Emre Firlar

Correlative electron and fluorescence microscopy of magnetotactic bacteria in liquid: toward in vivo imaging.

Magnetotactic bacteria biomineralize ordered chains of uniform, membrane-bound magnetite or greigite nanocrystals that exhibit nearly perfect crystal structures and species-specific morphologies. Transmission electron microscopy (TEM) is a critical technique for providing information regarding the organization of cellular and magnetite structures in these microorganisms. However, conventional TEM can only be used to image air-dried or vitrified bacteria removed from their natural environment. Here we present a correlative scanning TEM (STEM) and fluorescence microscopy technique for imaging viable cells of Magnetospirillum magneticum strain AMB-1 in liquid using an in situ fluid cell TEM holder. Fluorescently labeled cells were immobilized on microchip window surfaces and visualized in a fluid cell with STEM, followed by correlative fluorescence imaging to verify their membrane integrity. Notably, the post-STEM fluorescence imaging indicated that the bacterial cell wall membrane did not sustain radiation damage during STEM imaging at low electron dose conditions. We investigated the effects of radiation damage and sample preparation on the bacteria viability and found that approximately 50% of the bacterial membranes remained intact after an hour in the fluid cell, decreasing to ~30% after two hours. These results represent a first step toward in vivo studies of magnetite biomineralization in magnetotactic bacteria.

Direct Visualization of the Hydration Layer on Alumina Nanoparticles with the Fluid Cell STEM in situ.

Rheological behavior of aqueous suspensions containing nanometer-sized powders is of relevance to many branches of industry. Unusually high viscosities observed for suspensions of nanoparticles compared to those of micron size powders cannot be explained by current viscosity models. Formation of so-called hydration layer on alumina nanoparticles in water was hypothesized, but never observed experimentally. We report here on the direct visualization of aqueous suspensions of alumina with the fluid cell in situ. We observe the hydration layer formed over the particle aggregates and show that such hydrated aggregates constitute new particle assemblies and affect the flow behavior of the suspensions. We discuss how these hydrated nanoclusters alter the effective solid content and the viscosity of nanostructured suspensions. Our findings elucidate the source of high viscosity observed for nanoparticle suspensions and are of direct relevance to many industrial sectors including materials, food, cosmetics, pharmaceutical among others employing colloidal slurries with nanometer-scale particles.

Monitoring the Exocytosis and Full Fusion of Insulin Granules in Pancreatic Islet Cells via Graphene Liquid Cell-Transmission Electron Microscopy

Investigation of sub-cellular level activities has been of a great interest for the biological, medical and materials scientists for many years. Earlier approaches to monitor the live beta cell insulin granule trafficking fusion and exocytosis remained not fully accurate due to the sample preparation and imaging techniques used. With the electron microscopy techniques used till now, samples were either fixed with chemicals, stained, embedded and sectioned [1] or cryogenically fixed and imaged [2]. Liquid cell flow holder Transmission Electron Microscopy (TEM) imaging was also proposed but it had very high thickness, which reduced the imaging and chemical characterization resolution [3]. To that end, our approach for the investigation of live cell activities is to use Graphene Liquid Cells (GLC) in TEM at 80kV [4]. Encapsulating the liquid media in GLC helps to obtain high resolution in both TEM and Scanning Transmission Electron Microscopy (STEM) imaging. Via this proposed technique, we were able to visualize the effects of stimulators on insulin granule size, motion, exocytosis and trafficking. We have successfully imaged and recoded the sub-cellular phenomena in real-time as shown in Fig. 1: Dynamic full fusion, Fig. 2: Sequential Fusion events under 30 mM KCI and Fig 3: Exocytosis of MIN6 cells stimulated by 30 mM KCI. These were rarely ever observed by other conventional methodologies and will help generate novel drug development for the Diabetes treatment by comparing the healthy and pathological cells [5].

Electron Microscopy and Spectroscopy of Citrate Induced Calcium Oxalate Crystal Structure and Hydration State Changes, and Implications for Kidney Stones

In the USA alone, 20 million people are currently have kidney stones (KS) or are predicted to develop KS during their lifetimes. The most common KS symptom is excruciating pain, but symptoms often include nausea and emesis, blood in urine, difficulty urinating, and frequent urination. KS may require removal of the kidney and are associated with Chronic Kidney Disease as well as Cardiovascular Diseases [1]. Approximately 80% of KS contain Calcium Oxalate (CaOx) as the major phase with CaOx monohydrate (COM) as the most prominent hydration state [2]. Therefore, medical KS research primarily focuses on CaOx biomineralization and potential CaOx treatments.

Field-assisted self-assembly process: general discussion

Yugang Sun, Leonardo Scarabelli, Nicholas Kotov, Moritz Tebbe, Xiao-Min Lin, Ward Brullot, Lucio Isa, Peter Schurtenberger, Helmuth Moehwald, Igor Fedin, Orlin Velev, Damien Faivre, Christopher Sorensen, Regine Perzynski, Munish Chanana, Zhihai Li, Fernando Bresme, Petr Kral, Emre Firlar, David Schiffrin, Joao Batista Souza Junior, Andreas Fery, Elena Shevchenko, Ozgur Tarhan, Armand Paul Alivisatos, Sabrina Disch, Rafal Klajn and Suvojit Ghosh

Revealing the Iron Oxides Mineral Core in Ferritin due to the Variations in the H and L Subunits

Iron is an essential element involved in electron transfer processes in many biological reactions in the human body. Excess iron is stored and regulated in ferritin proteins during the biomineralization and demineralization processes. Cytosolic ferritin is 12nm in diameter and composed of a 24-subunit protein shell containing Heavy (H) and Light (L) chains and a ferritin iron core approximately 8nm in diameter. The H subunits are responsible for iron detoxification while L subunits are responsible for iron storage. Ferritin H and L subunit ratios are unique to each organ within the human body. H rich ferritins are prominent in organs such as human heart and brain while the L rich ferritins are primarily observed in storage organs such as the liver and spleen [1].

