Imaging Suter Lab 01/20/10

Microtubule and actin dynamics processing and analysis

Microtubule dynamics analysis

FIRST, CALIBRATE ALL IMAGES TO THE CORRECT CALIBRATION FILE,

Usually it is “Cascade II 1.5 x”

A) Image processing

Upload image: control, Q

Select specific images: stack  keep planes  low range=1st image used, step
size = 1, high range= last pic used

Microtubule images (stacks or images) are processed with spatial filters in Metamorph
7.0 in the following sequence:

(1) “Process” “Basic Filters” “Operations” “Low pass” filter width 4 x 4 pixels
And click plane button and select all planes
(2) “Process” “Detect Edges”  Filter “Laplace 2” And click plane button and select all
planes

(3) “Process” “Basic Filters” “Operations” “Low pass” filter width 3 x 3 pixels.
MAKE SURE CORRECT VID IS SELECTED

Alternatively, run the journal “MT and actin processing.jnl” on the timelapse stack. The
resulting stack is ready for MT speckle analysis.

B) Microtubule montages –don’t use

From the processed image stack, choose MTs exploring the P domain for speckle
analysis. A ‘good’ MT should have clear speckles against background, not cross paths
with other MTs or intrapodia (which would retain a lot of free tubulin and contribute to
noise), and preferably have distinct growth/collapse phases.

Crop out the MT: Use the box region tool to select the MT, and “Edit””Duplicate”
“Stack with zoom”. Keep only the planes where the selected MT is clearly seen by using
the “Stack””Keep planes” function. If the MT is skewed (example in figure below),
measure the skew angle with the multi-line tool and rotate the cropped MT stack using
“Display””Rotate” Linear Interpolation. Further crop the rotated MT stack so that the
MT is clearly in the box region.

“Stack with zoom” Rotated, and

further select
Measure region for
this angle cropping
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Make into montage

Montage the MT: “Stack””Montage” Rows: 1, Draw sequence number.

IMPORTANT NOTE ABOUT THE MONTAGE TOOL:
The resulting montage IS NOT CALIBRATED. You must apply calibration to the final
MT montage. Sometimes the montage tool makes the montage from your stack with “as
is” zoom, i.e. if your MT stack is at 200% zoom, the resulting montage will be 2x the size
(but shown as 100%). Since it is not calibrated, applying calibration at this point will
result in the WRONG calibration. To prevent this, it is best to always make your
montage from a 100% zoom MT stack.

C) MT assembly and translocation dynamics analysis

First, pick out clearly discernable speckles within the MT, use the line tool or multi-line
tool to trace the speckle from one frame to the other. This will be the internal reference
speckle to be used in both assembly and translocation dynamics.

To log translocation, measure the distance at which the reference speckle moves
relative to its position in the previous frame. Do this for every frame. If the slope of
internal speckle movement is less than 2 pixels between 2 sequential frames, I count it
as translocation pause.

To log assembly/disassembly, measure the length of distance from the plus end of the
MT, to an internal speckle for each frame. Be careful that a “true” assembly event
should see a new speckle at the plus end. Sometimes the tip-reference speckle
distance changes as a result of image processing of the speckles, and/or the MT is
being stretched /bent somehow. These do not count as assembly/disassembly events
and should only count as assembly pause.

Log translocation/assembly measurements from frame to frame for every frame. I do

this with pen and paper first, and then typed into excel.

Original montage and regions:

Imaging Suter Lab 01/20/10

Data typed into excel

“+” is assembly or forward translocation (also color shaded pink)
“-“ is disassembly or retrograde translocation (also color shaded pink)
“P” is assembly or translocation pause (color shaded yellow)
“Time 1” and “time 2” (in assembly log columns) are plus-end/internal speckle distance at
frames n and n+1, respectively.
“distance” (in translocation log columns) is the distance an internal reference speckle moved
over 1 frame.
Columns AZ and BG calculate the rates of assembly/disassembly and translocation rates over
each frame interval as microns/minute.

Assembly log Translocation log

Imaging Suter Lab 01/20/10

From this raw data table, one can calculate the following parameters:
MT assembly dynamics: 1) Assembly and disassembly rates (this must be time-
weighted) 2) percentage of total observed time spent in assembly/disassembly/pause.

MT translocation dynamics: 1) forward and retrograde translocation rates (this must be

time-weighted) 2) percentage of total observed time spent in translocating
forward/retrograde/pause.

Example excel sheet: “Dec_MT_dynamics_LATENCY_INTERACTION.xls”

For documentation, save all of the regions using “regions””save regions”. This way,
you may load the saved regions on the corresponding image for later use or future
reference. Also note the frame span of the analyzed MT from its original timelapse stack
(e.g. “MT #1 is from frame 9-22 in the original stack”).

Actin retrograde flow kymograph analysis –start here

A) actin image processing

Process the actin timelapse using the MT processing filters mentioned above (“MT and
actin processing.jnl”), or simply use a 4 x 4 pixel width low pass filter---whichever gives
less noise and better kymographs (play around). REMEMBER TO CALIBRATE
IMAGES.

Calibration: measure> calibrate distances > load from file > go to data depot, lab
organization, current camera calibration, cascade II (1.5=90X, 1=60X),

B) Linescans and Kymographs

Choose a region in the P-domain where actin speckles are clear (usually filopodium
have better actin signal/noise). Using the line tool, make a line on the actin bundle
starting from the leading edge to the C-domain*.

Open “Stack””Kymograph”. I typically use “line width 3”, “average” or “maximum”, and
background subtraction “minimum” for better kymograph signal/noise. Play around
these settings for best kymograph image quality. Press “create” while the line on the
stack is an active region.
Imaging Suter Lab 01/20/10

NOTE: The kymograph function in Metamorph 7.0 has a bug, please avoid the
following:
MetaMorph 7.0 introduced a problem with the Linescan and Kymograph functions
where the Linescan and Kymograph will measure incorrectly under the following
conditions:
The Line Width is greater than 1, and
The Direction of the line drawn is from Lower Right to Upper Left
http://support.meta.moleculardevices.com/docs/t20062.pdf (for more info)

On the created kymograph, choose clearly discernable slopes and place a line along
the slope from left to right. The data table in the kymograph box should automatically
show the velocity (as micron/sec), in this case this velocity is actin retrograde flow.
Open log (dynamic data exchange, i.e. excel), and log the data to excel using F9.

Create as many kymographs and measure slope (velocity) as needed. Note that the
kymograph function can only automatically measure velocity on the last created
kymograph, so do not make several kymographs at once and attempt to measure slope
velocity from each. Instead, make kymographs and measure velocities one by one.