Sensitive Biological Samples Research Articles

Stable Cu (I) Complexes for Intracellular Cu-Catalyzed Azide Alkyne Cycloaddition.

The copper-catalyzed azide-alkyne cycloaddition (CuAAC) has heralded a new era of chemical biology and biomedicine. However, caveats of CuAAC include formation of reactive oxygen species (ROS) and other copper-related toxicity. This limits utility in sensitive biological samples and matrices. Towards addressing these caveats, we synthesized and fully characterized two air and water stable trinuclear Cu (I) dimer complexes. The complexes were stable to oxidation in the presence of hydrogen peroxide, acid, base, and other chelators, which was reasoned to be due to the linear benzimidazole-Cu-benzimidazole geometry. Computational investigations of the catalytic cycle implicated two of the three coppers in the trimer complex as the active metal centers. The complexes were shown to catalyze the reaction at far below sub-toxic concentrations for intracellular click reactions to label triple negative breast cancer cells and compared to the current CuSO-4-THPTA standard.

Calibrating cryogenic temperature of TEM specimens using EELS

Cryogenic Scanning/Transmission Electron Microscopy has been established as a leading method to image sensitive biological samples and is now becoming a powerful tool to understand materials' behavior at low temperatures. However, achieving precise local temperature calibration at low temperatures remains a challenge, which is especially crucial for studying phase transitions and emergent physical properties in quantum materials. In this study, we employ electron energy loss spectroscopy (EELS) to measure local cryogenic specimen temperatures. We use the temperature-dependent characteristics of aluminum's bulk plasmon peak in EEL spectra, which shifts due to changes in electron density caused by thermal expansion and contraction. We successfully demonstrate the versatility of this method by calibrating different liquid nitrogen cooling holders in various microscopes, regardless of whether a monochromated or non-monochromated electron beam is used. Temperature discrepancies between the actual temperature and the setpoint temperatures are identified across a range from room temperature to 100 K. This work demonstrates the importance of temperature calibrations at intermediate temperatures and presents a straightforward, robust method for calibrating local temperatures of cryogenically-cooled specimens in electron microscopes.

OpenSTED: open-source dynamic intensity minimum system for stimulated emission depletion microscopy.

Stimulated emission depletion (STED) is a powerful super-resolution microscopy technique that can be used for imaging live cells. However, the high STED laser powers can cause significant photobleaching and sample damage in sensitive biological samples. The dynamic intensity minimum (DyMIN) technique turns on the STED laser only in regions of the sample where there is fluorescence signal, thus saving significant sample photobleaching. The reduction in photobleaching allows higher resolution images to be obtained and longer time-lapse imaging of live samples. A stand-alone module to perform DyMIN is not available commercially. In this work, we developed an open-source design to implement three-step DyMIN on a STED microscope and demonstrated reduced photobleaching for timelapse imaging of beads, cells, and tissue. The DyMIN system uses a fast multiplexer circuit and inexpensive field-programmable gate array controlled by Labview software that operates as a stand-alone module for a STED microscope. All software and circuit diagrams are freely available. We compared time-lapse images of bead samples using our custom DyMIN system to conventional STED and recorded a higher signal when using DyMIN after a 50-image sequence. We further demonstrated the DyMIN system for time-lapse STED imaging of live cells and brain tissue slices. Our open-source DyMIN system is an inexpensive add-on to a conventional STED microscope that can reduce photobleaching. The system can significantly improve signal to noise for dynamic time-lapse STED imaging of live samples.

Using XAS to monitor radiation damage in real time and post-analysis, and investigation of systematic errors of fluorescence XAS for Cu-bound amyloid-β.

X-ray absorption spectroscopy (XAS) is a promising technique for determining structural information from sensitive biological samples, but high-accuracy X-ray absorption fine structure (XAFS) requires corrections of systematic errors in experimental data. Low-temperature XAS and room-temperature X-ray absorption spectro-electrochemical (XAS-EC) measurements of N-truncated amyloid-β samples were collected and corrected for systematic effects such as dead time, detector efficiencies, monochromator glitches, self-absorption, radiation damage and noise at higher wavenumber (k). A new protocol was developed using extended X-ray absorption fine structure (EXAFS) data analysis for monitoring radiation damage in real time and post-analysis. The reliability of the structural determinations and consistency were validated using the XAS measurement experimental uncertainty. The correction of detector pixel efficiencies improved the fitting χ2 by 12%. An improvement of about 2.5% of the structural fitting was obtained after dead-time corrections. Normalization allowed the elimination of 90% of the monochromator glitches. The remaining glitches were manually removed. The dispersion of spectra due to self-absorption was corrected. Standard errors of experimental measurements were propagated from pointwise variance of the spectra after systematic corrections. Calculated uncertainties were used in structural refinements for obtaining precise and reliable values of structural parameters including atomic bond lengths and thermal parameters. This has permitted hypothesis testing.

