The way flow cytometers are usually configured is that the dimension of the flow orifice (50–100 μm) and the isotonic sheath fluid are optimized for the analysis of live human cells. Animal cells have similar sizes and osmolarities as human cells; therefore, they can be measured with the same setup and without substantial problems. By contrast, cytometric plant cell analysis is a challenge for several reasons. The rigid plant cell wall gives the cells a special, elongated form so that they can reach sizes that exceed the usual orifice size of a typical flow cytometer. Plant cell walls are highly organized fiber-laminateextracellular structures with strong anisotropy in their shape and growth, and thus, polarization characteristics have been widely used in revealing the anisotropic features of this complex cellulose-based structure (1). The removal of the wall leads to protoplasts with spherical form but the osmotic pressure of such cells is substantially higher than that of human cells. Isolated cell nuclei or chloroplasts and mitochondria from tissue homogenates are more tolerant for osmotic changes. DNA ploidy analysis is probably the most common flow cytometric assay used in plant science and breeding (2). The DNA pattern analysis of diploid and tetraploid calli and hairy roots make following the transformation process in tissue culture possible (3). The use of bead beating was suggested by Roberts to prepare suspensions of nuclei from fresh leaves, herbarium leaves, petals, and pollen for flow cytometry (4). Based on this technique, a relatively gentle homogenization procedure was developed by Cousin et al. (5) for releasing nuclei while leaving the leaf tissue largely intact to avoid producing in excess cellular debris. This is one part of the method that the authors developed or improved in order to assemble them to a whole work-flow of preparation and analysis for high-throughput DNA cytometry. Fast and efficient autofocusing is a prerequisite for automated imaging, slide-based cytometry, and high-content screening. Precise autofocus overcomes problems that include mechanical instability, movement of live specimens, variable thickness, drift (e.g., due to thermal expansion), and irregularities of biological substrates. Reflective positioning using laser-based methods (6) can be faster, but finding the best focus by measuring the resolution of the images is more direct and can produce sharper images, especially with higher NA objectives. Varga and colleagues (7) focused their work on an algorithm that selects the best focus position based on the sharpness value of the image. A higher sharpness value means a better focused image. Sharpness calculation is based on pixel value differences. Varga et al. have implemented a special algorithm in the slide-based microscope system to lower noise and cancel small artifacts (7). The image is shrunk by averaging a square of pixels. Then, the algorithm goes through the image and calculates the difference between every pixel and its neighbor to the right. The fifth power of every difference is calculated and added to the sharpness value of the image. If, for example, the difference of two pixels was 10, then 105 is added to the sharpness value. From two images with different exposure times but equivalent sharpness values, the one with shorter exposure was selected as the better focused. This was taken into account, the calculated sharpness values were divided by the exposure time. Weinigel et al. (8) used an alternative and elegant approach, namely, chromatic aberration. Chromatic aberration offers a potentially convenient method for simultaneously sampling the axial specimen space to speed autofocus. With color CCD cameras and common white light sources, the authors asked if axial chromatic aberration can be utilized to acquire multiple focal planes simultaneously and if it can be controlled through a range sufficient for practical use. For proof of concept, they theoretically and experimentally explored the focal differences between three narrow wavelength bands on a 3-chip color CCD camera. Coplanar glass inserts of various thicknesses and dispersions into the light path of the microscope were used to alter the amount of chromatic aberration and thus change the axial differences between the respective R-G-B focal planes. The authors state that chromatic aberration can be applied to speed up autofocus. Novel and inexpensive solid state laser technologies nowadays open the opportunity to build multiple laser flow cytometers that provide the optimal excitation wavelength for various fluorochromes. Habbersett et al. (9) demonstrated 2 years ago that commercial laser pointers can be used as light sources for FCM yielding acceptable quality of data. (The same group presents in this issue an inexpensive microcontrol-based data acquisition system (10)). The group of Telford explored last year, the applicability of solid state yellow and orange laser in flow cytometry (11). Now, the same group reports here of a new class of fiber lasers emitting at discrete wavelengths between 515 and 560 nm (12). Their specially built 550-nm green fiber laser has performance properties suitable for flow cytometry as they demonstrate by extensive evaluation in both cuvette and jet-in-air flow cytometers. Human regulatory T-cells, Tregs, are an important minor subpopulation of helper T-cells. There is a realistic hope that Tregs may render novel biological immunosuppressant that could sometime replace conventional drugs (13). Treg cells are often defined as CD31CD41CD251Foxp31 positive T-helper cells (14, 15). The expression of intracellular CTLA-4 and Foxp3 is positively correlated with surface CD25 expression. Higher levels of intracellular Foxp3 and CTLA-4 and lower levels of surface CD127 consistently distinguish CD25hi cells from CD25int/low and CD252 cells. More recent studies demonstrated that Treg cells also express low levels of CD127 marker (IL-7 receptor). Now Law et al. (16) made a comparison of six different anti-human Foxp3 clones (PCH101, 236A/E7, 3G3, 206D, 150D, and 259D/C7) to optimize Foxp3 staining. They also tested five different buffers from different vendors. This systematic review of commercially available antibody/buffer pairs showed that the variability of the staining depended not only on the clone of anti-Foxp3 antibody but also on the staining buffer composition and fluorochrome used. Dr. Jozsef Bocsi, Heart Center Leipzig, is acknowledged for his help with this editorial.
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Attila Tárnok (2009) studied this question.