Integrated genetic and computation methods for in planta cytometry
Fernán Federici, Lionel Dupuy, Laurent Laplaze, Marcus Heisler & Jim Haseloff
Nature Methods, Advance publication April 2012 (DOI 10.1038/nmeth.1940)
There is a critical need for improved techniques for the quantitative measurement of biological parameters within living systems. This new publication describes the integrated use of advanced genetic and imaging techniques for automated quantitative analysis of cell growth and genetic activity in living plant tissues. With this new technique, fluorescent protein markers can be used to identify cells, map cell geometries and provide ratiometric measurement of gene expression within intact tissues. The procedure, which we term in planta cytometry, allows the measurement of cellular properties in intact tissue, while retaining the cellular context for further studies.
The routine consists of four steps. (i) A genetic marker is used for the specific labeling of nuclei and cell membranes in transgenic plants. (ii) Fluorescence microscopy and image processing allows the counting of cells and extraction of positional information using a particle search algorithm (iii) Identified nuclei are used to initiate an active contour segmentation algorithm that uses a fluorescent signal located at the plasma membrane to extract information regarding size, shape and topology of cells. (iii) Cell-specific gene expression is quantified by ratiometric measurement of spectrally distinct nuclear fluorescent proteins expressed under the control of different regulated promoters. This new cytometry technique allows one to quantitatively measure cell activities within living organisms, and to precisely reconstruct cellular dynamics - and to produce a numerical description that can be used to inform computer models.
University of Cambridge.