New approaches for low phototoxicity imaging of living cells and tissues.

Fluorescence microscopy is a powerful tool used in scientific and medical research, but it is inextricably linked to phototoxicity. Neglecting phototoxicity can lead to erroneous or inconclusive results. Recently, several reports have addressed this issue, but it is still underestimated by many researchers, even though it can lead to cell death. Phototoxicity can be reduced by appropriate microscopic techniques and carefully designed experiments. This review focuses on recent strategies to reduce phototoxicity in microscopic imaging of living cells and tissues. We describe digital image processing and new hardware solutions. We point out new modifications of microscopy methods and hope that this review will interest microscopy hardware engineers. Our aim is to underscore the challenges and potential solutions integral to the design of microscopy systems. Simultaneously, we intend to engage biologists, offering insight into the latest technological advancements in imaging that can enhance their understanding and practice.

Laboratory system for optical coherence tomography (OCT) using a laser plasma source of soft x-rays and extreme ultraviolet and focusing ellipsoidal optics.

Optical coherence tomography (OCT) with the use of soft x-rays (SXR) and extreme ultraviolet (EUV) has been recently demonstrated [Fuchs et al. Sci. Rep.6, 20658 (2016)10.1038/srep20658; Fuchs et al. Optica4, 903 (2017)10.1364/OPTICA.4.000903]. This new imaging technique, named XCT, makes it possible to obtain cross-sectional and tomographic images of objects with nanometer spatial resolution. The article presents a newly developed laboratory system for XCT using a compact laser plasma light source operating in the SXR and EUV spectral ranges. The source is based on a gas puff target containing Kr gas or a Kr/Xe gas mixture irradiated with nanosecond laser pulses from an Nd:YAG laser. The use of the gas puff target enables efficient emission of SXR and EUV radiation without generating target debris associated with laser ablation when using a solid target. The system is equipped with an ellipsoidal mirror to collect radiation from the source and focus on the imaged object. The XCT measurements are made by processing the spectrum of the radiation reflected from the object recorded with a transmission grating spectrometer equipped with an identical focusing mirror and a CCD camera. The paper presents the characterization and optimization of the new XCT system and its application to the measurements of layered nanostructures.

Broadband soft X-ray source from a clustered gas target dedicated to high-resolution XCT and X-ray absorption spectroscopy.

The development of the broad-bandwidth photon sources emitting in the soft X-ray range has attracted great attention for a long time due to the possible applications in high-resolution spectroscopy, nano-metrology, and material sciences. A high photon flux accompanied by a broad, smooth spectrum is favored for the applications such as near-edge X-ray absorption fine structure (NEXAFS), extended X-ray absorption fine structure (EXAFS), or XUV/X-ray coherence tomography (XCT). So far, either large-scale facilities or technologically challenging systems providing only limited photon flux in a single shot dominate the suitable sources. Here, we present a soft, broad-band (1.5 nm - 10.7 nm) soft X-ray source. The source is based on the interaction of very intense laser pulses with a target formed by a cluster mixture. A photon yield of 2.4 × 1014 photons/pulse into 4π (full space) was achieved with a medium containing Xe clusters of moderate-size mixed with a substantial amount of extremely large ones. It is shown that such a cluster mixture enhances the photon yield in the soft X-ray range by roughly one order of magnitude. The size of the resulting source is not beneficial (≤500 µm but this deficit is compensated by a specific spectral structure of its emission fulfilling the specific needs of the spectroscopic (broad spectrum and high signal dynamics) and metrological applications (broad and smoothed spectrum enabling a sub-nanometer resolution limit for XCT).

1-keV emission from laser-plasma source based on an Xe/He double stream gas puff target.

Characterization of Xe emission in the spectral range between 1 and 1.5 keV is presented in the case when the laser-plasma is generated by nanosecond laser pulse irradiation of a double stream Xe/He gas-puff target. Gas target density was estimated using extreme ultraviolet (EUV) radiography. Emission spectral characteristics in the wavelength range from 0.8 to 5.2 nm were determined by using a flat field SXR spectrometer. Significant emission was recorded in two high-energy bands, the first one at wavelengths 0.8-1.6 nm (photon energy range 0.78-1.5 keV) and the second one at 1.6-2.5 nm (0.5-0.78 keV). Both plasma size and photon yield in each band were measured separately to individually assess radiation and source characteristics. Moreover, a proof-of-principle experiment for near-edge X-ray absorption fine structure spectroscopy of metallic sample near the L2,3 absorption edge was performed to show one of the applicability areas of such a compact source.

Adhesion of Triple-Negative Breast Cancer Cells under Fluorescent and Soft X-ray Contact Microscopy.

