Visiting the plant kingdom

Abstract

way flow cytometers are usually configured is that thedimension of the flow orifice (50–100 lm) and the isotonicsheath fluid are optimized for the analysis of live human cells.Animal cells have similar sizes and osmolarities as humancells; therefore, they can be measured with the same setup andwithout substantial problems. By contrast, cytometric plantcell analysis is a challenge for several reasons. The rigid plantcell wall gives the cells a special, elongated form so that theycan reach sizes that exceed the usual orifice size of a typicalflow cytometer. Plant cell walls are highly organized fiber-laminate extracellular structures with strong anisotropy intheir shape and growth, and thus, polarization characteristicshave been widely used in revealing the anisotropic features ofthis complex cellulose-based structure (1). The removal of thewall leads to protoplasts with spherical form but the osmoticpressure of such cells is substantially higher than that ofhuman cells. Isolated cell nuclei or chloroplasts and mitochon-dria from tissue homogenates are more tolerant for osmoticchanges. DNA ploidy analysis is probably the most commonflow cytometric assay used in plant science and breeding (2).The DNA pattern analysis of diploid and tetraploid calli andhairy roots make following the transformation process in tis-sue culture possible (3). The use of bead beating was suggestedby 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 homogenizationprocedure was developed by Cousin et al. (5) for releasingnuclei while leaving the leaf tissue largely intact to avoid pro-ducing in excess cellular debris. This is one part of the methodthat the authors developed or improved in order to assemblethem to a whole work-flow of preparation and analysis forhigh-throughput DNA cytometry.Fast and efficient autofocusing is a prerequisite for auto-mated imaging, slide-based cytometry, and high-contentscreening. Precise autofocus overcomes problems that includemechanical instability, movement of live specimens, variablethickness,drift(e.g.,duetothermalexpansion),andirregulari-ties of biological substrates. Reflective positioning using laser-based methods (6) can be faster, but finding the best focus bymeasuring the resolution of the images is more direct and canproducesharperimages,especiallywithhigherNAobjectives.Varga and colleagues (7) focused their work on an algo-rithm that selects the best focus position based on the sharp-ness value of the image. A higher sharpness value means abetter focused image. Sharpness calculation is based on pixelvalue differences. Varga et al. have implemented a specialalgorithm in the slide-based microscope system to lower noiseand cancel small artifacts (7). The image is shrunk byaveraging a square of pixels. Then, the algorithm goes throughthe image and calculates the difference between every pixeland its neighbor to the right. The fifth power of every differ-ence is calculated and added to the sharpness value of theimage. If, for example, the difference of two pixels was 10,then 10