The OT-AFM Combi-System pairs the exceptional surface force measurement and imaging capabilities of AFM with the ability of optical tweezers to apply and measure smallest forces in 3D.
The OT-AFM Combi-System pairs the exceptional surface force measurement and imaging capabilities of AFM with the ability of optical tweezers to apply and measure smallest forces in 3D. The combined setup fulfills the highest demands on mechanical stability, flexibility, and modularity. A specially designed OT-AFM ConnectorStage is the key to combining any AFM of the NanoWizard or CellHesion family with the NanoTracker optical tweezers on a research-grade inverted optical microscope.
The unique combination of 3D positioning, detection, and manipulation provided by OT and the high-resolution imaging and surface property characterization of AFM opens up a whole new spectrum of applications, such as cellular response, cell-cell or cell matrix interactions, immune response, infection or bacterial/virus/nanoparticle uptake processes, and more.
With a multitude of available handles, interaction, and detection sites, OT-AFM significantly extends the range of single-molecule applications.
1. DNA hairpin unzipping (AFM) while the optical trap can be used to suppress (high laser power) or to quantify rotation (low laser power).
2. Scanning of a decorated DNA molecule. The molecule with DNA binding proteins (green) is spanned between two optically trapped beads. A functionalized AFM tip (blue) scans along the molecule and whenever interactions between the DNA-attached proteins and the tip occur, these can be detected in the AFM and OT signals.
3. Monitoring of DNA-enzyme (e.g. polymerase, helicase) dynamics. With onestrand attached to an optically trapped particle, the step-wise motion can be tracked. Closed-loop force clamping allows maintaining a constant force on the single strand.
Cellular response, cell-cell or cell matrix interactions, immune response, infection or bacterial/virus/nanoparticle uptake processes are just a few of the examples that can be investigated with JPK’s new state of the art OT-AFM platform. JPKs proven AFM and OT core technologies, combined with fluorescence microscopy, have set the ultimate benchmark for live cell applications.
 + : Activation of cells with functionalized beads, parallel AFM measurement. Signaling molecules on the surface of a microparticle are brought in contact with the cell at defined positions and time points.
 + : A mechano-sensitive cell is stimulated by a periodic force, exerted by an optically trapped particle. Internal rearrangements of the cytoskeleton alter the mechanical properties of the cell. These properties are easily accessed with AFM methods like force mapping or JPK’s Quantitative Imaging Advanced (QI-Advanced).
 + : AFM can be used in parallel to monitor changes in the cell structure, e.g. by monitoring mechanical properties throughout the process or by molecular recognition force spectroscopy that investigates the distribution and mechanical behavior of membrane proteins.
Trigger cellular responses by using functionalized particles or modified microorganisms is a common method. The resulting changes in cellular structure, dynamics, and mechanical properties can be investigated using AFM-based methods. Delivering objects to specific regions of interest on the cell, however, is very difficult to achieve. OT provides the perfect tool for manipulating the sample and triggering cellular response, at a precise time and location. This significantly improves the throughput, flexibility, and reproducibility of these studies. In this application, the influence of signaling between dendritic cells (DCs) and regulatory T-cells (T reg) on the adhesion of conventional T-cells (Tconv) to the same DC is quantified by OT-AFM.
 Adhesion experiment with dendritic cells (DC) and conventional T-cells (Tconv). The Tconv is attached to a tipless cantilever, then approached to the surface-bound DC. The cantilever is pulled up and the adhesion forces are measured. A regulatory T-cell (Treg) is attached to and removed from the DC with optical tweezers to test its influence on the binding strength. + Measurement setup. The optical trap (red cross) moves the Treg while adhesion measurements are performed with a cantilever-attached Tconv.  Detachment work measured for the three situations. Treg attachment reduces DC-T conv interactions. After the Treg is removed, the adhesion level is almost restored. Sample courtesy of Yan Shi, University of Calgary/Tsinghua University, Beijing. The original experiment was designed by Yan Shi et al. (publication in print).
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