Understanding cancer cell adhesion could help to diminish tumor progression and metastasis. Adhesion mechanisms are currently the main therapeutic target of TNBC-resistant cells. This work shows the distribution and size of adhesive complexes determined with a common fluorescence microscopy technique and soft X-ray contact microscopy (SXCM). The results presented here demonstrate the potential of applying SXCM for imaging cell protrusions with high resolution when the cells are still alive in a physiological buffer. The possibility to observe the internal components of cells at a pristine and hydrated state with nanometer resolution distinguishes SXCM from the other more commonly used techniques for cell imaging. Thus, SXCM can be a promising technique for investigating the adhesion and organization of the actin cytoskeleton in cancer cells.

Effect of Extreme Ultraviolet (EUV) Radiation and EUV Induced, N2 and O2 Based Plasmas on a PEEK Surface's Physico-Chemical Properties and MG63 Cell Adhesion.

Polyetheretherketone (PEEK), due to its excellent mechanical and physico-chemical parameters, is an attractive substitute for hard tissues in orthopedic applications. However, PEEK is hydrophobic and lacks surface-active functional groups promoting cell adhesion. Therefore, the PEEK surface must be modified in order to improve its cytocompatibility. In this work, extreme ultraviolet (EUV) radiation and two low-temperature, EUV induced, oxygen and nitrogen plasmas were used for surface modification of polyetheretherketone. Polymer samples were irradiated with 100, 150, and 200 pulses at a 10 Hz repetition rate. The physical and chemical properties of EUV and plasma modified PEEK surfaces, such as changes of the surface topography, chemical composition, and wettability, were examined using atomic force microscopy (AFM), scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), and goniometry. The human osteoblast-like MG63 cells were used for the analysis of cell viability and cell adhesion on all modified PEEK surfaces. EUV radiation and two types of plasma treatment led to significant changes in surface topography of PEEK, increasing surface roughness and formation of conical structures. Additionally, significant changes in the chemical composition were found and were manifested with the appearance of new functional groups, incorporation of nitrogen atoms up to ~12.3 at.% (when modified in the presence of nitrogen), and doubling the oxygen content up to ~25.7 at.% (when modified in the presence of oxygen), compared to non-modified PEEK. All chemically and physically changed surfaces demonstrated cyto-compatible and non-cytotoxic properties, an enhancement of MG63 cell adhesion was also observed.

2-D nanometer thickness mapping applying a reduced bias soft X-ray NEXAFS approach.

We present a 2-D mapping of a sample thickness with nanometer accuracy employing a compact arrangement of near-edge X-ray absorption fine structure (NEXAFS) technique. A NEXAFS spectrum coupled with a scanning system was used to generate a 2-D thickness map of the TiO2 sample (anatase form) deposited on the top of a SiN membrane. The thickness values were retrieved from the experimental data by applying different methods of data processing. In the paper, the detailed analysis of the data processing methods and the identified sources of the errors show that the proposed procedure based on averaging two imperfect estimates reduces the error caused by the uncontrolled bias of the measured signals. This procedure was termed as the average one. The estimates from the proposed average approach and the standard absorption-jump ratio in the absorption edge vicinity were compared with the direct results obtained by applying scanning electron microscopy (SEM). The experimental arrangement of the NEXAFS spectroscopy system, the data acquisition method, as well as the possible error sources, are presented and discussed in detail.

Surface Modification of PLLA, PTFE and PVDF with Extreme Ultraviolet (EUV) to Enhance Cell Adhesion.

Recently, extreme ultraviolet (EUV) radiation has been increasingly used to modify polymers. Properties such as the extremely short absorption lengths in polymers and the very strong interaction of EUV photons with materials may play a key role in achieving new biomaterials. The purpose of the study was to examine the impact of EUV radiation on cell adhesion to the surface of modified polymers that are widely used in medicine: poly(tetrafluoroethylene) (PTFE), poly (vinylidene fluoride) (PVDF), and poly-L-(lactic acid) (PLLA). After EUV surface modification, which has been performed using a home-made laboratory system, changes in surface wettability, morphology, chemical composition and cell adhesion polymers were analyzed. For each of the three polymers, the EUV radiation differently effects the process of endothelial cell adhesion, dependent of the parameters applied in the modification process. In the case of PVDF and PTFE, higher cell number and cellular coverage were obtained after EUV radiation with oxygen. In the case of PLLA, better results were obtained for EUV modification with nitrogen. For all three polymers tested, significant improvements in endothelial cell adhesion after EUV modification have been demonstrated.

Compact system for near edge X-ray fine structure (NEXAFS) spectroscopy using a laser-plasma light source.

