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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.

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Narrow peaks are observed in the transmission spectra of p-polarized light passing through a thin gold film that is coated on the surface of a transparent diffraction grating. The spectral position and intensity of these peaks can be tuned over a wide range of wavelengths by simple rotation of the grating. The wavelengths where these transmission peaks are observed correspond to conditions where surface plasmon resonance occurs at the gold−air interface. Light diffracted by the grating couples with surface plasmons in the metal film to satisfy the resonant condition, resulting in enhanced light transmission through the film. Notably, this phenomenon is not observed at flat, gold-coated surfaces or uncoated gratings, where coupling to surface plasmons does not occur. The nature of the coupling and, thus, the details of light transmission are governed by the momentum matching conditions between the diffracted light and the surface plasmons. In the presence of bound analytes or surface films, the enhanced transmission peaks are red-shifted, making a simple, yet highly responsive sensing platform. The utility of this platform is demonstrated for ex situ sensing by analyzing thin films of various thicknesses and detecting a model immunoreaction between bovine serum albumin and anti-bovine serum albumin. This grating-based transmission surface plasmonic device represents a simple and sensitive platform, which can be readily tuned to enhance performance and be used in the study of a variety of surface adsorption processes or analysis of biomolecular interactions.

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We report a technique that utilizes surface plasmon resonance dispersion as a mechanism to provide multicolor contrast for imaging thin molecular films. Illumination of gold surfaces with p-polarized white light in the Kretschmann configuration produces distinct reflected colors due to excitation of surface plasmons and the resulting absorption of specific wavelengths from the source light. In addition, these colors transform in response to the formation of thin molecular films. This process represents a simple detection method for distinguishing between films of varying thickness in sensor applications. As an example, we interrogated a protein microarray formed by a commercial drop-on-demand chemical ink jet printer. Submonolayer films of a test protein (bovine serum albumin) were readily detected by this method. Analysis of the dispersion relations and absorbance sensitivities illustrate the performance and characteristics of this system. Higher detection sensitivity was achieved at angles where red wavelengths coupled to surface plasmons. However, improved contrast and spatial resolution occurred when the angle of incidence was such that shorter wavelengths coupled to the surface plasmons. Simplified optics combined with the robust microarray printing platform are used to demonstrate the applicability of this technique as a rapid and versatile, high-throughput tool for label-free detection of adsorbed films and macromolecules.

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