Biology Reference
In-Depth Information
2. For VAEM, cut a piece of agar containing the seedlings (width
1.5 cm, length according to the seedling size). We usually
observe two or three 5 days old plants per agar piece. Put a
drop of water on the chambered slide and put the piece of agar
placing the seedlings in contact with the glass, avoiding bub-
bles (the piece of agar helps to press the seedling tissues in
contact with the slide). Remove excess water by gently touch-
ing the edges of agar with paper tissue.
Follow the recommended standard procedures for your microscope.
3.2 Image
Acquisition
1. In case of CLSM, we record single slices (capturing an area of
the cell cortex adjacent to the cover slip) and Z series using the
following settings: excitation laser (488-nm argon) intensity
25 mW, detector window using the GFP preset values (
see
Note
8
), XY fi eld size 1,024 × 1,024 pixels, line averaging of 4-8
times, Z series interval 0.7-1
2. In case of VAEM, we use 400 nm peak excitation for GFP
constructs, 150-210 ms exposure time, frames taken in 0.5 s
intervals over the course of 2 min, and color depth of 8 bits.
μ
m, and color depth of 12 bits.
3.3 Measuring
Cytoskeletal Network
Density on CLSM
Stacks
1. Obtain serial optical sections (XYZ, i.e., Z-stack) of the corti-
cal cytoplasm of a cell expressing a suitable marker by CLSM.
In general, we aim towards imaging about 7-10 plants per
sample, with 5-10 cells per plant evaluated (
see
Note 9
).
2. Open the stack by dragging the microscope-generated *.lei
fi le onto the ImageJ window; use the “open as hyperstack”
option in the dialog box. Skeletonize the original serial optical
sections (Fig.
1a
) using Plugins > kbi > Kbi_Filter2d (set fi lter:
lineFilters and parms linemode: thinLine; Fig.
1b
). Generate a
Z projection (Image > Stacks > Zproject) using the maximum
intensity option and save the resulting image as a new 8 bits
*.tif fi le (Image > Type > 8bits; Fig.
1c
).
3. Select the area to be analyzed (a whole cell or a well-focused
region of the image) manually by ROI selection
(Plugins > ROI > Specify ROI). To specify multiple ROIs of
the same size and shape within an image, you may duplicate
the selected ROI (right mouse click > duplicate).
4. Evaluate the fi lament density within the ROI by estimation of
the GFP signal occupancy, i.e., the fraction of pixels constitut-
ing the skeletonized fi laments relative to the total pixel num-
ber of the ROI. Count pixel number of selected ROI using the
macro: hig_255counts.txt (Plugins > hig_255counts). The
occupancy value is proportional to the overall fi lament density
in the cell region of interest and was shown to serve as a useful
indicator to evaluate, e.g., the changes in the microfi lament
organization induced by physiological processes, treatments
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