Hi,
I have been recently trying to acquire 3d images from an N-STORM and was having trouble with the 3D calibration in thunderSTORM. I have a separate image file of crimson beads for the calibration process and have attached image files showing the large sigma x and sigma y values I get after running the analysis.
The sequence in which the frames were acquired by the instrument seems to be arbitrary and I think this may be causing the the large distribution in sigma values. I have outlined the sequence in which the frames are taken for the crimson bead file.
First 20 frames at focus point (round beads)
Moves to -800nm and then does 80 x 10nm steps towards focus
1 frame at focus point
80 x 10nm steps to +800nm
20 frames back at focus point
Could this sequence be causing the issue? If not, any sort of input is appreciated.
Thank-you,
Gopal.