Nikon N-STORM Operation Manual page 34

(4) Configure the N-STORM acquisition settings.
Specify the folder in which the ND2 file is saved, as
well as the file name.
Deselect the [Activation] checkbox. (During
calibration with fluorescent beads, the activation
laser is not used.)
Set the output of the imaging laser (647 nm) to about
10%.
(5) Click [Run Now] to acquire images.
The dialog box on the right is displayed. Do not
perform any operation with this dialog box until
image acquisition is completed.
201-frame image acquisition is automatically
performed according to the following steps.
• 20 frames at the focal position
• 161 frames from -400 nm to +400 nm with the
focal position regarded as being the origin (Z
stack)
• 20 frames at the focal position
(6) When image acquisition is completed, click [Cancel]
in the [N-STORM Z-Calibration] window to close the
window.
3 Click [Analysis GUI] to display the N-STORM analysis
window.
4 Click [File Open]
A dialog box for selecting a STORM image is displayed.
5 Select the image file (ND2 file) acquired for calibration
and then click [Open].
6 Make sure that the data is appropriate for calibration.
Check that the bright point image is not elliptical but
circular in the first and last 20 frames.
Automatically adjusting the contrast
The contrast is automatically adjusted by right-clicking
the image and then selecting [Auto Scale for Full Image].
If the bright point image is elliptical in the last 20
frames
If the bright point images are circular in the first 20
frames, but they are elliptical in the last 20 frames, this
may be because the specimen position has been shifted.
Make sure that the specimen is fixed properly and then
acquire images again.
Chapter 2
STORM Microscopy Operation
30