Correlative Microscopy Methods Research Articles

Correlative microscopy combining transmission electron microscopy and secondary ion mass spectrometry: A general review on the state-of-the-art, recent developments, and prospects

The development of advanced materials relies crucially on the availability of suitable high-resolution analytical characterization techniques. In this review, we discuss correlative microscopy methods combining Transmission Electron Microscopy (TEM) and Secondary Ion Mass Spectrometry (SIMS) for high-resolution high-sensitivity analysis. We review the literature on ex-situ TEM-SIMS correlation in materials science and beyond and motivate the need for in-situ TEM-SIMS. The instrument development aspects are discussed in detail followed by a computational analysis of the fundamental ion-solid interaction relevant for TEM-SIMS correlation. The correlative methodology and workflow are presented in detail together with a discussion on potential artifacts. Then, the strategies to enhance the performance characteristics such as the SIMS image resolution and sensitivities are discussed followed by example applications of the in-situ TEM-SIMS technique. Finally, the prospects of the TEM-SIMS correlative technique for solving challenging characterization problems are explored.

The Combination of Electron Microscopy, Raman Microscopy and Energy Dispersive X‐Ray Spectroscopy for the Investigation of Polymeric Materials

Polymers play an important role in materials science, because of their advantages over other types of materials. The huge variety in terms of the types, arrangements and combinations of monomers found in polymers leads to an extremely wide diversity of different polymeric materials. Although there is a wide range of analytical techniques to characterize polymeric materials, here the focus is laid on a novel correlative microscopy method, combining Raman microscopy, scanning electron microscopy (SEM) and energy dispersive X‐ray spectroscopy (EDXS). Therefore, three polymeric specimens were analysed by the new system RISE (Raman Imaging and Scanning Electron microscopy) in combination with EDXS. The aim of this contribution is to demonstrate the potential of the new system to characterize different polymeric layers, as well as fillers and additives in polymeric materials. In addition to the results, a methodical chapter deals with preparation aspects, investigation skills and imaging modes of the electron microscope. Here the so‐called variable pressure mode (VP) is mentioned, where nitrogen is used as imaging gas at pressures between 10 and 133 Pa. Furthermore, the low‐voltage mode in the high vacuum is discussed which gives a better surface resolution due to a smaller interaction volume of the electrons in the material and furthermore causes lower beam damage. Both modes work without a conductive coating on the specimen, which is a requirement for Raman investigations. Further capabilities of the RISE system in combination with the EDXS will be discussed afterwards.

High-speed near-field fluorescence microscopy combined with high-speed atomic force microscopy for biological studies

BackgroundHigh-speed atomic force microscopy (HS-AFM) has successfully visualized a variety of protein molecules during their functional activity. However, it cannot visualize small molecules interacting with proteins and even protein molecules when they are encapsulated. Thus, it has been desired to achieve techniques enabling simultaneous optical/AFM imaging at high spatiotemporal resolution with high correlation accuracy. MethodsScanning near-field optical microscopy (SNOM) is a candidate for the combination with HS-AFM. However, the imaging rate of SNOM has been far below that of HS-AFM. We here developed HS-SNOM and metal tip-enhanced total internal reflection fluorescence microscopy (TIRFM) by exploiting tip-scan HS-AFM and exploring methods to fabricate a metallic tip on a tiny HS-AFM cantilever. ResultsIn tip-enhanced TIRFM/HS-AFM, simultaneous video recording of the two modalities of images was demonstrated in the presence of fluorescent molecules in the bulk solution at relatively high concentration. By using fabricated metal-tip cantilevers together with our tip-scan HS-AFM setup equipped with SNOM optics, we could perform simultaneous HS-SNOM/HS-AFM imaging, with correlation analysis between the two overlaid images being facilitated. ConclusionsThis study materialized simultaneous tip-enhanced TIRFM/HS-AFM and HS-SNOM/HS-AFM imaging at high spatiotemporal resolution. Although some issues remain to be solved in the future, these correlative microscopy methods have a potential to increase the versatility of HS-AFM in biological research. General significanceWe achieved an imaging rate of ~3 s/frame for SNOM imaging, more than 100-times higher than the typical SNOM imaging rate. We also demonstrated ~39 nm resolution in HS-SNOM imaging of fluorescently labeled DNA in solution.

A targeted 3D EM and correlative microscopy method using SEM array tomography.

Using electron microscopy to localize rare cellular events or structures in complex tissue is challenging. Correlative light and electron microscopy procedures have been developed to link fluorescent protein expression with ultrastructural resolution. Here, we present an optimized scanning electron microscopy (SEM) workflow for volumetric array tomography for asymmetric samples and model organisms (Caenorhabditis elegans, Drosophila melanogaster, Danio rerio). We modified a diamond knife to simplify serial section array acquisition with minimal artifacts. After array acquisition, the arrays were transferred to a glass coverslip or silicon wafer support. Using light microscopy, the arrays were screened rapidly for initial recognition of global anatomical features (organs or body traits). Then, using SEM, an in-depth study of the cells and/or organs of interest was performed. Our manual and automatic data acquisition strategies make 3D data acquisition and correlation simpler and more precise than alternative methods. This method can be used to address questions in cell and developmental biology that require the efficient identification of a labeled cell or organelle.

