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.
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.
We report a SPR biosensor based on long-range (LR) and short-range (SR) surface plasmon (SP) modes excited
simultaneously on a diffraction grating. Employing the LRSP and SRSP in the grating-coupled SPR sensor offers several
interesting features such as extended probe depth of the LRSP and ability to distinguish sensor response caused by bulk
and surface refractive index changes. Prototype device based on wavelength interrogation of SPs was developed and be
tested in model refractometric experiment. This paper presents results of theoretical analysis and experimental
characterization of the sensor. Sensitivity of the laboratory prototype of the sensor agrees well with the theory. The
sensor is shown to be able to detect changes in the refractive index as small as 3.5 x 10-6
RIU (refractive index unit).
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