Different left and right laser power, sheet height and dimming during z-stack acquisition

[ucqfgor]

Hi all,

Issue 1: I'm noticing a difference in laser power between the lasers with the left lasers being dimmer, it's slightly different for different lasers lines too, what's the best way to fix this?

Issue 2: the left and right laser sheets are different heights with the left being audibly quieter (images below)

Issue 3: When using higher magnifications, I notice that the 488 laser power dips for some slices of a stack, the 561 and 647 seem OK though. This seems intermittent and can be repeated at even spacing throughout the sample or sometimes just be one set of slices that are dimmer. Any ideas why this might be?

Any help is much appreciated.

Peter

[nvladimus]

Hi, Peter, Issue 1: The most likely way to fix this is to balance the laser power between channels, physically. If you have 2 separate lasers, one for each arm, follow these tips. For single laser combiner and 2 fiber outputs, you need to measure the laser output (with power meter, or by looking at the beam on paper target), and tweak the fiber adapter knobs to equalize the outputs between two fibers. Double-check the balance between different laser wv, and at the fixed laser power (eg 10 mW).

Power can be also lost in the excitation path due to poor alignment, eg from vignetting, or by beam off-center at the galvo.

Issue 2: Offset of about 1V is quite high. I would suggest to set it to 0 and manually align the galvo rotation position until it is near the image center. Also, 5V amplitude is very high. I would expect it to be 3-4 V max. This is probably alignment and voltage clipping issue.

Issue 3: Well, the darker planes are bleached planes, that's why the effect is stronger for 488 laser. This happens when you look at the sample in Live mode for too long, or with too high laser power.