Free-electron Ramsey-type interferometry for enhanced amplitude and phase imaging of nearfields.

The complex range of interactions between electrons and electromagnetic fields gave rise to countless scientific and technological advances. A prime example is photon-induced nearfield electron microscopy (PINEM), enabling the detection of confined electric fields in illuminated nanostructures with unprecedented spatial resolution. However, PINEM is limited by its dependence on strong fields, making it unsuitable for sensitive samples, and its inability to resolve complex phasor information. Here, we leverage the nonlinear, overconstrained nature of PINEM to present an algorithmic microscopy approach, achieving far superior nearfield imaging capabilities. Our algorithm relies on free-electron Ramsey-type interferometry to produce orders-of-magnitude improvement in sensitivity and ambiguity-immune nearfield phase reconstruction, both of which are optimal when the electron exhibits a fully quantum behavior. Our results demonstrate the potential of combining algorithmic approaches with state-of-the-art modalities in electron microscopy and may lead to various applications from imaging sensitive biological samples to performing full-field tomography of confined light.

Real-time open-source FLIM analysis.

Fluorescence lifetime imaging microscopy (FLIM) provides valuable quantitative insights into fluorophores' chemical microenvironment. Due to long computation times and the lack of accessible, open-source real-time analysis toolkits, traditional analysis of FLIM data, particularly with the widely used time-correlated single-photon counting (TCSPC) approach, typically occurs after acquisition. As a result, uncertainties about the quality of FLIM data persist even after collection, frequently necessitating the extension of imaging sessions. Unfortunately, prolonged sessions not only risk missing important biological events but also cause photobleaching and photodamage. We present the first open-source program designed for real-time FLIM analysis during specimen scanning to address these challenges. Our approach combines acquisition with real-time computational and visualization capabilities, allowing us to assess FLIM data quality on the fly. Our open-source real-time FLIM viewer, integrated as a Napari plugin, displays phasor analysis and rapid lifetime determination (RLD) results computed from real-time data transmitted by acquisition software such as the open-source Micro-Manager-based OpenScan package. Our method facilitates early identification of FLIM signatures and data quality assessment by providing preliminary analysis during acquisition. This not only speeds up the imaging process, but it is especially useful when imaging sensitive live biological samples.

Fundamental nursing care focusing on older people’s needs and continuity of long-term care: a scoping review protocol

IntroductionKnowledge about long-term care services ability, regardless of if the service is home-based or facility-based, to provide an optimal and comprehensive fundamental nursing care (understood as focusing on physical, relational...

In Situ Dynamic Light Scattering Complementing Neutron Spin Echo Measurements on Protein Samples

Monitoring the state of the sample on the minute-time scale is crucial in case of sensitive soft matter or biological samples, given that neutron spin echo measurements take up to several days. Moreover, there is no method to interpret the normalized intermediate scattering function obtained by measuring the neutron spin echo if relevant sample properties change during measurement. Dynamic light scattering provides information on the diffusion constant of particles in solution (biological macromolecules, such as proteins, protein aggregates, polymer particles, etc.) with average hydrodynamic radii in a wide range from several nanometers up to several microns. This information can be obtained in a few minutes and it offers a good overview of the current sample state. Details on the novel in situ dynamic light scattering set-up with one fixed scattering angle and first results obtained on a molten globule state of apo-myoglobin are presented.

Electron Beam-Induced Transformation in High-Density Amorphous Ices

Amorphous ice is commonly used as a noncrystalline matrix for protecting sensitive biological samples in cryogenic electron microscopy (cryo-EM). The amorphization process of water is complex, and at least two amorphous states of different densities are known to exist, high- and low-density amorphous ices (HDA and LDA). These forms are considered to be the counterparts of two distinct liquid states, namely, high- and low-density liquid water. Herein, we investigate the HDA to LDA transition using electron diffraction and cryo-EM. The observed phase transition is induced by the impact of electrons, and we discuss two different mechanisms, namely, local heating and beam-induced motion of water molecules. The temperature increase is estimated by comparison with X-ray scattering experiments on identically prepared samples. Our results suggest that HDA, under the conditions used in our cryo-EM measurements, is locally heated above its glass-transition temperature.

