This challenge task is a step towards the target of creating an automated MM diagnostic tool.

Bi-directional Dual-flow-RootChip for Physiological Analysis of Plant Primary Roots Under Asymmetric Perfusion of Stress Treatments.

Due to technical limitations, research to date has mainly focused on the role of abiotic and biotic stress-signalling molecules in the aerial organs of plants, including the whole shoot, stem, and leaves. Novel experimental platforms including the dual-flow-RootChip (dfRC), PlantChip, and RootArray have since expanded this to plant-root cell analysis. Based on microfluidic platforms for flow stream shaping and force sensing on tip-growing organisms, the dfRC has further been expanded into a bi-directional dual-flow-RootChip (bi-dfRC), incorporating a second adjacent pair of inlets/outlet, enabling bi-directional asymmetric perfusion of treatments towards plant roots (shoot-to-root or root-to-shoot). This protocol outlines, in detail, the design and use of the bi-dfRC platform. Plant culture on chip is combined with guided root growth and controlled exposure of the primary root to solute changes. The impact of surface treatment on root growth and defence signals can be tracked in response to abiotic and biotic stress or the combinatory effect of both. In particular, this protocol highlights the ability of the platform to culture a variety of plants, such as Arabidopsis thaliana, Nicotiana benthamiana, and Solanum lycopersicum, on chip. It demonstrates that by simply altering the dimensions of the bi-dfRC, a broad application basis to study desired plant species with varying primary root sizes under microfluidics is achieved. Key features Expansion of the method developed by Stanley et al. (2018a) to study the directionality of defence signals responding to localised treatments. Description of a microfluidic platform allowing culture of plants with primary roots up to 40 mm length, 550μm width, and 500 μm height. Treatment with polyvinylpyrrolidone (PVP) to permanently retain the hydrophilicity of partially hydrophobic bi-dfRC microchannels, enabling use with surface-sensitive plant lines. Description of novel tubing array setup equipped with rotatable valves for switching treatment reagent and orientation, while live-imaging on the bi-dfRC. Graphical overview Graphical overview of bi-dfRC fabrication, plantlet culture, and setup for root physiological analysis.(a) Schematic diagram depicting photolithography and replica molding, to produce a PDMS device. (b) Schematic diagram depicting seed culture off chip, followed by sub-culture of 4-day-old plantlets on chip. (c) Schematic diagram depicting microscopy and imaging setup, equipped with a media delivery system for asymmetric treatment introduction into the bi-dfRC microchannel root physiological analysis under varying conditions.

Changes in the Observed Shape of H6TPPS J-Aggerates by the Polarisation of the Incoming Light

Samples of H6TPPS J aggregates and bundles, deposited on glass and aligned under nitrogen flow, were measured in a 2-photon microscopy setup. Changes in the polarization state of the incoming laser have shown a difference in the resulting 2-photon scanning of the same measured sample, revelling otherwise hidden features. In addition, tracing the response of certain areas under different polarisation can provide information about the arrangement of the dipoles in that area. This shows the significant role of polarisation in 2-photon measurement, and the need to consider such effects in the microscopy of biological samples.

Pyroptosis Induction and Visualization at the Single-Cell Level Using Optogenetics.

Pyroptosis has been identified as a pro-inflammatory form of programmed cell death. It can be triggered by different stimuli including pathogen invasion or cell stress/danger signals releasing hundreds of proteins upon lysis that cause complex responses in neighboring cells. Pyroptosis is executed by the gasdermin (GSDM) family of proteins which, upon cleavage by caspases, form transmembrane pores that release cytokines to induce inflammation. However, despite the importance of gasdermins in the development of inflammatory diseases and cancer, a lot is still to be understood in the downstream consequences of this cell death pathway. Currently, conventional methods, such as drug treatments or chemically forced oligomerization, are limited in the spatiotemporal analysis of pyroptosis signaling in the cellular population, since all cells are primed for undergoing pyroptosis. Here, we provide a protocol for the application of a novel optogenetics tool called NLS_PhoCl_N-GSDMD_mCherry that enables precise temporal and spatial pyroptosis induction in a confocal microscopy setup, followed by imaging of the cell death process and subsequent quantitative analysis of the experiment. This tool opens new opportunities for the study of pyroptosis activation and of its effects on the bystander cell responses.

