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. \ud 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

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 withx andy coordinates determined by the Sll + Sij and S11 - Sij 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

Autofocus and two-fold astigmatism correction in HAADF-STEM

A new simultaneous autofocus and two-fold 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 an image variance, which has already been used before as an image quality measure for different types of microscopy. In this paper we describe numerical simulations based on a classical HAADF-STEM linear image formation model showing that the modified variance reaches it's maximum for Scherzer focus and zero astigmatism. In order to find this maximum in a three-parameter space we employ the well-known Nelder-Mead simplex optimization algorithm. The method is implemented and tested on a FEI Tecnai F20.It successfully finds the optimal defocus and zero astigmatism with the time and accuracy, compared with the human operator. The method is iterative, and finding the optimal defocus and zero astigmatism requires obtaining typically 20-50 images

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 · 103 s-1, triplet state population rate k12 = 2 · 105 s-1, and depopulation rate k20 = 5 · 104 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