CONFOCOR
3
Carl Zeiss
Action Buttons
ConfoCor 3
112 M60-1-0025
e
02/2010
The data of the
Count rate scan
window can either be copied to the clipboard or written to a text file. A
click in the
Count rate scan
window with the right mouse button will open the context menu for line or
two dimensional scans.
•
Select the line
Copy data to clipboard
with a click of the mouse if you want to insert the data into
other WINDOWS programs directly via the clipboard. The Paste function enables you to insert these
data directly into the required program.
•
Select
Write text to file
to save the data in an external ASCII file (.txt).
You can zoom in by spanning with the mouse left mouse button held down a region of interest (not
available for a frame plot). You can rest the zoom by selecting the line plot by defining the zoom area
with.
Z-Scan for finding a labeled cell membrane
The Z-scan is especially appropriate to position the confocal volume on a cell membrane. Due to the
shape of the confocal volume, the membrane should not be approached from the side but instead rather
from the top of the cell. Please note that it is better to use the upper membrane for measurement, since
the lower membrane might be too close to the glass bottom surface resulting in disturbing reflections.
Besides using the
Z Scan
you can also position the membrane manually, albeit with less precision.
(1)
Manual Focusing on the Membrane
•
Acquire an image of the cell.
•
Place the focus above the cell and scan continuously.
•
Focus slowly down until you see a fluorescence signal.
•
Stop focusing.
Due to its high sensitivity ConfoCor 3 can still detect signals that cannot be imaged by the LSM. If the
signal is too low for imaging, the membrane can still be detected using the
Z Scan
feature.
(2)
Focusing on the Membrane by a Z Scan
The
Z Scan
measures the count rate at previously defined x, y positions. It is performed without table
movement, which is at the fixed position of the laser beam.
•
Acquire an image of the cell, focus through manually and select an equatorial section (middle of cell).
Optional: Perform a
Z Stack
that includes the membranes of the cell with the LSM and select an
equatorial section.
•
Go to the
Measure
tool to
Acquisition
and the
Positions
submenu. Position the region of interest at
the site of the fixed laser beam in
Current Position
using the
Crosshair
or use the crossline in the
LSM image
to mark the position. In the latter case,
Scanner
must be active, since the
XY-Stage
will
approach the marked position only during a measurement!
•
Go to the
X-Y-Z Scan
tool and activate
Z
. The
Z Scan
display shows the current Z position of the laser
beam.
•
Select the
Start
and
End
Position
and enter the
Step Distance
. If you have chosen an equatorial
section going 10 µm up and 10 mm down from the displayed current position are good values to start
with. Also a step distance of 0.5 µm should work well.