Multiphoton scanning laser microscope based on femtosecond fiber laser

We present a multiphoton scanning laser microscope based on a femtosecond frequency-doubled erbium-doped fiber laser. The laser used in the epi-illumination microscope setup generated 95 fs pulses at the wavelength of 780 nm with 44.3 mW average power at 100 MHz pulse repetition rate. The imaging process was controlled by custom software developed in the NI LabVIEW environment. Detection of two-photon fluorescence was proven by acquiring a series of images from various biological samples. Full Text: PDF ReferencesJ.W. Lichtman, J.A. Conchello, "Fluorescence microscopy", Nature methods 2(12), 910 (2005). CrossRef W. Zipfel, R. Williams, W. Webb, "Nonlinear magic: multiphoton microscopy in the biosciences", Nat. Biotechnol. 21, 1369 (2003). CrossRef Coherent, Chameleon Ultra Datasheet (2019). DirectLink J. Boguslawski et al., "In vivo imaging of the human eye using a 2-photon-excited fluorescence scanning laser ophthalmoscope", J. Clin. Invest. 132(2), e154218 (2022). CrossRef M.J. Marzejon et al., "Two-photon microperimetry with picosecond pulses", Biomed. Opt. Expr. 12, 462 (2021). CrossRef A. Fast et al., "Institutional Drivers Influence on CSR Engagement: A Comparison of Developed & Developing Economies", Sci. Rep. 10, 18093 (2020). CrossRef D. Stachowiak et al., "Femtosecond Er-doped fiber laser source tunable from 872 to 1075 nm for two-photon vision studies in humans", Biomed. Opt. Expr. 13, 1899 (2022). CrossRef MenloSystems, T-light Femtosecond Fiber Laser 1560 nm (2013). DirectLink J. Yao, L.V. Wang, "Photoacoustic microscopy", Laser and Photonics Rev. 7, 758 (2013). CrossRef D. Stachowiak et al., "Frequency-doubled femtosecond Er-doped fiber laser for two-photon excited fluorescence imaging", Biomed. Opt. Expr. 11, 4431 (2020). CrossRef B.R. Masters et al., "Mitigating thermal mechanical damage potential during two-photon dermal imaging", J. Biomed. Opt. 9, 1265 (2004). CrossRef

Structural and optical parameters of polyvinyl alcohol films reinforced with Mn2O3/reduced graphene oxide composite

The novel polyvinyl alcohol (PVA) films reinforced with varied concentrations of Mn2O3/reduced graphene oxide (rGO) nanoparticles (NP) are prepared via the casting technique. A hydrothermal approach methodology is used to prepare manganese oxide reduce graphene oxide (Mn2O3/rGO) composite. The X-ray diffraction (XRD), Fourier transform infrared (FTIR), scanning electron microscope (SEM), and optical microscope setups are used to study the impact of nanoparticles on the structure of the PVA matrix. The surface roughness was measured and found to increase with increasing NPs concentration in the polymer matrix. The UV–vis spectroscopy is used to investigate the optical absorption and transmission data for the prepared films. The addition of Mn2O3/rGO NP in the polymer matrix effects on the optical parameters like the absorption coefficient, optical bandgap, refractive index, and optical conductivity. The optical bandgap of PVA films with Mn2O3/rGO NP is lower than that of PVA pure. The refractive index and optical conductivity were tuned with the addition of Mn2O3/rGO NP. The PVA-Mn2O3/rGO films are promising material for various opto-electronic fields.