Transmission Electron Microscopy Studies of Calcium Phosphate Biomineralization

The continuous demineralization and remineralization processes continuously happen on the dental enamel. Once the balance between these two processes is broken, dental erosion or dentine hypersensitivity will happen. Therefore, elucidation of the biomineralization pathway in vitro will help dentists to find out a more precise strategy to maintain the oral health. In fact, the biomineralization of apatite, the core building block of the enamel, has been studied for decades, but due to resolution limit in the imaging, sub-micron level details of this process are still not clear. There are still several important questions to be answered: 1) How do the Ca and PO4 ions exist in aqueous solution, as ions or as metastable clusters, like Posner cluster (Ca9(PO4) 6), the smallest united in calcium phosphate?Posner and Betts [1] 2) How are these clusters or ions combined, as single ions attachment or as cluster aggregation? 3) How does the amorphous calcium phosphate (ACP) form the crystal structure[2], via direct phase transformation or via dissolution and recrystallization processes? Dynamical highresolution imaging is needed to be carried out for the investigation of the apatite mineralization pathway, so that we can answer these questions. Fortunately, in recent years, liquid cell (Scanning) Transmission Electron Microscopy ((S)TEM) has enabled the investigation of such dynamic biological processes in sub-micron scale due to the Z contrast. In this research, the liquid cell STEM imaging was used to investigate the biomineralization process. A mineral solution was encapsulated in liquid cell, and then it was imaged via STEM. Particles with 10-15 nm sizes nucleated from the solution and then attached onto each other to form a larger loosely bound cluster as shown in Figure 1 (b). The whole process was captured dynamically. Electron Energy Loss Spectroscopy (EELS) and Selected Area Electron Diffraction (SAED) were carried out to identify the chemical composition and crystal structure of the newly formed crystals, respectively. EELS confirmed the existence of calcium ions. Our result are partially disagreed the novel pathway for the crystallization of calcium phosphate, which insisted crystalline phase calcium phosphate formed by certain stages: metastable ion clusters, amorphous phase, and then the amorphous transform to crystal. From our research, we proposed a new pathway for the mineralization process, first some metastable cluster formed, because of the local fluctuation of ions, and then based on some driven force (interface energy), these metastable clusters formed some smaller crystal particles (10-15 nm) and finally these small particles aggregated to form crystal phase in a larger size (around hundreds nanometers). Moreover, for this test, no trace of existence of amorphous phase was observed, which dissented the proposed by other researchers [2, 3].

Spatially Resolved Electron Energy Loss Spectroscopy Studies in Graphene Liquid Cell for the Investigation of the Biomineralization Processes in Human Body

Biomineralization of matter in human body has been of a great interest for the biological, medical and materials scientists for many years. The motivation for this type of work remained to be mostly the similar, specifically, after the fully understanding of the biomineralization routes for these minerals, it has been aimed that 1) These minerals could be synthesized in vitro and 2) The cures to the diseases related with these minerals could be developed. Several different minerals with regard to the investigation of biomineralization routes have been studied during the past few decades, for instance, ferritins [1], hydroxyapatite [2], and magnetite [3]. The major challenges for the investigation of these biomineralization processes remained two fold, either not simulating the native environment of these minerals forming or not using the instruments with proper atomic resolution imaging and characterization capabilities. To that end, our approach for the investigation of biomineralization involves the utilization of the Graphene Liquid Cell (GLC) in Cs corrected Scanning Transmission Electron Microscope (STEM) at 80kV. Encapsulating the liquid media in GLC will help to obtain high resolution in both TEM and STEM imaging [4]. With the help of spatially resolved Electron Energy Loss Spectroscopy (EELS) and High Resolution Transmission Electron Microscopy (HRTEM) images, the chemistry of the mineral and structure changes will be visualized, respectively. The advantage of using GLC instead of cryogenic and in situ liquid cell imaging is that, the imaging will be dynamic and due to less sample thickness, analytical TEM studies will be easier, respectively. In this work, we carried out hydroxyapatite biomineralization experiments in GLC so as to have a deep understanding of this biomineralization process. The procedure to encapsulate liquid media and biological structures is as described in Shokuhfar et al. [5]. The chemical information from these minerals with regard to Ca L2,3, P L2,3 and O K edge changes with respect to the total electron dose will be reported via the spatially resolved EELS in HAADF-STEM mode. The formation of this mineral is seen in the inset of Figure 1. The EEL spectrum from the same particle verifies the formation of the calcium phosphate mineral with the presence of the fingerprints of Ca L edge, in conjunction to the C K edge from the graphene layer [6].

New Approach to Analysis of Noisy EELS Data

Electron Energy Loss Spectroscopy (EELS) is used to probe the chemical environment of materials at the nanometer scale. This analysis becomes challenging when dealing with embedded nanostructured materials, where small regions of interest residing in thick matrices yield noisy data. The data analysis can benefit from modeling the core loss spectra with the available reference peaks, using the experimental peak positions and ratio of the fitted peak areas to gauge the chemical binding in a material.