Defocus and twofold astigmatism correction in HAADF-STEM.

A new simultaneous autofocus and twofold astigmatism correction method is proposed for High Angle Annular Dark Field Scanning Transmission Electron Microscopy (HAADF-STEM). The method makes use of a modification of image variance, which has already been used before as an image quality measure for different types of microscopy, but its use is often justified on heuristic grounds. In this paper we show numerically that the variance reaches its maximum at Scherzer defocus and zero astigmatism. In order to find this maximum a simultaneous optimization of three parameters (focus, x- and y-stigmators) is necessary. This is implemented and tested on a FEI Tecnai F20. It successfully finds the optimal defocus and astigmatism with time and accuracy, compared to a human operator.

The determination of in vivo human tissue optical properties and absolute chromophore concentrations using spatially resolved steady-state diffuse reflectance spectroscopy.

A method is described for measuring optical properties and deriving chromophore concentrations from diffuse reflection measurements at the surface of a turbid medium. The method uses a diffusion approximation model for the diffuse reflectance, in combination with models for the absorption and scattering coefficients. An optical fibre-based set-up, capable of measuring nine spectra from 400 to 1050 nm simultaneously, is used to test the method experimentally. Results of the analyses of phantom and in vivo measurements are presented. These demonstrate that in the wavelength range from 600 to 900 nm, tissue scattering can be described as a simple power dependence of the wavelength and that the tissue absorption can be accurately described by the addition of water, oxy- and deoxyhaemoglobin absorption.

Experimental and model investigations of bleaching and saturation of fluorescence in flow cytometry.

We investigated the fluorescence emission from three fluorophores commonly used for labeling cells in flow cytometry. We have demonstrated that the fluorescence emission from cells labeled with fluorescein-isothiocyanate (FITC), phycoerythrin (PE), and allophycocyanin (APC) is considerably saturated and bleached in standard flow cytometric conditions. Therefore, for optimization of fluorescence detection in a flow cytometer, it is important to know the emission kinetics in detail. We made a mathematical model of the optical processes involved: absorption, fluorescence emission, nonradiative decay, photodestruction, and triplet state occupation. The validity of the model was experimentally tested with a set of averaged fluorescence pulses, measured in a large range of intensities and illumination times. The fluorescence of APC could be completely described by the model and produced the following rate constants: photodestruction rate kb1 = 6 x 10(3) s(-1), triplet state population rate k12 = 2 x 10(5) s(-1), and depopulation rate k20 = 5 x 10(4) s(-1). The fluorescence kinetics of FITC- and PE-labeled cells could not be fitted with only three parameters over the entire range, indicating that other optical processes are involved. We used the model to determine the sensitivity of our flow cytometer and to calculate the optimum conditions for the detection of APC. The results show that in principle a single APC molecule on a cell can be detected in the presence of background, i.e., autofluorescence and Raman scattering by water.

Elastic light-scattering measurements of single biological cells in an optical trap.

We have developed an instrument for determination of the angular light scattering of beads and biological cells. The instrument uses radiation pressure for levitation of particles inside a cuvette. The setup consists of two 780-nm diode lasers in a vertical double-beam trapping configuration. In the horizontal direction a weakly focused 633-nm probe beam is used to illuminate the trapped particle. One can detect scattered light over the range of from - 150 to 150 deg with an angular resolution of 0.9 deg using an avalanche photodiode. With this setup light scattering from polystyrene beads was measured, and the obtained scattering patterns were compared with theoretical scattering patterns from Lorenz-Mie theory. The results show that the setup is stable, gives reproducible patterns, and qualitatively agrees with the calculations. Trapping of biological cells is more difficult than trapping of beads, because smaller forces result from smaller refractive indices. We present an angular scattering pattern measured from a human lymphocyte measured from 20 to 60 deg.

Lissajous-like patterns in scatter plots of calibration beads.

