Surface-modified complex SU-8 microstructures for indirect optical manipulation of single cells

We introduce a method that combines two-photon polymerization (TPP) and surface functionalization to enable the indirect optical manipulation of live cells. TPP-made 3D microstructures were coated specifically with a multilayer of the protein streptavidin and non-specifically with IgG antibody using polyethylene glycol diamine as a linker molecule. Protein density on their surfaces was quantified for various coating methods. The streptavidin-coated structures were shown to attach to biotinated cells reproducibly. We performed basic indirect optical micromanipulation tasks with attached structure-cell couples using complex structures and a multi-focus optical trap. The use of such extended manipulators for indirect optical trapping ensures to keep a safe distance between the trapping beams and the sensitive cell and enables their 6 degrees of freedom actuation. (C)2015 Optical Society of Americ

Localization STED (LocSTED) microscopy with 15 nm resolution

We present localization with stimulated emission depletion (LocSTED) microscopy, a combination of STED and single-molecule localization microscopy (SMLM). We use the simplest form of a STED microscope that is cost effective and synchronization free, comprising continuous wave (CW) lasers for both excitation and depletion. By utilizing the reversible blinking of fluorophores, single molecules of Alexa 555 are localized down to ~5 nm. Imaging fluorescently labeled proteins attached to nanoanchors structured by STED lithography shows that LocSTED microscopy can resolve molecules with a resolution of at least 15 nm, substantially improving the classical resolution of a CW STED microscope of about 60 nm. LocSTED microscopy also allows estimating the total number of proteins attached on a single nanoanchor

Statistical analysis of 3D localisation microscopy images for quantification of membrane protein distributions in a platelet clot model.

We present the software platform 2CALM that allows for a comparative analysis of 3D localisation microscopy data representing protein distributions in two biological samples. The in-depth statistical analysis reveals differences between samples at the nanoscopic level using parameters such as cluster-density and -curvature. An automatic classification system combines multiplex and multi-level statistical approaches into one comprehensive parameter for similarity testing of the compared samples. We demonstrated the biological importance of 2CALM, comparing the protein distributions of CD41 and CD62p on activated platelets in a 3D artificial clot. Additionally, using 2CALM, we quantified the impact of the inflammatory cytokine interleukin-1β on platelet activation in clots. The platform is applicable to any other cell type and biological system and can provide new insights into biological and medical applications

An Improved Transwell Design for Microelectrode Ion-Flux Measurements

The microelectrode ion flux estimation (MIFE) is a powerful, non-invasive electrophysiological method for cellular membrane transport studies. Usually, the MIFE measurements are performed in a tissue culture dish or directly with tissues (roots, parts of the plants, and cell tissues). Here, we present a transwell system that allows for MIFE measurements on a cell monolayer. We introduce a measurement window in the transwell insert membrane, which provides direct access for the cells to the media in the upper and lower compartment of the transwell system and allows direct cell-to-cell contact coculture. Three-dimensional multiphoton lithography (MPL) was used to construct a 3D grid structure for cell support in the measurement window. The optimal polymer grid constant was found for implementation in transwell MIFE measurements. We showed that human umbilical vein endothelial cells (HUVECs) efficiently grow and maintain their physiological response on top of the polymer structures

An Improved Transwell Design for Microelectrode Ion-Flux Measurements

The microelectrode ion flux estimation (MIFE) is a powerful, non-invasive electrophysiological method for cellular membrane transport studies. Usually, the MIFE measurements are performed in a tissue culture dish or directly with tissues (roots, parts of the plants, and cell tissues). Here, we present a transwell system that allows for MIFE measurements on a cell monolayer. We introduce a measurement window in the transwell insert membrane, which provides direct access for the cells to the media in the upper and lower compartment of the transwell system and allows direct cell-to-cell contact coculture. Three-dimensional multiphoton lithography (MPL) was used to construct a 3D grid structure for cell support in the measurement window. The optimal polymer grid constant was found for implementation in transwell MIFE measurements. We showed that human umbilical vein endothelial cells (HUVECs) efficiently grow and maintain their physiological response on top of the polymer structures

Localization Microscopy of Actin Cytoskeleton in Human Platelets

Here, we measure the actin cytoskeleton arrangement of different morphological states of human platelets using a new protocol for photo-switching of rhodamine class fluorophores. A new medium composition was established for imaging the cytoskeleton using Alexa Fluor 488 conjugated to phalloidin. Morphological states of platelets bound to a glass substrate are visualized and quantified by two-dimensional localization microscopy at nanoscopic resolution. Marker-less drift correction yields localization of individual Alexa 488 conjugated to phalloidin with a positional accuracy of 12 nm

Aminosilane-based functionalization of two-photon polymerized 3D SU-8 microstructures.

There is an increasing interest in functionalized complex 3D microstructures with sub-micrometer features for micro- and nanotechnology applications in biology. Depending primarily on the material of the structures various methods exist to create functional layers of simple chemical groups, biological macromolecules or metal nanoparticles. Here an effective coating method is demonstrated and evaluated on SU-8 based 3D microstructures made by two-photon polymerization. Protein streptavidin and gold nanoparticles (NP) were bound to the microstructures utilizing acid treatment-mediated silane chemistry. The protein surface density, quantified with single molecule fluorescence microscopy revealed that the protein forms a third of a monolayer on the two-photon polymerized structures. The surface coverage of the gold NPs on the microstructures was simply controlled with a single parameter. The possible degrading effect of the acid treatment on the sub-micrometer features of the TPP microstructures was analyzed. Our results show that the silane chemistry-based method, used earlier for the functionalization of large-area surfaces can effectively be adapted to coat two-photon polymerized SU-8 microstructures with sub-micrometer features