Practical Guidelines for Optimization and Characterization of the Beckman Coulter CytoFLEX™ Platform.

Cytometer characterization is critical to define operational bounds within which the data generated are reliable and reproducible. Existing instrument optimization and characterization protocols were developed for cytometers relying on photomultiplier tubes (PMTs) for photon detection. Recently, instrument manufacturers have begun incorporating avalanche photodiodes (APDs) in place of PMTs. Differences in noise and signal amplification properties of the two detector types make many of the established PMT characterization protocols inappropriate for APD-based instruments. In this article, we tested (three machines on two different sites) a variety of approaches to determine the best method for APD optimization on the Beckman Coulter CytoFLEX™ (CytoFLEX). From this, we propose easy-to-implement guidelines for CytoFLEX characterization and operation. These protocols are not designed to compare APD versus PMT based systems, nor are they designed to directly compare different CytoFlex instruments. Following these protocols will allow CytoFLEX users to characterize their instruments and help to identify optimized settings that allow for the generation of consistent and reproducible data. © 2020 International Society for Advancement of Cytometry.

Introducing a non-pixelated and fast centre of mass detector for differential phase contrast microscopy.

With the advent of probe corrected STEM machines it became possible to probe specimens on a scale of less than 50 pm resolution. This opens completely new horizons for research, as it is e.g. possible to probe the electrostatic fields between individual rows of atoms, using differential phase contrast (DPC). However, in contrast to conventional DPC, where one deals with extended fields which can be assumed constant across the electron probe, this is not possible for sub-atomic probes in DPC. For the latter case it was shown [1,2], that the strongly inhomogeneous field distribution within the probe diameter, which usually is caused by the nuclear potentials of an atomic column, leads to a complicated intensity redistribution within the diffraction disk. The task is then to determine the intensity weighted centre of the diffraction disk pattern (frequently also called centre of mass, COM), which is proportional to the average lateral momentum gained by the average electron, transmitted through the probe diameter. In first reported measurements, the determination of this COM was achieved using a pixelated detector in combination with a software-based evaluation of the COM. This suffers from two disadvantages: first, the nowadays available pixelated detectors are still not very fast (approximately 1000 fps) and quite expensive, and second, the amount of data to be processed after acquisition is comparatively huge. In this paper we report on an alternative to a pixelated detector, which is able to directly deliver the COM of a diffraction disk's intensity distribution with frequencies up to 200 kHz. We present measurements on the sensitivity of this detector as well as first results from DPC imaging. From these results we expect the detector also to serve well in sub-atomic DPC field sensing, possibly replacing today's segmented or pixelated detectors.

On detector linearity and precision of beam shift detection for quantitative differential phase contrast applications.

Differential phase contrast is a STEM imaging mode where minute sideways deflections of the electron probe are monitored, usually by using a position sensitive device (Chapman, 1984 [1]; Lohr et al., 2012 [2]) or, alternatively in some cases, a fast camera (Müller et al., 2012 [3,4]; Yang et al., 2015 [5]; Pennycook et al., 2015 [6]) as a pixelated detector. While traditionally differential phase contrast electron microscopy was mainly focused on investigations of micro-magnetic domain structures and their specific features, such as domain wall widths, etc. (Chapman, 1984 [1]; Chapman et al., 1978, 1981, 1985 [7-9]; Sannomiya et al., 2004 [10]), its usage has recently been extended to mesoscopic (Lohr et al., 2012, 2016 [2,12]; Bauer et al., 2014 [11]; Shibata et al., 2015 [13]) and nano-scale electric fields (Shibata et al., 2012 [14]; Mueller et al., 2014 [15]). In this paper, the various interactions which can cause a beam deflection are reviewed and expanded by two so far undiscussed mechanisms which may be important for biological applications. As differential phase contrast microscopy strongly depends on the ability to detect minute beam deflections we first treat the linearity problem for an annular four quadrant detector and then determine the factors which limit the minimum measurable deflection angle, such as S/N ratio, current density, dwell time and detector geometry. Knowing these factors enables the experimenter to optimize the set-up for optimum performance of the microscope and to get a clear figure for the achievable field resolution error margins.

An investigation of the matrix sensitivity of refinery gas analysis using gas chromatography with flame ionisation detection.

The response of a flame ionisation detector (FID) on a gas chromatograph to methane, ethane, propane, i-butane and n-butane in a series of multi-component refinery gas standards was investigated to assess the matrix sensitivity of the instrument. High-accuracy synthetic gas standards, traceable to the International System of Units, were used to minimise uncertainties. The instrument response exhibited a small dependence on the component amount fraction: this behaviour, consistent with that of another FID, was thoroughly characterised over a wide range of component amount fractions and was shown to introduce a negligible bias in the analysis of refinery gas samples, provided a suitable reference standard is employed. No significant effects of the molar volume, density and viscosity of the gas mixtures on the instrument response were observed, indicating that the FID is suitable for the analysis of refinery gas mixtures over a wide range of component amount fractions provided that appropriate drift-correction procedures are employed.