Setup of a TIRF Microscope With Attached Microfluidics for the Detection of Fluorescently Labeled Single Molecules

At Laserinstitut Hochschule Mittweida (LHM), single molecule fluorescence microscopy is to be established for biophotonic RNA research. The aim of this work is to establish an objective-based total internal reflection fluorescence microscope (TIRFM) with alternating laser excitation (ALEX) in the millisecond range. We have designed a suitable detection beam path for color channel-selected imaging with a scientific CMOS camera. The two detection channels allow distance changes to be measured at the molecular level using Förster resonance energy transfer (FRET). In addition to the microscope setup, this work details the imaging and color channel selection of the fluorescently labeled samples and the modulated laser excitation.

Comprehensive Investigation of Parameters Influencing Fluorescence Lifetime Imaging Microscopy in Frequency- and Time-Domain Illustrated by Phasor Plot Analysis.

Having access to fluorescence lifetime, researchers can reveal in-depth details about the microenvironment as well as the physico-chemical state of the molecule under investigation. However, the high number of influencing factors might be an explanation for the strongly deviating values of fluorescent lifetimes for the same fluorophore reported in the literature. This could be the reason for the impression that inconsistent results are obtained depending on which detection and excitation scheme is used. To clarify this controversy, the two most common techniques for measuring fluorescence lifetimes in the time-domain and in the frequency-domain were implemented in one single microscopy setup and applied to a variety of fluorophores under different environmental conditions such as pH-value, temperature, solvent polarity, etc., along with distinct state forms that depend, for example, on the concentration. From a vast amount of measurement results, both setup- and sample-dependent parameters were extracted and represented using a single display form, the phasor-plot. The measurements showed consistent results between the two techniques and revealed which of the tested parameters has the strongest influence on the fluorescence lifetime. In addition, quantitative guidance as to which technique is most suitable for which research task and how to perform the experiment properly to obtain consistent fluorescence lifetimes is discussed.

Label-Free Non-Linear Optics for the Study of Tubulin-Dependent Defects in Central Myelin.

The satisfactory visualization of cytoskeletal components in the brain is challenging. The ubiquitous distribution of the networks of microtubules, microfilaments, and intermediate filaments in all the neural tissues, together with the variability in the outcomes of fluorescent protein fusion strategies and their limited applicability to dynamic studies of antibodies and drugs as chromophore vehicles, make classical optical approaches not as effective as for other proteins. When tubulin needs to be studied, the label-free generation of second harmonics is a very suitable option due to the non-centrosymmetric organization of the molecule. This technique, when conjugated to microscopy, can qualitatively describe the volumetric distribution of parallel bundles of microtubules in biological samples, with the additional advantage of working with fresh tissues that are unfixed and unpermeabilized. This work describes how to image tubulin with a commercial second harmonic generation microscopy setup to highlight microtubules in the tubulin-enriched structures of the oligodendrocytes, asin hypomyelination with atrophy of the basal ganglia and cerebellum (H-ABC) tubulinopathy, a recently described myelin disorder.

Direct observation of internal short circuits by lithium dendrites in cross-sectional lithium-ion in situ full cells

Li metal deposition and internal short circuits (ISC) are directly observed in full cells using a cross-sectional in situ optical microscopy set-up. The cell chemistries under investigation are graphite/LFP and graphite/NMC 622. Direct observations are made on (i) lithiation gradients without Li metal deposition, (ii) Li metal deposition without ISC, (iii) Li metal deposition with ISC by a dendrite growing above the separator, and (iv) Li metal deposition with ISC induced by mechanical closing of separator pores. The speed of the lithiation fronts in graphite/LPF cells was determined to be 2,736 ± 47 μm2 min−1 for LiC12 and 1,619 ± 51 μm2 min−1 for LiC6. By image processing (binarization) of in situ microscopy image sequences, the accumulated observable Li area as well as the projected two-dimensional areal growth (+1,215 ± 41 μm2 min−1) and dissolution rate (−676 ± 30 μm2 min−1), along with the turning point between deposition and dissolution were estimated. Li dendrite dissolution, re-intercalation, and reorganization under different conditions (CC vs. CV-charging), as well as self-discharge, the fuse-effect, self-healing of dendrites, the importance of overhang areas, and the effect of closed separator pores are discussed in this publication.