Flow cytometric measurements of light scattering of polystyrene calibration beads revealed remarkable Lissajous-like loops in two-parameter scatter plots. The existence of such loops is shown to be in qualitative agreement with Lorenz-Mie scattering theory of homogeneous spheres. The occurrence of these patterns reflects the extreme particle size dependency of perpendicular light scattering of homogeneous spheres. These effects may give an explanation for the frequently observed phenomenon that polystyrene spheres reveal a relatively large CV in the perpendicular light scattering signals, whereas the CV of the forward light scattering signal is small. We conclude that one should be careful to optimize the perpendicular light scattering channel by minimizing the CV, because there is no linear relationship between instrument alignment quality and the CV of the perpendicular light scattering signals of calibration beads.

ANALYSIS: software for graphical analysis of multidimensional flow cytometric list mode data.

A computer program for graphical analysis of multidimensional flow cytometric list mode data is described. The program offers one-, two-, and three-dimensional inspection of an amount of data that is only limited by disk space. Subpopulations within the original data set can be identified by setting one or more two-dimensional AND gates around them. The order of measurement can be used as a parameter for evaluation of time-dependent processes. Other new parameters can be made by zooming in on a parameter, logarithmic transformation, or division of two parameters. The program is written in Turbo Pascal and it can run on any MS-DOC PC with an EGA/VGA resolution screen.

Visible diode lasers can be used for flow cytometric immunofluorescence and DNA analysis.

This report describes a feasibility study concerning the use of a visible diode laser for two important fluorescence applications in a flow cytometer. With a 3 mW 635 nm diode laser, we performed immunofluorescence measurements using the fluorophore allophycocyanin (APC). We have measured CD8 positive lymphocytes with a two-step labeling procedure and the resulting histograms showed good separation between the negative cells and the dim and the bright fluorescent subpopulations. As a second fluorescence application, we chose DNA analysis with the recently developed DNA/RNA stains TOTO-3 and TO-PRO-3. In our setup TO-PRO-3 yielded the best results with a CV of 3.4%. Our results indicate that a few milliwatts of 635 nm light from a visible diode laser is sufficient to do single color immunofluorescence measurements with allophycocyanin and DNA analysis with TO-PRO-3. The major advantages of using a diode laser in a flow cytometer are the small size, the low price, the high efficiency, and the long lifetime.

Another face of Lorenz-Mie scattering: monodisperse distributions of spheres produce Lissajous-like patterns.

The complete scattering matrix S of spheres was measured with a flow cytometer. The experimental equipment allows simultaneous detection of two scattering-matrix elements for every sphere in the distribution. Two-parameter scatterplots with x and y coordinates determined by the S(ll) + S(ij) and S(ll)-S(ij) values are measured. Samples of spheres with very narrow size distributions (< 1%) were analyzed with a FlowCytometer, and they produced unexpected two-parameter scatterplots. Instead of compact distributions we observed Lissajous-like loops. Simulation of the scatterplots, using Lorenz-Mie theory, shows that these loops are due not to experimental errors but to true Lorenz-Mie scattering. It is shown that the loops originate from the sensitivity of the scattered field on the radius of the spheres. This paper demonstrates that the interpretation of rare events and hidden features in flow cytometry needs reconsideration.

White blood cell differentiation using a solid state flow cytometer.

A flow cytometer using a solid state light source and detector was designed and built. For illumination of the sample stream two types of diode lasers (670 nm and 780 nm) were tested in a set-up designed to differentiate human leukocytes by means of light scattering. The detector is an avalanche photodiode, which was used to detect the weak scattered light in the orthogonal direction. The new flow cytometer set-up is very small, relatively cheap and yields similar results as a standard flow cytometer set-up using a helium-neon laser and photomultipliers.

Simple delay monitor for droplet sorters.

We have constructed a simple device by which the optimal delay time between optical measurement of a cell and the application of the droplet charging pulse can be determined directly in a flow sorter. The device consists of a stainless steel chamber in which the sorted droplets are collected. In the collection chamber the collected droplets run through a capillary where a continuous fluorescence measurement is made. With a sample of fluorescent particles, the delay time is optimal when the measured fluorescence is maximal. The measuring volume is always filled with the last droplets sorted (about 3,000). With this device, the setting of the delay time can be done in a few seconds without the need for microscopical verification. The fluorescence in the collection chamber is excited and detected via optical fibers using about 10% of the light of the existing laser from the flow cytometer and an extra photomultiplier.