Dear Confocal Microscopists,
 
Barry Burbach recently left a message regarding the alignment of his
Bio-Rad MRC-600 microscope.  When he examined the PSF of sub-resolution
beads he found them to be tilted to one side.
 
Since the PSF is tilted to one side, it sounds like the beam is
entering the microscope off-axis.  The microscope may be aligned
correctly internally, and the scanhead may be aligned correctly
internally, but the "marriage" between the scanhead and the microscope
is off-axis relative to each other.
 
The way to remedy the situation is to re-align the scanhead so that the
beam is exiting the scanhead off-axis.  This can be done by using an
alignment tool that screws onto the eyepiece in the scanhead.  This
alignment tool has two pinholes in it, one slightly larger than the
other because the beam is diverging as it leaves the scanhead.  The
internal optics in the scanhead (mirror M1 and the three point
adjustment on the filter block closest to the laser) can be adjusted so
that the beam is leaving the scanhead on axis, i.e. it is centered
through the two pinholes.  This is done while the beam is parked (one
easy way to park the beam is to use a Zoom=999.  This will both park
the beam and center it.).
 
Once the beam is leaving the scanhead on axis, the scanhead and the
microscope must be matched up so that the laser beam is centered into
the turret prism on the microscope, without making any adjustments to
the internal optics of the scanhead.  This involves repositioning the
angle of the scanhead on it's mounting bracket.  If instead mirror M1
is adjusted to bring the beam into the center of the turret prism, the
sytem will no longer be scanning on axis.
 
Since most people do not have the alignment tool that screws onto the
eyepiece in the scanhead, there is an alternative:  when the system is
staged in production the galvos are installed so that the laser beam is
centered on both galvos.  At this point the galvos are also adjusted so
the beam is on axis.  If you can recreate this situation by adjusting
the internal optics of the scanhead so the beam is centered on the
galvos, it is most likely that the beam will also be on axis.
 
If anyone has more questions they can send an e-mail to my address at:
[log in to unmask]  Or call me at 510-741-6780.
 
Best regards,
 
Jim Hayden
Bio-Rad
Confocal Imaging Product Specialist