Extracellular vesicles (EVs) are nanoparticles released by cells and have high potential as disease biomarkers. EVs are studied by flow cytometers, which measure scattering in arbitrary units. With Mie theory, arbitrary units can be related to diameter when the particle refractive index (RI) is known. However, a setup to determine the RI of nanoparticles in a traceable manner, including uncertainty, is lacking. Therefore, we have developed the first metrological flow cytometer, utilizing Laguerre-Gauss illumination, multi-angle light scattering, artificial intelligence data processing, and a calibrated syringe pump, to traceably determine the size, RI, and concentration of single nanoparticles in liquid.
Refractive index (RI) measurements are highly dependent on environmental conditions, which are often not reported, resulting in non-traceable measurements. Additionally, the RI of buffer solutions, such as phosphate-buffered saline (PBS), is unknown. We built a new optical set-up based on the minimum deviation angle to traceably measure the RI of solids and liquids under controlled environmental conditions. We measured the RI of fused silica, 1.470091, and PBS, 1.344599, at 405 nm, 20.00 °C with an expanded uncertainty of 1.4e-6. Our results differ from previously assumed RIs and therefore have practical implications for nanoparticle flow cytometry measurements.
The need for fast and accurate inspection of small sample features is eminent considering the developments in micro and nano technology. The scanning probe microscope (SPM) offers extreme resolution and even accuracy when properly calibrated but the principle of operation result in inherently slow acquisition of the measurement data. Therefore scanning probe microscopes are rarely deployed in industrial in-line inspection and quality control where the time aspect usually is critical. We propose an alternative mode of operation that can considerably speed up SPM measurements. In this mode only the areas of interest are probe with maximum accuracy while the rest of the imaging is ignored. The measuring mode is best suited when a-priori information of the surface is available, like in an industrial production line.
We will report on the progress of our project to realize a traceable Scanning Probe Microscope at the Van Swinden Laboratorium of the Nederlands Meetinstituut in the Netherlands. The traceable Atomic Force Microscope (AFM) is constructed from a separate AFM head, a 3D translation stage and an accurate 3D laser interferometer system. Nanometer uncertainty can be maintained in the entire scanning volume of 100 μm × 100 μm × 20 μm. Apart from providing direct traceability to the SI unit of length, the coordinates provided by the laser interferometer are also used in a closed loop position feedback controller to realize accurate positioning at arbitrary locations within the volume provided by the translation stage. In this paper we will emphasize the development of the control system.
KEYWORDS: Digital signal processing, Laser stabilization, Modulation, Human-machine interfaces, Absorption, Photodiodes, Frequency combs, Process control, Signal processing, Iodine cells
We have developed a digital controller to stabilize one of NMi's iodine stabilized helium-neon lasers using the third harmonic technique. The controller is proven to be a suitable replacement of the analogue electronics, as demonstrated by internal comparisons and a calibration against the frequency comb of the Bureau International des Poids et Mesures.
The calibration of our interference microscope is currently performed by an elaborate multi step process. As a result the total uncertainty of a measurement performed with the interference microscope is much larger than the intrinsic repeatability of the microscope which is of the order of 1 nm. A major contribution to the total uncertainty is a length dependent factor, resulting from a calibration step using gauge blocks that finally yields 8 nm uncertainty for a step height of 2 micrometers . In order to reduce the total uncertainty we propose a novel step-height standard and calibration procedure. The standard is adjustable and can be simultaneously measured with the interference microscope and a laser interferometer allowing calibration of the entire dynamic range of the microscope with a single artefact. The new calibration method eliminates the process that contributes most to the total uncertainty budget in the current procedure. A possible implementation of the step-height standard is presented.
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