Large cluster ions: soft local probes and tools for organic and bio surfaces.

Ionised cluster beams have been produced and employed for thin film deposition and surface processing for half a century. In the last two decades, kiloelectronvolt cluster ions have also proved to be outstanding for surface characterisation by secondary ion mass spectrometry (SIMS), because their sputter and ion yields are enhanced in a non-linear fashion with respect to monoatomic projectiles, with a resulting step change of sensitivity for analysis and imaging. In particular, large gas cluster ion beams, or GCIB, have now become a reference in organic surface and thin film analysis using SIMS and X-ray photoelectron spectroscopy (XPS). The reason is that they induce soft molecular desorption and offer the opportunity to conduct damageless depth-profiling and 3D molecular imaging of the most sensitive organic electronics and biological samples, with a nanoscale depth resolution. In line with these recent developments, the present review focuses on rather weakly-bound, light-element cluster ions, such as noble or other gas clusters, and water or alcohol nanodroplets (excluding clusters made of metals, inorganic salts or ionic liquids) and their interaction with surfaces (essentially, but not exclusively, organic). The scope of this article encompasses three aspects. The first one is the fundamentals of large cluster impacts with surfaces, using the wealth of information provided by molecular dynamics simulations and experimental observations. The second focus is on recent applications of large cluster ion beams in surface characterisation, including mass spectrometric analysis and 2D localisation of large molecules, molecular depth-profiling and 3D molecular imaging. Finally, the perspective explores cutting edge developments, involving (i) new types of clusters with a chemistry designed to enhance performance for mass spectrometry imaging, (ii) the use of cluster fragment ion backscattering to locally retrieve physical surface properties and (iii) the fabrication of new biosurface and thin film architectures, where large cluster ion beams are used as tools to transfer biomolecules in vacuo from a target reservoir to any collector substrate.

Live-imaging of Bioengineered Cartilage Tissue using Multimodal Non-linear Molecular Imaging

Coherent anti-Stokes Raman scattering (CARS) and second harmonic generation (SHG) are non-linear techniques that allow label-free, non-destructive and non-invasive imaging for cellular and tissue analysis. Although live-imaging studies have been performed previously, concerns that they do not cause any changes at the molecular level in sensitive biological samples have not been addressed. This is important especially for stem cell differentiation and tissue engineering, if CARS/SHG microscopy is to be used as a non-invasive, label-free tool for assessment of the developing neo-tissue. In this work, we monitored the differentiation of human fetal-femur derived skeletal cells into cartilage in three-dimensional cultures using CARS and SHG microscopy and demonstrate the live-imaging of the same developing neo-tissue over time. Our work conclusively establishes that non-linear label-free imaging does not alter the phenotype or the gene expression at the different stages of differentiation and has no adverse effect on human skeletal cell growth and behaviour. Additionally, we show that CARS microscopy allows imaging of different molecules of interest, including lipids, proteins and glycosaminoglycans, in the bioengineered neo-cartilage. These studies demonstrate the label-free and truly non-invasive nature of live CARS and SHG imaging and their value and translation potential in skeletal research, regeneration medicine and tissue engineering.

Absolute ozone densities in a radio-frequency driven atmospheric pressure plasma using two-beam UV-LED absorption spectroscopy and numerical simulations

The efficient generation of reactive oxygen species (ROS) in cold atmospheric pressure plasma jets (APPJs) is an increasingly important topic, e.g. for the treatment of temperature sensitive biological samples in the field of plasma medicine. A 13.56 MHz radio-frequency (rf) driven APPJ device operated with helium feed gas and small admixtures of oxygen (up to 1%), generating a homogeneous glow-mode plasma at low gas temperatures, was investigated. Absolute densities of ozone, one of the most prominent ROS, were measured across the 11 mm wide discharge channel by means of broadband absorption spectroscopy using the Hartley band centred at λ = 255 nm. A two-beam setup with a reference beam in Mach–Zehnder configuration is employed for improved signal-to-noise ratio allowing high-sensitivity measurements in the investigated single-pass weak-absorbance regime. The results are correlated to gas temperature measurements, deduced from the rotational temperature of the N2 (C 3 B 3, υ = 0 2) optical emission from introduced air impurities. The observed opposing trends of both quantities as a function of rf power input and oxygen admixture are analysed and explained in terms of a zero-dimensional plasma-chemical kinetics simulation. It is found that the gas temperature as well as the densities of O and O2(b) influence the absolute O3 densities when the rf power is varied.