We present a compact laboratory system for near edge soft X-ray fine structure (NEXAFS) spectroscopy that was developed using a laser-plasma light source. The source is based on a double stream gas puff target. The plasma is formed by the interaction of a laser beam with the double stream gas puff target approach. The laser plasma source was optimized for efficient soft X-ray emission from a krypton/helium target in the range of 1.5 to 5 nm wavelength. This emission is used to acquire simultaneously the emission and absorption spectra of soft X-ray light from the source and from the investigated sample using a grazing incidence spectrometer. The measurements in the transmission mode reveal the features near the carbon K-α absorption edge of thin PET film. From those features, the composition of the sample was successfully obtained. The data are in agreement with synchrotron measurements. In the paper, the detailed information about the source, its optimization, the system, spectral measurements and the results are presented and discussed.

A stand-alone compact EUV microscope based on gas-puff target source.

We report on a very compact desk-top transmission extreme ultraviolet (EUV) microscope based on a laser-plasma source with a double stream gas-puff target, capable of acquiring magnified images of objects with a spatial (half-pitch) resolution of sub-50 nm. A multilayer ellipsoidal condenser is used to focus and spectrally narrow the radiation from the plasma, producing a quasi-monochromatic EUV radiation (λ = 13.8 nm) illuminating the object, whereas a Fresnel zone plate objective forms the image. Design details, development, characterization and optimization of the EUV source and the microscope are described and discussed. Test object and other samples were imaged to demonstrate superior resolution compared to visible light microscopy.

A Compact "Water Window" Microscope with 60 nm Spatial Resolution for Applications in Biology and Nanotechnology.

Short illumination wavelength allows an extension of the diffraction limit toward nanometer scale; thus, improving spatial resolution in optical systems. Soft X-ray (SXR) radiation, from "water window" spectral range, λ=2.3-4.4 nm wavelength, which is particularly suitable for biological imaging due to natural optical contrast provides better spatial resolution than one obtained with visible light microscopes. The high contrast in the "water window" is obtained because of selective radiation absorption by carbon and water, which are constituents of the biological samples. The development of SXR microscopes permits the visualization of features on the nanometer scale, but often with a tradeoff, which can be seen between the exposure time and the size and complexity of the microscopes. Thus, herein, we present a desk-top system, which overcomes the already mentioned limitations and is capable of resolving 60 nm features with very short exposure time. Even though the system is in its initial stage of development, we present different applications of the system for biology and nanotechnology. Construction of the microscope with recently acquired images of various samples will be presented and discussed. Such a high resolution imaging system represents an interesting solution for biomedical, material science, and nanotechnology applications.

Recording oscillations of sub-micron size cantilevers by extreme ultraviolet Fourier transform holography.

We recorded the fast oscillation of sub-micron cantilevers using time-resolved extreme ultraviolet (EUV) Fourier transform holography. A tabletop capillary discharge EUV laser with a wavelength of 46.9 nm provided a large flux of coherent illumination that was split using a Fresnel zone plate to generate the object and the reference beams. The reference wave was produced by the first order focus while a central opening in the zone plate provided a direct illumination of the cantilevers. Single-shot holograms allowed for the composition of a movie featuring the fast oscillation. Three-dimensional displacements of the object were determined as well by numerical back-propagation, or "refocusing" of the electromagnetic fields during the reconstruction of a single hologram.

Extreme ultraviolet tomography using a compact laser-plasma source for 3D reconstruction of low density objects.

A tomographic method for three-dimensional reconstruction of low density objects is presented and discussed. The experiment was performed in the extreme ultraviolet (EUV) spectral region using a desktop system for enhanced optical contrast and employing a compact laser-plasma EUV source, based on a double stream gas puff target. The system allows for volume reconstruction of transient gaseous objects, in this case gas jets, providing additional information for further characterization and optimization. Experimental details and reconstruction results are shown.

Structural Stability and Disorder Level of Moderately Reduced Paper-like Graphene Oxide Investigated with Micro-Raman Analysis.

This paper discusses the results of the micro-Raman analysis performed on paper-like graphene oxide (GO) samples consisting of many functionalised graphene layers and annealed at moderate temperatures (≤500 °C) under vacuum conditions (p ≃ 10-4 mbar). The analysis of the standalone samples revealed that the obtained material is characterised by a noticeable disorder level but still stays below the commonly accepted threshold of high or total disorder. GO formed in a simple way showed two spectral bands above 1650 cm-1 recorded very rarely or not at all and their origin has been discussed in detail. The results also confirmed the metastable character of multilayer GO after the annealing process at moderate temperatures as the C/O ratio was kept between 2 and 3 and the spectral features were stable within the annealing temperature range.

Tabletop single-shot extreme ultraviolet Fourier transform holography of an extended object.