Endoscopic imaging of white matter fiber tracts using polarization-sensitive optical coherence tomography

Polarization sensitive optical coherence tomography (PSOCT) has been shown to image and delineate white matter fibers in a label-free manner by revealing optical birefringence within the myelin sheath using a microscope setup. In this proof-of-concept study, we adapt recent advancements in endoscopic PSOCT to perform depth-resolved imaging of white matter structures deep inside intact porcine brain tissue ex-vivo, through a small, rotational fiber probe. The probe geometry is comparable to microelectrodes currently used in neurosurgical interventions. The presented imaging system is mobile, robust, and uses biologically safe levels of optical radiation making it well suited for clinical translation. In neurosurgery, where accuracy is imperative, endoscopic PSOCT through a narrow-gauge fiber probe could provide intra-operative feedback on the location of critical white matter structures.

Automated staging of zebrafish embryos using machine learning

The zebrafish (Danio rerio), is an important biomedical model organism used in many disciplines, including development, disease modeling and toxicology, to better understand vertebrate biology. The phenomenon of developmental delay in zebrafish embryos has been widely reported as part of a mutant or treatment-induced phenotype, and accurate characterization of such delays is imperative. Despite this, the only way at present to identify and quantify these delays is through manual observation, which is both time-consuming and subjective. Machine learning approaches in biology are rapidly becoming part of the toolkit used by researchers to address complex questions. In this work, we introduce a machine learning-based classifier that has been trained to detect temporal developmental differences across groups of zebrafish embryos. Our classifier is capable of rapidly analyzing thousands of images, allowing comparisons of developmental temporal rates to be assessed across and between experimental groups of embryos. Finally, as our classifier uses images obtained from a standard live-imaging widefield microscope and camera set-up, we envisage it will be readily accessible to the zebrafish community, and prove to be a valuable resource.

Novel In-Situ Observation of the Grease Constituents in Elastohydrodynamic Contacts by Fluorescence Microscopy

Grease is important lubricant for ball bearings as it is used much more often than oil. However, mechanism of the lubrication is not completely clear, especially concerning the role of the thickener in the lubricating process. It was shown that contribution of the thickener to film thickness build-up varies with operating conditions. Also, its influence on the resistive torque of the bearing was proved. However, all studies were in-direct showing its effects. This paper presents new in-situ fluorescence method of the grease constituents’ observation, where both, thickener, and base oil can be observed independently. Two different fluorescence dyes are used to distinguish grease constituents with the use of different microscope setup. Moreover, the new method was tested on two different test rigs. First is typical ball on disc test rig widely used for study of elastohydrodynamic lubrication. Second is ball on ring test rig which more closely represents radial ball bearing conditions. It was shown that thickener engagement is very different for each geometrical configuration.

Calcite-Assisted Localization and Kinetics (CLocK) Microscopy.

Localization-based super-resolution imaging techniques have improved the spatial resolution of optical microscopy well below the diffraction limit, yet encoding additional information into super-resolved images, such as anisotropy and orientation, remains a challenge. Here we introduce calcite-assisted localization and kinetics (CLocK) microscopy, a multiparameter super-resolution imaging technique easily integrated into any existing optical microscope setup at low cost and with straightforward analysis. By placing a rotating calcite crystal in the infinity space of an optical microscope, CLocK microscopy provides immediate polarization and orientation information while maintaining the ability to localize an emitter/scatterer with <10 nm resolution. Further, kinetic information an order of magnitude shorter than the integration time of the camera is encoded in the unique point spread function of a CLocK image, allowing for new mechanistic insight into dynamic processes such as single-nanoparticle dissolution and single-molecule surface-enhanced Raman scattering.