Highly sensitive detection of urinary protein variations using tilted fiber grating sensors with plasmonic nanocoatings

Surface plasmon resonance (SPR) optical fiber biosensors can be used as a cost-effective and relatively simple-to-implement alternative to well established bulky prism configurations for high sensitivity biological sample measurements. The miniaturized size and remote operation ability offer them a multitude of opportunities for single-point sensing in hard-to-reach spaces, even possibly in vivo. The biosensor configuration reported in this work uses a tilted fiber Bragg grating (TFBG) in a commercial single mode fiber coated with a nanometer scale silver film. The key point is that by reducing the silver film thickness to around 20–30nm (rather than 50nm for optimal SPR excitation), different modes of the TFBG spectrum present very high but opposite sensitivities to refractive index (RI) changes around the TFBG. Experimental results obtained with the coated TFBG embedded inside a microfluidic channel show an amplitude sensitivity greater than 8000dB/RIU (Refractive Index Unit) and a limit of detection of 10−5RIU. Using this device, the effect of different concentrations of protein in rat urine was clearly differentiated between healthy samples, nephropatic samples and samples from individuals under treatment, with a protein concentration sensitivity of 5.5dB/(mg/ml) and a limit of detection of 1.5×10−3mg/ml. Those results show a clear relationship between protein outflow and variations in the RI of the urine samples between 1.3400 and 1.3408, pointing the way to the evaluation and development of new drugs for nephropathy treatments. The integration of TFBGs with microfluidic channels enables precise measurement control over samples with sub-microliter volumes and does not require accurate temperature control because of the elimination of the temperature cross-sensitivity inherent in TFBG devices. Integration of the TFBG with a hypodermic needle on the other hand would allow similar measurements in vivo. The proposed optical fiber/microfluidic plasmonic biosensor represents an appealing solution for rapid, low consumption and highly sensitive detection of analytes at low concentrations in medicine as well as in chemical and environmental monitoring.

Fast single‐beam‐CARS imaging scheme based on in silico optimization of excitation phases

A novel scheme for the detection of known species with single‐beam coherent anti‐Stokes Raman scattering spectroscopy is presented in theory and simulations. The introduction of a simulation tool based on fast Fourier transforms allows a purely numerical optimization of excitation phases ex situ, to which we refer to as in silico optimization. Such an optimal phase is based on the encoding of vibrational resonances in specific spectral detection windows using multichannel measurements. The presented detection scheme offers a route to fast chemical selective imaging with high contrast and simplified data acquisition, thereby avoiding photodamage of sensitive biological samples. Copyright © 2015 John Wiley & Sons, Ltd.

Electron cryotomography of vitrified cells with a Volta phase plate.

Electron cryotomography provides a means of studying the three dimensional structure of pleomorphic objects, such as organelles or cells, with a resolution of 1-3nm. A limitation in the study of radiation sensitive biological samples is the low signal-to-noise ratio of the tomograms which may obscure fine details. To overcome this limitation, the recently developed Volta phase plate (VPP) was applied in electron cryotomographic studies of a wide range of cellular structures, from magnetotactic bacteria to primary cultured neurons. The results show that the VPP improves contrast significantly and consequently the signal-to-noise ratio of the tomograms, moreover it avoids disturbing fringing artifacts typical for Zernike phase plates. The contrast improvement provided by the VPP was also confirmed in projection images of relatively thick (∼400nm) samples. In order to investigate the respective contributions of the VPP and the energy filter, images acquired with different combinations of the two were compared. Zero-loss energy filtering reduced the background noise in thicker areas of the sample and improved the contrast of features such as poly-β-hydroxybutyrate granules in magnetotactic bacteria, whereas the VPP provided an overall contrast improvement for all sample areas. After 3D reconstruction, tomograms acquired with the combination of a VPP and an energy filter showed structural features in neuronal processes with outstanding clarity. We also show that the VPP can be combined with focused ion beam milling to examine structures embedded deeply inside cells. Thus, we expect that VPP will become a standard element of the electron cryotomography workflow.

