High magnification imaging of glomerular structures.

<p>(A) Detail of a glomerular podocyte showing a secondary projection and interdigitating foot processes (GA-fixation, extended methanol freeze-substitution dehydration protocol). Many tubular projections with more bulbous ends (white arrows) emerge from the podocyte membrane. Small (20–30 nm) irregularities of unknown nature can be seen on the external surface of the podocyte membrane (black arrows). Bar = 120 nm. (B) Detail of four “filtration” regions (slit diaphragms) between five adjacent podocyte foot processes. Numerous cross-bridging filaments extend at regular intervals across the space between adjacent foot processes (smaller arrows). In some regions, these delicate structures appear damaged, revealing another structure below, which may represent the glomerular basement membrane (larger arrows). Bar = 100 nm.</p

Imaging of renal collecting duct.

<p>(A) Luminal surface of an outer medullary collecting duct (GA-fixation, dehydration using the rapid graded methanol procedure) showing principal and intercalated cells. Each principal cell (PC) has one long, solitary cilium (arrows) and numerous short, stubby microvilli. The intercalated cell (IC) has numerous elaborate apical microplicae and no cilium. Bar = 2 µm. (B) High magnification view of a principal cell cilium (Bar = 200 nm). At its base, a concentric pattern of surface protrusions (arrows) can be seen in the position of the ciliary necklace. A similar structure is shown on another principal cell cilium in the inset (arrows, Bar = 100 nm). A principal cell cilium from the same kidney was also imaged by conventional SEM without sputter coating, using an in-lens detector (C). Structural details of the cilium, ciliary necklace, microvilli, and membrane indentations are more clearly distinguishable in the HIM than in the SEM images. Bar = 300 nm.</p

Imaging of renal proximal convoluted tubule.

<p>(A) Lower magnification showing GA-fixed proximal tubule (dehydrated using the extended methanol freeze-substitution protocol) and its extensive brush border (BB). Bar = 5 µm. (B) shows a lateral section of modified PLP-fixed proximal tubule dehydrated as in (A), demonstrating the apical brush border (BB) and the extensive basolateral plasma membrane infoldings and invaginations (arrows) that are characteristic of the S1 segment of the proximal tubule. Bar = 1 µm. (C) shows the tightly packed, slender brush border microvilli in greater detail (GA fixation, extended methanol freeze-substitution dehydration protocol). Bar = 0.5 µm. (D) Brush border microvilli at high magnification showing that their surface membrane has numerous micropits of unknown significance (arrows). Bar = 100 nm. Similar regions from the same kidney were also imaged by conventional SEM after coating using the in-lens detector and are shown at lower (E, bar = 0.5 µm) and higher magnification (F, bar = 100 nm). The conventional images have considerable less clarity and surface detail than the HIM-imaged brush border region.</p

Glomerular podocyte slit diaphragms from the same kidney as shown by HIM in Fig. 2, imaged by conventional scanning electron microscopy (SEM).

<p>(A) Sample imaged without sputter coating, using an in-lens detector. (B, C) Coated samples imaged using either the standard SE2 detector (B) or an in-lens detector (C). Structural details of the slit diaphragm are less well defined than in the HIM image shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057051#pone-0057051-g002" target="_blank">Fig. 2B</a>. Bar = 100 nm.</p

HIM imaging of external gold labeling in the kidney.

<p>(A) Lower magnification of a modified PLP-fixed proximal tubule with its brush border after labeling of surface glycoproteins (and/or glycolipids) with gold-conjugated WGA. The tissue was dehydrated using the rapid graded methanol procedure. The gold particles appear as discrete, white globular entities associated with the external surface of brush border microvilli and other parts of the cell surface adjacent to the microvilli. Bar = 1 µm. The gold label can be seen more easily at higher magnification (B - arrows), where it extends along the entire length of the microvilli. Bar = 200 nm. The inset in panel B shows a modified PLP-fixed proximal tubule brush border that has been immunolabeled with a monoclonal anti-megalin antibody followed by a secondary, gold-conjugated anti-mouse antibody. In this case, the pale gold particles (arrows) are concentrated towards the base of the microvilli and do not extend along their entire length (inset; Bar = 200 nm). (C) The apical surface of a collecting duct principal cell from the same kidney immunolabeled with the anti-megalin antibody and the respective gold-conjugated secondary antibody. The image shows no gold particles, attesting to the specificity of the proximal tubule megalin binding. Bar = 500 nm.</p

Detail from Fig. 6A showing the principal cell (PC) and an intercalated cell (IC) at higher magnification.

<p>The apical membrane of the intercalated cell has a highly complex organization that is formed of many microplicae and membrane furrows between these structures. Bar = 1 µm. (B) Higher magnification image of the elaborate intercalated cell apical membrane microplicae showing the deep infoldings of this membrane domain. Bar = 200 nm. (C) Apical membrane of a principal cell showing surface features that may represent exocytotic or endocytotic events. These depressions were frequently seen at the base of the short microvilli - a location in which clathrin mediated endocytosis often occurs. Bar = 200 nm.</p

HIM imaging of glomerular endothelial cells.

<p>(A) Two adjacent endothelial cells from a glomerular capillary (GA-fixed, dehydrated using the extended methanol freeze-substitution protocol), imaged from the luminal side. The most striking features of these cells are the numerous, round fenestrations that are present over the entire cell surface. The raised ridges (arrows) represent the location of the tight junction between the two cells. Bar = 175 nm. (B) Higher magnification showing details of the fenestrations. In some of them, a substructure consisting of faint spokes like a bicycle wheel can be seen (arrows). Bar = 80 nm.</p

Toward Plasmonics with Nanometer Precision: Nonlinear Optics of Helium-Ion Milled Gold Nanoantennas

Plasmonic nanoantennas are versatile tools for coherently controlling and directing light on the nanoscale. For these antennas, current fabrication techniques such as electron beam lithography (EBL) or focused ion beam (FIB) milling with Ga⁺-ions routinely achieve feature sizes in the 10 nm range. However, they suffer increasingly from inherent limitations when a precision of single nanometers down to atomic length scales is required, where exciting quantum mechanical effects are expected to affect the nanoantenna optics. Here, we demonstrate that a combined approach of Ga⁺-FIB and milling-based He⁺-ion lithography (HIL) for the fabrication of nanoantennas offers to readily overcome some of these limitations. Gold bowtie antennas with 6 nm gap size were fabricated with single-nanometer accuracy and high reproducibility. Using third harmonic (TH) spectroscopy, we find a substantial enhancement of the nonlinear emission intensity of single HIL-antennas compared to those produced by state-of-the-art gallium-based milling. Moreover, HIL-antennas show a vastly improved polarization contrast. This superior nonlinear performance of HIL-derived plasmonic structures is an excellent testimonial to the application of He⁺-ion beam milling for ultrahigh precision nanofabrication, which in turn can be viewed as a stepping stone to mastering quantum optical investigations in the near-field