Digital holographic cytometry (DHC) is a state-of-the-art quantitative phase imaging (QPI) method that permits time-lapse imaging of cells without induced cellular toxicity. DHC platforms equipped ...
In environmental scanning electron microscopy (ESEM) electrical insulating, wet and biological samples can be investigated without additional sample preparation. The imaging gas inside the chamber suppresses charging and outgassing of the sample but it also decreases the signal to noise ratio (SNR) [1]. Especially applications in the kPa regime are limited by poor image quality (e.g. wetting experiments). Recent publications on high pressure capabilities of state of the art microscopes have shown that they are working far away from physical limits and that there is plenty of room for improvements [2]. The key to high image quality at high pressures is to reduce scattering of the primary beam electrons inside the imaging gas as far as possible while maintaining ideal operation conditions for the SE‐detector [3]. In the FEI Quanta 600 ESEM the gaseous environment in the sample chamber is separated by a differential pumping system and two pressure limiting apertures (PLA) from the high vacuum inside the electron column. Nevertheless, a lot of gas streams through the PLA upwards and a significant amount of scattering takes place even before the electron beam is entering the sample chamber [2]. Based on the insights of Monte Carlo and finite element simulations a new aperture holder was designed that significantly reduces this gas flow and therefore also the primary beam scattering. The PLAs are exchangeable and smaller diameters further increase the SNR at the expense of a smaller field of view. In a conventional ESEM the secondary electron detector is a positively biased electrode which attracts and accelerates the secondary electrons. On their way to the detector the secondary electrons undergo collision ionization which amplifies the signal and generates positively biased gas ions. With increasing chamber pressure this SE signal amplification strongly decreases because the electron mean free path decreases and the SEs do not gain enough energy between collisions to ionize the imaging gas anymore. By replacing the position and modifying the shape of the detector it can be optimized for high pressure applications. Nearby a needle detector with very small tip radius (R < 10 µm) the electric field is strong enough for SE amplification and by positioning the needle on the sample table it operates at ideal conditions regardless of pressure and working distance. The distance sample to PLA and sample to detector is no longer coupled. A by‐product of this design is that the conventional position of the backscatter electron detector (BSE) at the end of the column is no longer blocked by the SE detector. With this outstanding signal to noise ratio at high chamber pressures the limits of conventional ESEM technology can be crossed. Wetting experiments at low acceleration voltages and low dwell times are possible as well as imaging liquid samples without cooling (see figure 1,2). In figure 3 a BSE image of gold nanoparticle in oil at 10 kPa chamber pressure can be seen and the overall improvements are shown in figure 4.
The mechanical properties of cells play an important role in cell function and behavior. This paper presents recent developments that have enabled the use of laser-generated phonons (ultrasound) with sub-optical wavelengths to look inside living cells. The phonons reveal contrast from changes in the elasticity of the cell and can provide high resolution three dimensional images.
In this contribution table-top, lab-based microscopes in the extreme ultraviolet (EUV) in transmission and reflection mode as well as in the soft x-ray will be presented. The application for the transmission microscope is the investigation of thin film nanostructures and particles, the application of the reflection microscope is mask blank inspection for EUV lithography. The soft x-ray water window microscope has been developed for investigations of organic samples and nanocrystals in liquids.
In nearly all cell culture applications it is critical to control cell growth and cell status regularly. Due to the tiny dimensions of cells the morphologic assessment has to be assisted by microscopy. A very common and widespread technique for making ceils that are almost transparent visible is phase cantrast microscopy. Ceils are usually cultured in nutrient medium on transparent plastic material for example microtiter plates (MTPs). Such a plate which has a footprint of about 128 mm x 85 mm contains several weils which are separate compartments for the ceils to grow in. Microscopic analysis directly takes place in these microtiter plates on inverted microscopes. In order to get a good overview of the ceil culture status it is helpful to image the whole content of an entire weil of a microtiter plate. This is especiaily true because cell colanies tend to grow preferably in the border regions of a weil. Usually it is not just one single weil on a MTP that needs to be imaged but the whole plate. As the weils are closely adjacent to each other the weil bottarn area covers almost the whole plate's footprint. As a consequence, to examine a completely filled microtiter plate under a microscope means imaging
In recent years the atomic force microscope (AFM) has evolved from a high resolution imaging tool to an enabling platform for physical studies at the nanoscale including quantitative mapping of mechanical characteristics of surfaces providing simultaneous topography and mechanical property maps across the length scales. In the work presented here peak force tapping AFM was utilized to elaborate the nanoscale mechanical performance of phase separated polyurethanes (PUs) and the mechanical properties of lysozyme molecules adsorbed to mica substrates.
Studying biological samples with scanning electron microscopy has specific requirements for their preparation. Sample drying is a particularly critical operation for objects such as cultured cells. The requirement for damaging drying step can be eliminated using environmental scanning electron microscopy. This study compares dried and wet samples of cultured human embryonic stem cells. It points to the advantages of both methods and to the complementarity of the information that they provide.
Electron channeling contrast imaging (ECCI) is a powerful technique for the quantitative characterization of deformation structures in the SEM. The coupling of ECCI with EBSD provides an efficient method to attain enhanced diffraction contrast in the SEM. The EBSD-based ECCI set-up allows the imaging of dislocation and nano-twin substructures in the SEM. Some examples of quantitative microstructural characterization on structural materials are provided.
