ROCS microscopy, a label-free super-resolution technique, provides high spatial (150nm) and temporal (10ms) resolution, ideal for live-cell imaging. By rotating a blue laser beam at large angles, ROCS captures interference patterns, yielding high-contrast images without post-processing and minimal laser power. This method allows rapid acquisition of thousands of images of live cell dynamics. In the ROCS Bright-field (BF) mode, coherent amplification of scattered light enables imaging at lower optical powers, facilitating prolonged tracking of cellular interactions. Utilized for Fibroblast-born tunneling nanotubes (TNTs), which play a vital role in cardiac cell communication, ROCS employs total internal reflection (TIR) and dark-field (DF) modes to measure TNT growth with exceptional contrast. With the capability to reach depths of up to 5 micrometers in Non -TIR mode, ROCS effortlessly switches between modes, establishing itself as a versatile tool to study biological samples.
Particle uptake is of huge interest in pharmacology and medicine, but a user-controlled uptake of particles into GUVs has been technically demanding. Here, we indent the membrane of differently composed GUVs with optically trapped particles until particle uptake. By continuous 1MHz back-focal-plane interferometric tracking and autocorrelating the particle’s positions within 30µs delays for different indentations, the fluctuations’ amplitude, the damping, the mean forces, and the energy profiles were obtained.
The study demonstrates that soft matter can interact completely differently on different temporal and spatial frequency modes, which are excited by the environmental thermal noise.
Photonic Force Microscopy (PFM) uses optically trapped particles to measure fluctuation based forces and potential landscapes. This feature makes it a very soft probing technique that is well suited to investigate surface interactions with biological samples. In addition, PFMs can be used in scan mode to create surface-profiles with a resolution better than the optical diffraction limit. In this talk we present an improved intermittent contact (tapping) mode PFM and a revised data analysis for more detailed surface height profiles.
Photonic Force Microscopy (PFM) uses optically trapped nanoparticles to measure forces in the sub-piconewton range. This makes it a very soft probing technique that is perfectly suited to investigate surface interactions with biological samples. In addition, PFMs can be used in scan mode to create surface-profiles with a resolution better than the optical diffraction limit. A common problem for probing techniques that operate in contact mode is sticking of the probe to the sample. To overcome this problem, we present an intermittent contact mode PFM to improve the technique’s robustness by reducing contact times and binding between sample and probe.
The transfer of mechanical signals through cells is a complex phenomenon. To uncover a new mechanotransduction pathway, we study the frequency-dependent transport of mechanical stimuli by single microtubules and small networks in a bottom-up approach using optically trapped beads as anchor points. We interconnect microtubules to linear and triangular geometries to perform micro-rheology by defined oscillations of the beads relative to each other, which are measured with 3D back focal plane interferometry. We find a substantial stiffening of single filaments above a characteristic transition frequency of 1-30 Hz depending on the filament’s molecular composition. Below this frequency, filament elasticity only depends on its contour and persistence length. Interestingly, this elastic behavior is transferable to small networks, where we found the surprising effect that linear two filament connections act as transistor-like, angle dependent momentum filters, whereas triangular networks act as stabilizing elements. These observations implicate that cells can tune mechanical signals by temporal and spatial filtering stronger and more flexibly than expected. In addition, we integrate a novel label-free microscopy techniques, capable of imaging freely-diffusing microtubules in real-time and independent of their orientation. We show that rotating coherent scattering (ROCS) microscopy in dark-field mode provides strong contrast also for structures far from the coverslip such as arrangements of isolated MTs and networks. We could acquire thousands of images over up to 30 minutes without loss in image contrast or visible photo damage.
Optical potential landscapes for diffusing particles can be generated by time-multiplexing (scanning) an optical trap and additionally modulating the trapping laser power. We show that it is possible to determine the lateral and axial positions of several particles in parallel with some 10 nm precision and at kHz rates by using dynamic back focal plane interferometry. This allows measuring the interaction of diffusing, (non-) functionalized particles in a confined volume. The scan frequency of the optical trap can be optimized for interaction measurements with high dynamic or temporal resolution, given a data sampling frequency of 1MHz.
Functionalized surfaces can affect (bio-) chemical reactions and control spatially the affinity for various binding partners. By either distributing specific binding points, by using biological reconstituted systems on the surface or by investigating whole cells, a surface phase relief is introduced. In the proximity of a trapped particle, these surface scatterers will both disturb the optical trap and the position tracking signal by changing the wave front of the trapping laser. We investigate the influence of an additional scatterer on trapping force, detection sensitivity and local viscous drag by scanning an optically trapped bead (probe) across a structured surface. Using a photonic force microscope, the probe's fluctuation traces are recorded interferometrically in three dimensions with nm precision and at scan rates of several hundred kilohertz with a quadrant photodiode. The phase disturbance located underneath the optical trap alters the interferometric probe position signals and can lead to incorrect interaction measurements. We propose and test a procedure to correct for the phase disturbance of the surface structure. In a roll over experiment, where one nano-sphere rolls over another, we prove the applicability of our phase correction approach. In addition we investigate the influence of small gold dots on the coverslip on trapping parameters which are relevant for specific interaction measurements in biotechnology.
The Photonic Force Microscope is a novel scanning probe microscope based on optical tweezers to hold a probe, which fluctuates in its position due to thermal noise. The three-dimensional position of the probe is detected with high temporal resolution and spatial precision by analyzing the interference of unscattered and forward scattered light. We present the theoretical framework of the optical forces acting on the probe, as well as of the detection signal due to coherent scattering and describe thermal noise position fluctuations by a Brownian dynamics simulation. As an application we simulate the temporal and spatial behavior of a probe tethered to the coverslip (according to the molecular kinesin/microtubule system) at different laser powers and different anchor positions.
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