Bernhard Goetze

Helium Ion Microscopy (HIM) for the imaging of biological samples at sub-nanometer resolution

Scanning Electron Microscopy (SEM) has long been the standard in imaging the sub-micrometer surface ultrastructure of both hard and soft materials. In the case of biological samples, it has provided great insights into their physical architecture. However, three of the fundamental challenges in the SEM imaging of soft materials are that of limited imaging resolution at high magnification, charging caused by the insulating properties of most biological samples and the loss of subtle surface features by heavy metal coating. These challenges have recently been overcome with the development of the Helium Ion Microscope (HIM), which boasts advances in charge reduction, minimized sample damage, high surface contrast without the need for metal coating, increased depth of field, and 5 angstrom imaging resolution. We demonstrate the advantages of HIM for imaging biological surfaces as well as compare and contrast the effects of sample preparation techniques and their consequences on sub-nanometer ultrastructure.

High Resolution Helium Ion Scanning Microscopy of the Rat Kidney

Helium ion scanning microscopy is a novel imaging technology with the potential to provide sub-nanometer resolution images of uncoated biological tissues. So far, however, it has been used mainly in materials science applications. Here, we took advantage of helium ion microscopy to explore the epithelium of the rat kidney with unsurpassed image quality and detail. In addition, we evaluated different tissue preparation methods for their ability to preserve tissue architecture. We found that high contrast, high resolution imaging of the renal tubule surface is possible with a relatively simple processing procedure that consists of transcardial perfusion with aldehyde fixatives, vibratome tissue sectioning, tissue dehydration with graded methanol solutions and careful critical point drying. Coupled with the helium ion system, fine details such as membrane texture and membranous nanoprojections on the glomerular podocytes were visualized, and pores within the filtration slit diaphragm could be seen in much greater detail than in previous scanning EM studies. In the collecting duct, the extensive and striking apical microplicae of the intercalated cells were imaged without the shrunken or distorted appearance that is typical with conventional sample processing and scanning electron microscopy. Membrane depressions visible on principal cells suggest possible endo- or exocytotic events, and central cilia on these cells were imaged with remarkable preservation and clarity. We also demonstrate the use of colloidal gold probes for highlighting specific cell-surface proteins and find that 15 nm gold labels are practical and easily distinguishable, indicating that external labels of various sizes can be used to detect multiple targets in the same tissue. We conclude that this technology represents a technical breakthrough in imaging the topographical ultrastructure of animal tissues. Its use in future studies should allow the study of fine cellular details and provide significant advances in our understanding of cell surface structures and membrane organization.

Structure brings clarity:Structured illumination microscopy in cell biology

Biological samples are three dimensional and, therefore, optical sectioning is mandatory for microscopic images to precisely show the localization or function of structures within biological samples. Today, researchers can choose from a variety of methods to obtain optical sections. This article focuses on structured illumination microscopy, which is a group of techniques utilizing a combination of optics and mathematics to obtain optical sections: A structure is imaged onto the sample by optical means and the additional information thereby encoded in the image is used to calculate an optical section from several acquired images. Different methods of structured illumination microscopy (mainly grid projection and aperture correlation) are discussed from a practical point of view, concentrating on advantages, limitations and future prospects of these techniques and their use in cell biology. Structured illumination can also be used to obtain super‐resolution information if structures of higher frequency are projected onto the sample. This promising approach to super‐resolution microscopy is also briefly discussed from a users perspective.

High-resolution helium ion microscopy of epididymal epithelial cells and their interaction with spermatozoa

We examined the rat and mouse epididymis using helium ion microscopy (HIM), a novel imaging technology that uses a scanning beam of He(+) ions to produce nanometer resolution images of uncoated biological samples. Various tissue fixation, sectioning and dehydration methods were evaluated for their ability to preserve tissue architecture. The cauda epididymidis was luminally perfused in vivo to remove most spermatozoa and the apical surface of the epithelial lining was exposed. Fixed epididymis samples were then subjected to critical point drying (CPD) and HIM. Apical stereocilia in principal cells and smaller apical membrane extensions in clear cells were clearly distinguishable in both rat and mouse epididymis using this technology. After perfusion with an activating solution containing CPT-cAMP, a permeant analog of cAMP, clear cells exhibited an increase in the number and size of membrane ruffles or microplicae. In contrast, principal cells did not exhibit detectable structural modifications. High-resolution HIM imaging clearly showed the ultrastructure of residual sperm cells, including the presence of concentric rings on the midpiece, and of cytoplasmic droplets in some spermatozoa. Close epithelium-sperm interactions were also detected. We found a number of sperm cells whose heads were anchored within the epididymal epithelium. In certain cases, the surface of the sperm cytoplasmic droplet was covered with vesicle-like structures whose size is consistent with that of epididymosomes. In conclusion, we describe here the first application of HIM technology to the study of the structure and morphology of the rodent epididymis. HIM technology represents a major imaging breakthrough that can be successfully applied to study the epididymis and spermatozoa, with the goal of advancing our understanding of their structure and function.

