Recently, we have done some tests of azidoblebbistatin on hela cells using A1R multiphoton microscopy (Nikon Ti-E). This instrument own 800 nm wavelength with 2700mW power and 900nm with 1900mW. We have test the two parameters with different stimulate time from 1s to 8 min. But we did not observe the stimulate area to turn green. So we want you give us a detailed protocol, which containing the concentration of the drug, stimulate time, the wavelength and its power.
(Anna Rauscher, Phd answers)
Based on recent experiments the suggested wavelength for 2-photon irradiation is 860 nm. Based on the dimensions of the 2-photon laser focus, suggested step size is 0.1 um/ pixel.
In most cases fast scanning is recommended.
It is necessary to optimize the followings:
- intensity of laser and irradiation time
- azidoblebbistatin concentration
Below you find an example of a working protocol on HeLa cells for a 2.4 um x 10-15 um area:
5 uM azidoblebbistatin (incubation time: 15 mins)
3.5% laser intensity (~20 mW in the focal spot) 860 nm
166.1um/ms scanning speed
0.1 um/ pixel step size
500 ms scanning of the area, repeat 10 times, every 2 sec
It is important to check if your cells tolerate this laser intensity to make sure that no photodamage occurs. If your cells tolerate more, you may need to increase the intensity. In the above example 5 uM azidoblebbistatin was applied, which fully inhibited myosin in the irradiated area after irradiation. Without irradiation, 20 uM para-nitroblebbistatin had to be used for full inhibition.