To analyze the tubulin dynamics during spindle formation, Drosophila S2 cells were transfected with mEos2-labled tubulin. By local irradiation with UV laser light (405nm) the green fluorescent mEos2 photoconverts to a red fluorescent dye. In contrast to photobleaching, where the fluorescent signal is locally depleted, the photoswitching allowed the independent observation of two different tubulin fractions in space and time.

 

 

 

 

 

Movie1: Photoconversion of mEos2-labeled tubulin in the spindle of a Drosophila S2 cell. The video is sped up 20-fold from real time.

Setup:

• Microscope: Standard widefield microscope
• Objective: 100x NA 1.4
• 405 & 473 nm diode laser

Rapp OptoElectronic Components:

• UGA-40 – point scanning device (integrated in µ-manager)

 

 

Data taken from:
Kurt’s Microscopy Blog
http://nic.ucsf.edu/blog/2014/04/photobleaching-and-photoactivation/

Kurt Thorn (1) & Nico Stuurman (2)

(1) Nikon Imaging Center (NIC) at University of California – San Francisco (UCSF)
(2) Vale Lab at University of California – San Francisco (UCSF)