Manual Mass
Measurement
on
Earthworm
Heamoglobin
and
TMV Standard
|
-
Start PCMassXX.exe and verify that image NK737537 is read in. The header should be
displayed on a green background in the bottom left portion of the image.
-
Strike the <2> key. Observe that all particles and a significant area of
the background are masked out in blue. The 'picture background' is computed
using the remaining pixels. The results are tabulated along the bottom of the image,
showing mean, SD and num pts for each of 16 sub-areas for both large angle and small
angle detectors. This image has a relatively clean background, so there are no
complications to deal with at this point.
-
Move the mouse pointer. The screen should clear and three concentric circles
should appear with a size specified by the default model number 35, earthworm hemoglobin
face-on. Continue moving the mouse pointer until it is centered on one of the round,
slightly hexagonal particles. After a brief delay, the particle of interest should
appear in the zoom window below the image. Below and to the right of the zoom window
should be a fitted model and immediately to the right of the zoom window should appear
a difference image.
-
If the circle does not appear to be centered, move it left or right and up and down
with the arrow keys.
-
Strike the <t> and <y> keys to decrease or increase the circle radii.
Note the effect on the measured mass, listed below the zoom window.
-
Press the left mouse button, strike <ENTER> or hit the <=> key to enter a
measurement. A single circle should be drawn with the sequential measurement number
and the mass to the right of the particle.
-
Repeat this for the remaining face-on particles which are separated from other objects.
Stop after 10 if you find this tedious.
-
Strike the <m> key to select a new model. Move the mouse until model number 21 (TMV)
is highlighted and press the left mouse button.
-
Select TMV segments as above. The automatic search should align the box at the correct
angle if it is fairly close to the centerline. If the circle markings are obscuring your view,
strike the <0> (zero) key to restore the screen.
-
Strike the <m> key to select a new model. Move the mouse until model number 49
(EHb side) is highlighted and press the left mouse button.
-
Select side views as above. The automatic search should align the box at the correct angle
if it is fairly close to the centerline. Note that the ends of the box are now closed and
total mass is measured (instead of mass per unit length).
-
Strike the <i> key to list the measurements to date. Strike it a second time
if there are more than 25. Scroll using the up/down arrow keys. The particle corresponding
to the first measurement in the list should be marked in the image with the circle or rectangle
used for the measurement. Particles can be 'removed' by hitting the <delete> key
which changes the mass to a negative value, removing them from the averaging process.
(A second hit on <delete> switches the mass back to positive.)
-
Strike the <o> key to observe the results. Only the first three rows should
be populated. The column headings identify the models. The first row is all measurements
you entered, giving mean, SD in percent and number of measurements. The second row is a
subset passing a test for clean background around the particle or along the sides of the segment.
The third row summarizes particles or segments passing a pre-defined set of quality tests.
Move the highlight with the arrow keys. A histogram for that class of measurements will appear
on the right panel, along with scattergrams describing the population.
-
Strike <ENTER> to mark all particles of the selected type. Hold down
<SHIFT> and hit <ENTER> mark the particles and list masses.
-
Hold down the space bar and move the mouse to the right panel. A pointer should appear
under the large histogram. Particles in the bin above the pointer should be circled in white
while the remainder should be circled in red.
-
In the file menu, select save *.smm. This saves all current measurements. The same happens
when you go to another file or shut down the program. With a text editor such as Notepad, open
\\Mass_Meas\nk737537.smm and view the tabulated measurements. These are suitable for import
into a plotting program such as SigmaPlot. Note that the masses are not scaled and are in kDa
for particles, Da/A for mass per unit length and Da/A**2 for mass per unit area
-
Go on to the next file by striking the <1> key. Return to NK737537 by striking the
<-> key. Strike <i> or <o> to verify that the measurements
were read back in.
|
