Novel Microscopic Techniques for Podocyte Research

Abstract

Together with endothelial cells and the glomerular basement membrane, podocytes form the size-specific filtration barrier of the glomerulus with their interdigitating foot processes. Since glomerulopathies are associated with so-called foot process effacement-a severe change of well-formed foot processes into flat and broadened processes-visualization of the three-dimensional podocyte morphology is a crucial part for diagnosis of nephrotic diseases. However, interdigitating podocyte foot processes are too narrow to be resolved by classic light microscopy due to Ernst Abbe's law making electron microscopy necessary. Although three dimensional electron microscopy approaches like serial block face and focused ion beam scanning electron microscopy and electron tomography allow volumetric reconstruction of podocytes, these techniques are very time-consuming and too specialized for routine use or screening purposes. During the last few years, different super-resolution microscopic techniques were developed to overcome the optical resolution limit enabling new insights into podocyte morphology. Super-resolution microscopy approaches like three dimensional structured illumination microscopy (3D-SIM), stimulated emission depletion microscopy (STED) and localization microscopy [stochastic optical reconstruction microscopy (STORM), photoactivated localization microscopy (PALM)] reach resolutions down to 80-20 nm and can be used to image and further quantify podocyte foot process morphology. Furthermore, in vivo imaging of podocytes is essential to study the behavior of these cells in situ. Therefore, multiphoton laser microscopy was a breakthrough for in vivo studies of podocytes in transgenic animal models like rodents and zebrafish larvae because it allows imaging structures up to several hundred micrometer in depth within the tissue. Additionally, along with multiphoton microscopy, lightsheet microscopy is currently used to visualize larger tissue volumes and therefore image complete glomeruli in their native tissue context. Alongside plain visualization of cellular structures, atomic force microscopy has been used to study the change of mechanical properties of podocytes in diseased states which has been shown to be a culprit in podocyte maintenance. This review discusses recent advances in the field of microscopic imaging and demonstrates their currently used and other possible applications for podocyte research.

Keywords: STED microscopy; atomic force microscopy; light-sheet imaging; multiphoton imaging; podocyte nephropathy; serial block-face scanning electron microscopy (SBFSEM); structured illumination microscopy; superresolution microscopy.

Figure 1

The Jablonski diagram in (A) shows the energy levels of fluorophore electrons upon single-photon excitation (1-PE) and two-photon excitation (2-PE). For fluorescence emission, electrons are elevated from the S₀ to the S₁ level where they fall back to S₀ while emitting a photon. Dense temporal concentration of exciting photons of longer wavelengths compared with 1-PE elevates fluorophores up on the S₁ level with subsequent emission of a photon. (B) shows a scheme of 1-PE and 2-PE in a homogenously fluorescent liquid. Compared to 1-PE the fluorescence emission (red) of 2-PE excitation is spatially restricted to a small point within the focal plane. (C) shows an in vivo multiphoton micrograph of a transgenic zebrafish larva (5 days past fertilization) with expression of mCherry in podocytes and 78 kDa gc-eGFP fusion protein in the blood plasma. The magnification in (D) shows interdigitating major processes on the face of the glomerular capillaries. The scale bars indicate 50 μm in (C), 10 μm in the magnification of (C) and 5 μm in (D).

Figure 2

The scheme in (A) shows the interference of an illumination pattern in three different angles with the probe. In the first phase, vertical Moiré patterns are clearly visible and resolvable by widefield microscopy. (B) shows a histological section of a murine glomerular capillary stained for nephrin. The inserts show the gain of resolution compared from widefield microscopy and 3D-SIM. Clearly, individual interdigitating foot processes are resolvable on the glomerular capillary. The scale bar indicates 1 μm.

Figure 3

Dual color 3D-SIM of rodent (A) in mouse and (B) in rat) paraffin kidney sections shows the interdigitating foot process pattern of podocytes. Foot processes and the slit diaphragm were labeled using anti-synaptopodin and anti-nephrin antibodies, respectively. Although both proteins are in close structural vicinity the individual staining patterns do not overlap and therefore demonstrates the high resolution accomplished by 3D-SIM. All scale bars represent 1 μm.

Figure 4

Scheme (A) presents the basic principle of localization microscopy. Labeled structures are cyclically imaged and positions of individual fluorophores calculated. For further explanations see the main text. (B) shows a dSTORM image of a murine glomerulus labeled with an antibody for the glomerular basement membrane component laminin detected by secondary antibodies conjugated to Alexa Fluor 647. Ten thousand imaging cycles were recorded in the presence of a redox buffering system containing 2-mercaptoethylamine and in absence of oxygen accomplished by glucose oxidase and catalase. Superresolution is demonstrated in comparison with the corresponding WF picture. The scale bar indicates 200 nm in the magnification. (C) shows the microtubule cytoskeleton of a murine cultured podocyte detected by anti α-tubulin antibodies. The magnification demonstrates localizations of individual antibodies on microtubules. The scale bar indicates 500 nm.