I am currently working on measuring the 3D PSF(stack several 2D images on different focal plane) of the confocal microscope by using PZT stage to scan the sample. However, it seems that there are shifting between image and image(see following GIF). To be specific, the psf pattern should be in the same position as I change the Z axes position. However, in my result , the psf pattern is running around . I already checked the servo of the PZT stage, it is working normally. Has anyone encounter this kind of problem before? Or is there a proper way to solve this problem?
My equipment are listed below:
-0.1 micrometer latex beads as a specimen
-473nm light source
-100x,0.8NA Air lens
-Piezo stage to scan the specimen
-Single-photon avalanche diode as the detector
Thank you all for reading.