Morphological Analysis of Cell Growth Mutants in Physcomitrella - Plant Cell Morphogenesis: Methods and Protocols - page 199

Fig. 1 Representative images of a GUS-RNAi control plant ( a ) and a plant under-
going myosin XI-RNAi ( b ). The images were acquired with a fl uorescence stereo
microscope and 1× lens. This imaging confi guration allows to visualize both the
chlorophyll auto-fl uorescence and the GFP signal, which in this case is absent
from the cell nucleus. Scale bar 100 μ m

GUS-RNAi control plants (121031GUS-RNAi.tif). The second
data set has 30 images from a myosin XI-RNAi transformation
(121031MyosinXI-5UTRi.tif). The myosin XI-RNAi plants are
smaller and do not show polarized growth. These plants represent
an extreme example of polarized growth inhibition, but they also
remain alive showing strong chlorophyll fl uorescence that is help-
ful when performing morphological analysis.

3.6 Morphology
Analysis Macro

To quantify the morphology of the regenerated plants, an ImageJ
macro is provided. In brief, the macro takes a stack of images cor-
responding to one RNAi condition and quantifi es each individual
plant to yield data including area, perimeter, solidity, circularity,
convex hull area, and convex hull perimeter. Although the macro
is mostly automated, there are some important manual functions
that the user will need to input in order for the data processing to
be completed.

1. Make image stack. The user must fi rst have the images in an
ordered stack. This can be done by dragging a folder contain-
ing individual fi les from a window into image J and creating an
image stack. The macro works on all image types including
RGBs, RGB stacks, and 8-32 bit grey-scaled images.
2. Name your image stack. Since some of the macro's code is
name sensitive, it is important to name your fi le in compliance
with the following format. Include the date as the fi rst six
characters of the fi le name. The remaining characters can be
whatever that suits the user and will be displayed on the results
table. Although this format is recognized by the code, it is not
essential for data processing.