Abstract:We present a new super-resolution technique, Re-scan Confocal Microscopy (RCM), based on standard confocal microscopy extended with an optical (re-scanning) unit that projects the image directly on a CCDcamera. This new microscope has improved lateral resolution and strongly improved sensitivity while maintaining the sectioning capability of a standard confocal microscope. This simple technology is typically useful for biological applications where the combination high-resolution and highsensitivity is required. Toomre, and J. Bewersdorf, "Video-rate nanoscopy using sCMOS camera-specific single-molecule localization algorithms," Nat. Methods 10(7), 653-658 (2013).

Tunneling membrane nanotubes (TnTs) are membrane protrusions connecting nearby or distant cells in vitro and in vivo. Functions of TnTs in cellular processes are various and rely on TnT structure, which also depends on cytoskeletal composition. In the present study, we focused on the organization of microtubules (MTs) and intermediate filaments (IFs) in TnTs of urothelial cells. We analysed TnTs of normal porcine urothelial cells, which morphologically and physiologically closely resemble normal human urothelial cells, and of cancer cells derived from invasive human urothelial neoplasm. Wide-field fluorescence, confocal and super-resolution microscopy techniques, together with image analyses and 3D reconstructions enlightened specific MT-IF organization in TnTs, and for the first time revealed that MTs and IFs co-occur in the majority of normal and cancer urothelial cell TnTs. Our findings show that in the initiation segment of TnTs, MTs are cross-linked with each other into filamentous network, however in the middle and the attaching segment of TnT, MTs can helically enwrap IFs, the phenomenon that has not been shown before within the TnTs. In this study, we assess MT-IF co-occurrence in TnTs and present evidence that such helical organization of MTs enwrapping IFs is only occurring in a minority of the TnTs. We also discuss the possible cell-biological and physiological reasons for helical organization of MTs in TnTs.

The new high-sensitive and high-resolution technique, Re-scan Confocal Microscopy (RCM), is based on a standard confocal microscope extended with a re-scan detection unit. The re-scan unit includes a pair of re-scanning mirrors that project the emission light onto a camera in a scanning manner. The signal-to-noise ratio of Re-scan Confocal Microscopy is improved by a factor of 4 compared to standard confocal microscopy and the lateral resolution of Re-scan Confocal Microscopy is 170 nm (compared to 240 nm for diffraction limited resolution, 488 nm excitation, 1.49 NA). Apart from improved sensitivity and resolution, the optical setup of Re-scan Confocal Microscopy is flexible in its configuration in terms of control of the mirrors, lasers and filters. Because of this flexibility, the Re-scan Confocal Microscopy can be configured to address specific biological applications. In this paper, we explore a number of possible configurations of Re-scan Confocal Microscopy for specific biomedical applications such as multicolour, FRET, ratio-metric (e.g. pH and intracellular Ca measurements) and FRAP imaging.

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. Technological advances have identified these structures as key elements in neuron connectivity and synaptic plasticity. The quantitative analysis of spine morphology using light microscopy remains an essential problem due to technical limitations associated with light's intrinsic refraction limit. Dendritic spines can be readily identified by confocal laser-scanning fluorescence microscopy. However, measuring subtle changes in the shape and size of spines is difficult because spine dimensions other than length are usually smaller than conventional optical resolution fixed by light microscopy's theoretical resolution limit of 200 nm.Several recently developed super resolution techniques have been used to image cellular structures smaller than the 200 nm, including dendritic spines. These techniques are based on classical far-field operations and therefore allow the use of existing sample preparation methods and to image beyond the surface of a specimen. Described here is a working protocol to apply super resolution structured illumination microscopy (SIM) to the imaging of dendritic spines in primary hippocampal neuron cultures. Possible applications of SIM overlap with those of confocal microscopy. However, the two techniques present different applicability. SIM offers higher effective lateral resolution, while confocal microscopy, due to the usage of a physical pinhole, achieves resolution improvement at the expense of removal of out of focus light. In this protocol, primary neurons are cultured on glass coverslips using a standard protocol, transfected with DNA plasmids encoding fluorescent proteins and imaged using SIM. The whole protocol described herein takes approximately 2 weeks, because dendritic spines are imaged after 16-17 days in vitro, when dendritic development is optimal. After completion of the protocol, dendritic spines can be reconstructed in 3D from series of SIM image stacks using specialized software.

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