Helium ion microscopy of enamel crystallites and extracellular tooth enamel matrix

An unresolved problem in tooth enamel studies has been to analyze simultaneously and with sufficient spatial resolution both mineral and organic phases in their three dimensional (3D) organization in a given specimen. This study aims to address this need using high-resolution imaging to analyze the 3D structural organization of the enamel matrix, especially amelogenin, in relation to forming enamel crystals. Chemically fixed hemi-mandibles from wild type mice were embedded in LR White acrylic resin, polished and briefly etched to expose the organic matrix in developing tooth enamel. Full-length amelogenin was labeled with specific antibodies and 10 nm immuno-gold. This allowed us to use and compare two different high-resolution imaging techniques for the analysis of uncoated samples. Helium ion microscopy (HIM) was applied to study the spatial organization of organic and mineral structures, while field emission scanning electron microscopy (FE-SEM) in various modes, including backscattered electron detection, allowed us to discern the gold-labeled proteins. Wild type enamel in late secretory to early maturation stage reveals adjacent to ameloblasts a lengthwise parallel alignment of the enamel matrix proteins, including full-length amelogenin proteins, which then transitions into a more heterogeneous appearance with increasing distance from the mineralization front. The matrix adjacent to crystal bundles forms a smooth and lacey sheath, whereas between enamel prisms it is organized into spherical components that are interspersed with rod-shaped protein. These findings highlight first, that the heterogeneous organization of the enamel matrix can be visualized in mineralized en bloc samples. Second, our results illustrate that the combination of these techniques is a powerful approach to elucidate the 3D structural organization of organic matrix molecules in mineralizing tissue in nanometer resolution.

Imaging with the Helium Ion Microscope

A newly developed technology, the helium ion microscope (HIM), provides high-resolution imaging with several benefits compared to the standard scanning electron microscope (SEM). First, the images provide high resolution because the helium beam can be brought to a focused probe size that can be as small as 0.25 nm. Second, the images provide contrast mechanisms that are often markedly different from the SEM. These contrast mechanisms can reveal topographic, composition, and other types of information about the sample. Third, compared to the SEM, the HIM images tend to be more surface-specific – revealing information about the surface without the confusing subsurface information. Fourth, the HIM can obtain high-resolution images even of insulating samples that would otherwise charge excessively in the SEM. The HIM is still in its infancy compared to the SEM, having only been commercially available for 7 years; however, it has already provided several unique advantages for the imaging of biological materials.

Freeze Drying Method with Gaseous Nitrogen to Preserve Fine Ultrastructure of Biological Organizations for Scanning Electron Microscopy, Helium Ion Beam Microscopy and Fluorescence Microscopy

Electron Microscopy Resource Center, Laboratory of Bacterial Pathogenesis and Immunology, Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, New York, USA; Center for Biomedical Research, Population Council, New York, New York, USA, Department of Physiology and Cellular Biophysics, The Clyde and Helen Wu Center for Molecular Cardiology, Columbia University Medical Center, New York, New York, USA., Department of Molecular Pharmacology & Physiology, University of South Florida Morsani College of Medicine, Tampa, Florida, USA., Ion Microscopy Innovation Center, Zeiss Microscopy LLC, Peabody, Massachusetts, USA., Department of Infectious Diseases, Kings College London School of Medicine, Guys Hospital, Great Maze Pond, London SE1 9RT, UK

Advantages of Helium and Neon Ion Beams for Intelligent Imaging

Scanning electron microscopy (SEM) has become a crucial tool to visualize the exterior morphology and surface structures for samples in both biology and material science. Conventional SEMs are usually limited by relative low resolution and charging effects. Carbon or metal coating is required for insulator samples to minimize charging effects. However, the coating layer can sometimes obscure miniscule surface details and the coating procedure may generate artifacts, which could be confused with the true ultrastrcuture.

Freeze Drying Method with Gaseous Nitrogen for Biological Application of Helium Ion Microcopy

1 Electron Microscopy Resource Center, 2 Center for the Study of Hepatitis C, Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, New York, USA; 3 Department of Infectious Diseases, King’s College London, London SE1 9RT, United Kingdom, 4 Department of Physiology, Columbia University, New York, NY, 10032, 5 Department of Molecular Pharmacology & Physiology, University of South Florida, Morsani College of Medicine, Tampa, FL 33612, 6 Carl Zeiss Microscopy, LLC., Ion Microscopy Innovation Center, MA 01960 USA

Advanced metrology and inspection solutions for a 3D world

Semiconductor devices and packages have firmly moved in to an era where scaling is driven by 3D architectures. However, most of the metrology and inspection technologies in use today were developed for 2D devices and are inadequate to deal with 3D structures. An additional complication is the need for specific structural and defect information that may be buried deep within a 3D structure. We present concepts and technologies that allow for 3D imaging as well as tomography, enabling engineers to view structural information with unprecedented clarity, detail and speed.