We demonstrate single and multi-shot Fourier transform holography with the use of a tabletop extreme ultraviolet laser. The reference wave was produced by a Fresnel zone plate with a central opening that allowed the incident beam to illuminate the sample directly. The high reference wave intensity allows for larger objects to be imaged compared to mask-based lensless Fourier transform holography techniques. We obtain a spatial resolution of 169 nm from a single laser pulse and a resolution of 128 nm from an accumulation of 20 laser pulses for an object ~11x11µm(2) in size. This experiment utilized a tabletop extreme ultraviolet laser that produces a highly coherent ~1.2 ns laser pulse at 46.9 nm wavelength.

A 50 nm spatial resolution EUV imaging-resolution dependence on object thickness and illumination bandwidth.

In this paper we report a desk-top microscopy reaching 50 nm spatial resolution in very compact setup using a gas-puff laser plasma EUV source. The thickness of an object and the bandwidth of illuminating radiation were studied in order to estimate their quantitative influence on the EUV microscope spatial resolution. EUV images of various thickness objects obtained by illumination with variable bandwidth EUV radiation were compared in terms of knife-edge spatial resolution to study the bandwidth/object thickness parasitic influence on spatial resolution of the EUV microscope.

High numerical aperture tabletop soft x-ray diffraction microscopy with 70-nm resolution.

Light microscopy has greatly advanced our understanding of nature. The achievable resolution, however, is limited by optical wavelengths to approximately 200 nm. By using imaging and labeling technologies, resolutions beyond the diffraction limit can be achieved for specialized specimens with techniques such as near-field scanning optical microscopy, stimulated emission depletion microscopy, and photoactivated localization microscopy. Here, we report a versatile soft x-ray diffraction microscope with 70- to 90-nm resolution by using two different tabletop coherent soft x-ray sources-a soft x-ray laser and a high-harmonic source. We also use field curvature correction that allows high numerical aperture imaging and near-diffraction-limited resolution of 1.5lambda. A tabletop soft x-ray diffraction microscope should find broad applications in biology, nanoscience, and materials science because of its simple optical design, high resolution, large depth of field, 3D imaging capability, scalability to shorter wavelengths, and ultrafast temporal resolution.

Demonstration of Near Edge X-ray Absorption Fine Structure Spectroscopy of Transition Metals Using Xe/He Double Stream Gas Puff Target Soft X-ray Source.

A near 1-keV photons from the Xe/He plasma produced by the interaction of laser beam with a double stream gas puff target were employed for studies of L absorption edges of period 4 transitional metals with atomic number Z from 26 to 30. The dual-channel, compact NEXAFS system was employed for the acquisition of the absorption spectra. L1-3 absorption edges of the samples were identified in transmission mode using broadband emission from the Xe/He plasma to show the applicability of such source and measurement system to the NEXAFS studies of the transition metals, including magnetic materials.

Sub-70 nm resolution tabletop microscopy at 13.8 nm using a compact laser-plasma EUV source.

We report the first (to our knowledge) demonstration of a tabletop, extreme UV (EUV) transmission microscope at 13.8 nm wavelength with a spatial (half-pitch) resolution of 69 nm. In the experiment, a compact laser-plasma EUV source based on a gas puff target is applied to illuminate an object. A multilayer ellipsoidal mirror is used to focus quasi-monochromatic EUV radiation onto the object, while a Fresnel zone plate objective forms the image. The experiment and the spatial resolution measurements, based on a knife-edge test, are described. The results might be useful for the realization of a compact high-resolution tabletop imaging systems for actinic defect characterization.

Physico-Chemical Surface Modifications of Polyetheretherketone (PEEK) Using Extreme Ultraviolet (EUV) Radiation and EUV-Induced Nitrogen Plasma.

In this work, the effect of extreme ultraviolet (EUV) radiation and the combination of EUV radiation and low-temperature nitrogen plasma on the physico-chemical properties of polyetheretherketone (PEEK) surfaces were presented. The laser-plasma EUV source based on a double gas puff target was used in this experiment to irradiate PEEK surfaces with nanosecond pulses of EUV radiation and to produce low-temperature plasma through the photoionization of nitrogen with EUV photons. The changes in surface morphology on irradiated polymer samples were examined using atomic force microscopy (AFM) and scanning electron microscopy (SEM). Chemical changes of the PEEK surfaces were analysed using X-ray photoelectron spectroscopy (XPS). EUV radiation and nitrogen plasma treatment caused significant changes in the topography of modified PEEK's surfaces and an increase in their average roughness. Strong chemical decomposition, appearance of new functional groups as well as incorporation of nitrogen atoms up to ~17 at.% on the PEEK's surface were observed.