Calibration For 3D-Storm - Nikon N-STORM Operation Manual

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
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