Local Stress States and Microstructural Damage Response Associated with Deformation Twins in Hexagonal Close Packed Metals

The current work implements a correlative microscopy method utilizing electron back scatter diffraction, focused ion beam and digital image correlation to accurately determine spatially resolved stress profiles in the vicinity of grain/twin boundaries and tensile deformation twin tips in commercially pure titanium. Measured local stress gradients were in good agreement with local misorientation values. The role of dislocation-boundary interactions on the buildup of local stress gradients is elucidated. Stress gradients across the twin-parent interface were compressive in nature with a maximum stress magnitude at the twin boundary. Stress profiles near certain grain boundaries initially display a local stress minimum, followed by a typically observed “one over square root of distance” variation, as was first postulated by Eshelby, Frank and Nabarro. The observed trends allude to local stress relaxation mechanisms very close to the grain boundaries. Stress states in front of twin tips showed tensile stress gradients, whereas the stress state inside the twin underwent a sign reversal. The findings highlight the important role of deformation twins and their corresponding interaction with grain boundaries on damage nucleation in metals.

Correlative scanning-transmission electron microscopy reveals that a chimeric flavivirus is released as individual particles in secretory vesicles.

The intracellular morphogenesis of flaviviruses has been well described, but flavivirus release from the host cell remains poorly documented. We took advantage of the optimized production of an attenuated chimeric yellow fever/dengue virus for vaccine purposes to study this phenomenon by microscopic approaches. Scanning electron microscopy (SEM) showed the release of numerous viral particles at the cell surface through a short-lived process. For transmission electron microscopy (TEM) studies of the intracellular ultrastructure of the small number of cells releasing viral particles at a given time, we developed a new correlative microscopy method: CSEMTEM (for correlative scanning electron microscopy - transmission electron microscopy). CSEMTEM analysis suggested that chimeric flavivirus particles were released as individual particles, in small exocytosis vesicles, via a regulated secretory pathway. Our morphological findings provide new insight into interactions between flaviviruses and cells and demonstrate that CSEMTEM is a useful new method, complementary to SEM observations of biological events by intracellular TEM investigations.

Correlative microscopy methods that maximize specimen fidelity and data completeness, and improve molecular localization capabilities

Correlative microscopy techniques interrogate biological systems more thoroughly than is possible using a single modality. This is particularly true if disparate data types can be acquired from the same specimen. Recently, there has been significant progress towards combining the structural information obtained from soft X-ray tomography (SXT) with molecular localization data. Here we will compare methods for determining the position of molecules in a cell viewed by SXT, including direct visualization using electron dense labels, and by indirect methods, such as fluorescence microscopy and high numerical aperture cryo-light microscopy. We will also discuss available options for preserving the in vivo structure and organization of the specimen during multi-modal data collection, and how some simple specimen mounting concepts can ensure maximal data completeness in correlative imaging experiments.

Correlative Microscopy Study of FIB Patterned Stainless Steel Surfaces as Novel Nano-Structured Stents for Cardiovascular Applications

ABSTRACTCoronary artery disease is a major problem worldwide causing 7.2 million deaths worldwide annually, resulting from vascular occlusion, myocardial infarction and its complications. Stent implantation is a percutaneous interventional procedure that mitigates vessel stenosis, providing mechanical support within the artery. However, stenting causes physical damage to the arterial wall. It is well accepted that a valuable route to reduce in-stent re-stenosis can be based on promoting cell response to nano-structured stainless steel (SS) surfaces such as, for example, by patterning nano-pits in SS. In this regard patterning by Focussed Ion-Beam (FIB) milling offers several advantages for flexible prototyping (i) practically any substrate material that is able to withstand high vacuum conditions of the microscope chamber can be used, (ii) there is high flexibility in the obtainable shapes and geometries by modulating the ion beam current and the patterning conditions, (iii) reduced complexity of the pattering process e.g. it is a single-step process with a possibility of real-time monitoring of the milling progression. On the other hand FIB patterning of polycrystalline metals is greatly influenced by channelling effects and re-deposition. Correlative microscopy methods present an opportunity to study such effects comprehensively and derive structure-property understanding that is important for developing improved pattering. In this report we present a FIB patterning protocol for nano-structuring features (concaves) ordered in rectangular arrays on pre-polished 316L Stainless Steel (SS) surfaces. An investigation based on correlative microscopy approach of the size, shape and depth of the developed arrays in relation to the crystal orientation of the underlying SS domains, is presented. The correlative microscopy protocol is based on cross-correlation of top-view Scanning Electron Microscopy (SEM), Electron Backscattered Diffraction (EBSD), and Atomic Force Microscopy (AFM).Various dose tests were performed, aiming at improved productivity by preserving nano-size accuracy of the patterned process. The optimal FIB patterning conditions for achieving reasonably high throughput (patterned rate of about 0.03 mm2 per hour) and nano-size accuracy in dimensions and shapes of the features, are discussed as well.

