Hello. I have a question about the relationship between laser alignment and the resulted image resolution.
I imaged DAPI channel of a fixed brain section at 20x/0.8 on a point scanning confocal. It’s 1 AU on the green channel, not Nyquist sampling, but good enough resolution to see sharp edge of the nuclear. Then, I imaged the same slide a few weeks later, reused the imaging setting, and the nuclear edge became blurry. I thought something is wrong with my sample. Then, I tried a different point scanning confocal, and use the same pixel size, and similar laser intensity/other setting. The nuclear edge was sharp again. The screenshot below illustrates the resolution difference (L, sharp edge; R, blurry). The service engineer diagnosed the situation and realigned the 405 laser on the first confocal. That fixed the issue.
In order to take advantage of the full NA of the objective, the collimated laser source must be centered and slightly overfill the back aperture. So if your laser is off-axis (mis-aligned) then it will under fill and reduce the overall NA.
The alignment of the confocal aperture can also have this effect, though most modern confocals have a fixed aperture assembly and use some upstream alignment mirrors to correct for the different laser couplings. Most separate the 405 laser path from the rest of the visible lasers because it is more cost effective to combine that many wavelengths at the dichroic vs. using lenses to do it on a single beampath.
The 880 system in your example has user control of this in the Maintain menu under ‘Pinhole Alignment’. Here you are actually adjusting the laser alignment onto the pinhole rather than the positioning the pinhole itself. Other systems like the Leica I believe have to adjust this at the fiber coupling into the scan head (service only procedure) - but others can correct me if I am wrong on that.
Thanks so much. Got it, this is caused by reduced NA from laser under filling the objective. My problematic confocal is leica, the 880 is the reference in this case.
Sorry I will get off topic here. If I have two invisible lasers, 405 and 730, for the Airyscan alignment, is the 980 align them one by one using the same mechanism (laser rather than pinhole)? I have some conflict information from my service engineer and application scientist.
