Nikon N-STORM Kullanım Kılavuzu - Sayfa 33
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2.5
Calibration for 3D-STORM
To perform 3D-STORM analysis, it is necessary to carry out calibration in advance so that the positions in the Z-axis direction
are analyzed correctly. For calibration, a dataset (ND2 file) acquired through a predetermined method is used. This
calibration associates the positions in the Z-axis direction with the ellipticity ratio of an unfocused bright point image, allowing
the analysis of positions in the Z-axis direction.
1
Make preparations for acquiring 3D-STORM images
according to "2.1 Preparation of the N-STORM
System."
A fluorescent bead specimen is used for calibration.
Recommended fluorescent beads:
TetraSpeck Microspheres, 0.1 µm Fluorescent
Blue/Green/Orange/Dark Red T-7279 (Molecular
Probes)
2
Acquire images for calibration.
(1)
Click [Z-Calibration] in the [N-STORM] control
window. (If [Z-Calibration] is not displayed, it is
displayed by clicking [Advanced].)
(2)
Turn off the PFS.
Click [PFS] in the [N-STORM Z-Calibration] dialog
box to gray out the button.
PFS settings
To perform calibration, it is necessary to turn off the PFS.
(3)
Click [Up] and [Down] in [Piezo Z Movement] to set
the focus.
Chapter 2
STORM Microscopy Operation
29
2-(4)
2-(1)
2-(2)
2-(3)
2-(5)