Discovery of a new RNA‐containing nuclear structure in UVC‐induced apoptotic cells by integrated laser electron microscopy

Treatment of cells with UVC radiation leads to the formation of DNA cross-links which, if not repaired, can lead to apoptosis. gamma-H2AX and cleaved caspase 3 are proteins formed during UVC-induced DNA damage and apoptosis respectively. The present study sets out to identify early morphological markers of apoptosis using a new method of correlative microscopy, ILEM (integrated laser electron microscopy). Cleaved caspase 3 and gamma-H2AX were immunofluorescently labelled to mark the cells of interest. These cells were subsequently searched in the fluorescence mode of the ILEM and further analysed at high resolution with TEM (transmission electron microscopy). Following the treatment of HUVECs (human umbilical vein endothelial cells) with UVC radiation, in the majority of the cells gamma-H2AX was formed, whereas only in a subset of cells caspase 3 was activated. In severely damaged cells with high levels of gamma-H2AX a round, electron-dense nuclear structure was found, which was hitherto not identified in UV-stressed cells. This structure exists only in nuclei of cells containing cleaved caspase 3 and is present during all stages of the apoptotic process. Energy-loss imaging showed that the nuclear structure accumulates phosphorus, indicating that it is rich in nucleic acids. Because the nuclear structure did not label for DNA and was not affected by regressive EDTA treatment, it is suggested that the UV-induced nuclear structure contains a high amount of RNA. Because the UV-induced nuclear structure was only found in cells labelled for cleaved caspase 3 it is proposed as an electron microscopic marker for all stages of apoptosis. Such a marker will especially facilitate the screening for early apoptotic cells, which lack the well-known hallmarks of apoptosis within a cell population. It also raises new questions on the mechanisms involved in the UV-induced apoptotic pathway.

Correlative light/electron microscopy for the investigation of microbial mats from Black Sea Cold Seeps

In several fields of cell biology, correlative microscopy is applied to compare the structure of objects at high resolution under the electron microscope with low resolution light microscopy images of the same sample. It is, however, difficult to prepare samples and marker systems that are applicable for both microscopic techniques for the same specimen at the same time. In our studies, we used microbial mats from Cold Seep communities for a simple and rapid correlative microscopy method. The mats consist of bacterial and archaeal microorganisms, coupling reverse methanogenesis to the reduction of sulfate. The reverse methanogenic pathway also generates carbonates that precipitate inside the mat and may be the main reason for the formation of a microbial reef. The mat shows highly differentiated aggregates of various organisms, tightly interconnected by extracellular polysaccharides. In order to investigate the role of EPS as adhesive mucilage for the biofilm and as a precipitation matrix for carbonate minerals, samples were embedded in a hydrophilic resin (Lowicryl K4 M). Sections were suitable for light as well as electron microscopy in combination with lectins, either labeled with a fluorescent marker or with colloidal gold. This allows lectin mapping at low resolution for light microscopy in direct comparison with a highly resolved electron microscopic image.

Identification of Focal Viral Infections by Confocal Microscopy for Subsequent Ultrastructural Analysis

A correlative microscopy method for the ultrastructural analysis of focal viral tissue infections is presented. Using a confocal scanning laser microscope, foci of infection are identified in tissue sections prior to embedment; a variety of techniques can be employed for viral detection, including staining with standard histochemical reagents and fluorescently labeled antibodies. Areas of infection identified using confocal microscopy are excised from the tissue sections, embedded, and examined by transmission electron microscopy. Applications of this technique in both diagnostic and basic research settings are described.

Correlative microscopy techniques for material science

The microscopy center at Clemson University recently invested funds to provide a computer network system that incorporates all of its microscopes. The facility connects SEM, TEM, STM/AFM, Auger Microprobe and the light microscope to Sun workstations equipped with chemical analysis and imaging programs. Images from the network system microscopes can be sent to any of the workstations. I should like to review a few applications of correlative microscopy techniques related to material science; this is a technology that allows the acquisition of multiple data from a given sample. Often a given technique can be augmented by the use of complimentary microscopy technique. Since electron microscopy is subject to interpretation, correlative microscopy methods will prove to be useful in reaching conclusions regarding the image micrographs. Additionally, more than one type of information may be necessary for a given material and this can be found with the different systems of microscopy. In my presentation I will discuss instrumentation and methods by demonstrating advantages and disadvantages of applications as they apply to materials such as polymers, ceramics, microstructures and textiles.