Interferometric scattering microscopy is a breakthrough in ultrasensitive, label-free detection of biomolecules enabled by fast and sensitive imaging of light scattered by weakly scattering objects. Our research focuses on discerning subtle fluctuations in the scattering signal to describe biomolecular interactions and processes hidden deep within the subdiffractional volume of the probe beam. We demonstrate how the fluctuation in the scattering amplitude can be associated with conformation changes taking place at the level of a single, or a few unlabeled biomolecules and open new possibilities for the next generation of super-resolution microscopy techniques. We further combine the ultrasensitive detection of single molecules with real-time Raman spectroscopy to monitor the structural fluctuations on the single-molecule level. Understanding the dynamics of biological matter opens new avenues in label-free super-resolution microscopy and ultimately sensitive detection and identification of biomolecular samples.
We combine ultrasensitive microscopy with a novel, highly precise, and fast phase control to enable quantitative phase imaging of weakly scattering objects such as single microtubules. We demonstrate 3D mapping of microtubule networks and real-time 3D localization of single proteins labeled with small metallic nanoparticles. In particular, the 3D trajectories of microtubule-associated proteins acquired at the kHz rate revealed a complex trajectory of the protein diffusion on the surface of microtubules. The fast and accurate phase shaping technique combined with interferometric scattering microscopy pushes the limits of the quantitative phase imaging deep into the subdiffraction regime.
Biology is a fundamental scientific field which has made significant progress over the course of recent centuries and with the help of modern microscopy techniques, major discoveries are still being made today. The time span of processes such as protein dynamics ranges from slow to extremely fast. That is why high temporal resolution has recently become one of the desired parameters in biological experiments. The improvement of ultrafast image acquisition technology can help us to achieve higher temporal resolutions than before and detailed biological processes of rapid nature can now be observed. With these possibilities comes a desire to determine the noise characteristics of ultrafast cameras to set the limitations in localization precision in tracking of biological objects and their labels, which is the focus of this manuscript.
The understanding of nanoscale biological processes is limited by the level of details we can achieve when observing their dynamics. Addressing molecules of interest using fluorescent labels is the most common contrast mechanism in biological nano-imaging. However, the complex photophysics of fluorescent labels limits the localization precision as well as observation times in practical experiments. As an alternative to fluorescence-based microscopy interferometric scattering microscopy (iSCAT) was recently introduced. It is an optical microscopy technique allowing to detect and track nanoscale objects with sub-nanometre localization precision. The basic concept of this technique is the interference of light scattered on the particle with a reference wave light partially reflected at the microscopic slide. Recent advancements pushed the sensitivity and high-speed tracking down to a level of a single unlabelled protein by balancing the amplitudes of scattering and reference waves. This is often achieved by optimizing the reference wave, e.g. via placing a partially transparent mask near the back focal plane of a high numerical aperture microscope. In this contribution we introduce and demonstrate an innovative layout of the iSCAT microscope with optimized reference wave and minimized interferometric artefacts. We benchmark the detection capabilities of the new layout using series of extremely small spherical gold nanoparticles and demonstrate possible applications of the novel detection scheme.
Novel methods aiming at understanding complex biophysical processes allow revealing the dynamics and behaviour in extreme detail down to a single protein. Developments of fluorescence-based super-resolution microscopy and nanoscopic tracking techniques helped to reach a spatial resolution in length scales below 10 nm. These advances rely on the efficient collection of fluorescence at single-molecule levels. However, complex photophysics and saturation of fluorescent labels limit the temporal resolution to milliseconds timescales. To overcome the spatiotemporal limitations of fluorescent-based techniques we are employing interferometric scattering microscopy (iSCAT). iSCAT is an optical microscopy technique which allows for the detection and localization of extremely low scattering signals. It is based on interference of light scattered on the particle with a reference wave, e.g. light partially reflected at a glass coverslip. The sensitivity of iSCAT was previously proven in detection experiments with small nanoparticles as well as unlabelled single proteins. Here, we show that scattering labels can be imaged and localized with a nanometer precision and a few microseconds temporal resolution. We investigate the limits of fast tracking of scattering labels and identify pitfalls of high-speed collection for which the tracking fidelity drops rapidly due to fluctuations in the label position.
We report a novel high-throughput surface plasmon resonance (SPR) biosensor for rapid and parallelized detection of
protein biomarkers. The biosensor is based on a high-performance SPR imaging sensor with polarization contrast and
internal referencing which yields a considerably higher sensitivity and resolution than conventional SPR imaging
systems (refractive index resolution 2 × 10-7 RIU). We combined the SPR imaging biosensor with microspotting to
create an array of antibodies. DNA-directed protein immobilization was utilized for the spatially resolved attachment of
antibodies. Using Human Chorionic Gonadotropin (hCG) as model protein biomarker, we demonstrated the potential for
simultaneous detection of proteins in up to 100 channels.
We report a compact multi-channel biosensor based on diffraction grating-coupled SPR for the most demanding
detection applications in the field or home environments. The sensor utilizes special diffraction grating (referred
to as surface plasmon coupler and disperser - SPRCD) for coupling light into the surface plasmon and its
simultaneous wavelength dispersion through a different diffraction order. This approach combines most of the
optical instrumentation on a single SPR chip produced by stamper hot-embossing technique which is fully
compatible with mass production. The sensor consists of a disposable cartridge (SPR chip and microfluidics) and
a compact SPR instrument with the footprint which includes optical system of SPR sensor, supporting and data
acquisition electronics, microfluidics delivering sample into six independent sensing channels in the cartridge,
and temperature stabilization. We demonstrate that the sensor is able to measure changes in the refractive index
as low as 2x10-7 refractive index units (RIU) and to detect the binding of antibodies to the antigen-coated sensor
surface.
This contribution reviews the present state of the art in the development of surface plasmon resonance (SPR) (bio)sensor
technology, discusses emerging trends, and presents recent results of research into SPR biosensors at the Institute of
Photonics and Electronics, Prague. The developments discussed in detail include a high-performance SPR sensor for
parallelized observation of biomolecular interactions, a miniature fiber optic SPR sensor for localized measurements, and
sensing based on localized surface plasmons on gold nanoparticles.
We study changes in the polarization of a light induced by the interaction of light with a surface plasmon. Based on the
results of the study, a novel polarization control scheme for surface plasmon resonance (SPR) sensors is proposed.
Theoretical model of an SPR sensor employing the new polarization control scheme is presented. The theoretical
analysis suggests that the proposed polarization control scheme can significantly improve sensitivity and operating range
of SPR sensors. Results of the theoretical analysis are validated experimentally using a laboratory SPR imaging sensor
system with the proposed polarization control.
We present an optical sensor based on excitation of surface plasma waves in optical fiber structure consisting of a side-polished single-mode polarization-maintaining fiber and a metal overlayer. We describe two modes of operation of the sensor in which variations in the refractive index of the sample are determined by measuring changes in the transmitted optical power at a fixed wavelength (amplitude mode) and by measuring changes in the wavelength at which the resonant attenuation of the fiber mode occurs (spectral mode). We demonstrate that this design allows suppressing sensitivity of the sensor to deformation of the fiber yielding an improved stability and resolution.
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