In-situ detection of density alteration in non-physiological cells with polarimetric tilted fiber grating sensors

Tilted fiber Bragg grating (TFBG) biosensors can be used as a cost-effective and relatively simple-to-implement alternative to well established biosensor platforms for high sensitivity biological sample measurements in situ or possibly in vivo. The fiber biosensor presented in this study utilizes an in-fiber 12° tilted Bragg grating to excite a strong evanescent field on the surface of the sensor over a large range of external medium refractive indices. The devices have minimal cross-sensitivity to temperature and their fabrication does not impact the structural integrity of the fiber and its surface functionalization. Human acute leukemia cells with different intracellular densities and refractive index (RI) ranging from 1.3342 to 1.3344 were clearly discriminated in-situ by using the differential transmission spectrum between two orthogonal polarizations for the last guided mode resonance before “cut-off”, with an amplitude variation sensitivity of 1.8×104dB/RIU, a wavelength shift sensitivity of 180nm/RIU, and a limit of detection of 2×10−5RIU. The detection process was precisely controlled with a micro-fluidic chip which allows the measurement of nL-volumes of bio-samples. The proposed in-fiber polarimetric biosensor is an appealing solution for rapid, sub-microliter dose and highly sensitive detection of analytes at low concentrations in medicine, chemical and environmental monitoring. @ 2013 Elsevier B.V. All rights reserved.

Rapid 3D light-sheet microscopy with a tunable lens

The in-vivo investigation of highly dynamic biological samples, for example the beating zebrafish heart, requires high-speed volume imaging techniques. Light-sheet microscopy is ideal for such samples as it records high-contrast images of entire planes within large samples at once. However, in order to obtain images of different planes, it has been necessary to move the sample relative to the fixed focal plane of the detection objective lens. This mechanical movement limits speed, precision and may be harmful to the sample. We have built a light-sheet microscope that uses remote focusing with an electrically tunable lens (ETL). Without moving specimen or objective we have thereby achieved flexible volume imaging at much higher speeds than previously reported. Our high-speed microscope delivers 3D snapshots of sensitive biological samples. As an example, we imaged 17 planes within a beating zebrafish heart at 510 frames per second, equivalent to 30 volume scans per second. Movements, shape changes and signals across the entire volume can be followed which has been impossible with existing reconstruction techniques.

High-sensitive and temperature-self-calibrated tilted fiber grating biological sensing probe

High sensitivity biological sample measurement has been achieved by using a 10° tilted fiber Bragg grating sensing probe. Human acute leukemia cells with different intracellular densities were clearly discriminated by identifying their slight refraction index (RI) perturbations in the range from 1.3342 to 1.3344, combining with a temperature self-calibration property. We studied the relationship between the intracellular density of cells (S50 and S60) and their RIs, the experimental results provide a potential way to verify the hypothesis for “density alteration in non-physiological cells (DANCE)”.

Focus on vacuum and cryogenics

Focus on vacuum and cryogenics

Atomic spectrometry update-X-ray fluorescence spectrometry

This review offers the reader a wealth of information published between April 2010 and March 2011 concerning analytical endeavours using the range of conventional and hyphenated XRF techniques that encourage the user to ensure the potential for high spectral sensitivity and, where appropriate, spatial resolution is achieved. The development of advanced micro-beam set ups and new X-ray optics driven by third generation synchrotron based XRF techniques provide nano-imaging and the detection of nano-particles on single cells whilst TXRF coupled with GIXRF and GEXRF offer great potential for non-destructive investigations of thin layers on reflecting surfaces as well as depth profiling of implants. A new portable XRF system is described as an alternative for the traditionally applied K-X-ray fluorescence technology for in vivo measurements of lead in bone. Cryogenic cooling of heat sensitive biological samples is offered as a method to mitigate possible damage by the use of the more powerful μ-XRF technique. Other new preparation methods are also reviewed for the presentation and analysis of industrial, environmental and archaeological samples. One of the more unusual contributions available this year in the characterisation and use of industrial minerals showed that a semi-precious stone, amethyst, is more effective at shielding radiation than concrete.