Morphological Analysis of Cell Growth Mutants in Physcomitrella - Plant Cell Morphogenesis: Methods and Protocols - page 201

have to select a unique fi le path for each fi le they select.
Important: clicking cancel while selecting the fi le path will
abort the macro.
9. Selecting the fi les to save. Once the saving details have been
determined, a dialogue box will appear asking the user, “What
fi les would you like to save?” These fi les include Binary
Montage (8 bit Tiff fi le that displays each thresholded plant
sorted by area in a montage), Normal Montage (8 bit Tiff fi le
that displays plants prior to thresholding sorted by area in
montage), Phenotype Results Table (text fi le that contains all
the image computations including circularity, solidity, thresh-
old, perimeter, convexity, convex hull area, convex hull perim-
eter; see Note 14 ), Sorted Image Stack (8 bit Tiff fi le that
contains the images sorted by area), and Sorted Binary Image
Stack (8 bit Tiff fi le that contains the binary images sorted by
area). Following the completion of the macro, all of these fi les
will be closed so it is important to save all of those the user
fi nds relevant.
10. Macro confi rmation. After the fi les have been selected, Image
J will display a message saying “Macro is complete.” This
statement appears once the fi les have been saved and the macro
is complete. Be sure to review the data and image fi les to
ensure that the binary images are representative of the plant's
area and make sure the macro has run properly.

1. For routine analysis, three independent transformations for
each construct or complementation set are suggested. This
should provide measurements for 100-200 plants.
2. In our experience the variance between experiments does not
change signifi cantly and the values for the individual plants can
be averaged for their comparisons [ 3 , 5 ].
3. When more than two conditions are compared, we use an
ANOVA test using a Tukey post hoc test for the comparison
between means. For the comparison of only two conditions, a
t -test can be used.
4. For the comparison of area values, we divide the total area of
each plant by the average area of the RNAi control plants for
each experiment. This provides a consistent value that is not
altered by changes in fl uorescence intensity resulting from
day-to-day fl uctuations.
5. The area and circularity values follow a log normal distribu-
tion, so the standard statistical analyses such as the t -test and
ANOVA should be performed on the natural logarithm of the
data which results in a normal distribution.
6. The solidity values follow more closely a normal distribution
and can be analyzed without conversion.

3.7 Statistics