Hi. My name is Geoff Lambright
and I am going to walk you through the steps to align
a Sutter two-photon imaging system
particularly a Sutter Movable Objective Microscope.
For our Sutter Movable Objective Microscope, we sell an
alignment kit to assist you in aligning your microscope.
This kit consists of an alignment laser
that fits into the objective holder of the MOM,
as well as a pinhole for the Tube Lens
and a pinhole for the Scan Lens that will enable us
to center the alignment beam through the microscope.
We can then setup pinholes on the table
using the centered alignment beam.
Then, as a final step, we will run the imaging laser
through the table pinholes and into the system.
With a final tweak for centering the beam,
the system will be ready and optimized for imaging.
Step One
Assemble the alignment tool with the objective holder
so it can replace the objective in your system.
Step Two
Remove the front cover of the MOM,
and very carefully thread on the pinhole for the Tube Lens.
You'll notice the mirrors on the head of the microscope
are very close to where you are working.
Please be extremely careful not to touch, bump, scratch
or even look at them too closely
They are very delicately mounted on their brackets
in order to reduce stress on the optical elements,
so they are quite easy to break off.
Step Three
Very carefully remove the scan lens cover,
and slide the unthreaded Scan Lens pinhole
over the front of the scan lens.
Now both pinholes are in place
which will be used to align the alignment beam
through the center of the optical path.
If your CCD mirror is in the way,
loosen the scan lens set screw
and slide the scan lens back
just enough to insert the pinhole.
Remember to be careful to return the scan lens
back to its exact position after removing the pinhole later.
Step Four
Use both the translational adjustment
(the top thumb screws)
as well as the angular adjustment
(the bottom thumb screws)
to guide the laser beam through the pinholes.
At this point, the alignment beam should be going
through both pinholes and onto the scanning mirrors.
On the Galvo scanner mirrors,
you can see the beam positioned on the mirrors.
On Sutter's enclosed Resonant Scanner,
or on Vidrio's Rapid Multi Region scanner,
these will be inside the enclosed box
but you will still be able to see the beam
exiting the periscope once the scanners are centered.
It is important to note at this point,
that the scanning mirrors MUST BE CENTERED for this to work!
If the scanning mirrors are not centered,
we will not have anything close to a good beam alignment
when we finish.
So, Step 5 is to center the scanning mirrors.
For centering Galvo-Galvo mirrors
start with the controller powered off.
On the back of the controller,
unplug the BNC cables for the X and Y control,
but make sure the grey scanner cables
that go from the controller to the scanner stay connected.
Now, power the controller back up
and the mirrors should make a little jump
to their centered position
about a second or so after flipping the controller on.
For Resonant scanners,
also start with the controller powered off.
On the back of the MDR-R controller,
unplug the Y BNC cable;
then additionally unplug the resonant scanner cable
from the back of the controller.
Then power it up
and the scanners should jump to their centered position
about one second after flipping the switch back on.
At this point,
With the scan mirrors sitting
in their centered position,
the beam should be pointing straight down the periscope tube
coming out of the center of the periscope tube,
and reflecting off the mirror at the bottom of the tube
across the table
toward a mirror that would direct it then
towards the imaging laser.
It may be interesting to note that if this was a new install,
this is when the MOM main rail height would be adjusted
so that the beam coming out of the back of the periscope
was exactly at the same height
as the beam coming out of the imaging laser.
Step Seven
Now we can put the pinholes on the table,
marking our alignment beam coming out of the periscope.
Generally, the 45-degree mirror
on the bottom of the periscope is not adjusted,
unless the beam is not coming
straight out the back and horizontal.
Tighten the pinholes precisely around the center of the beam.
At this point, it is likely that the alignment beam
is reflecting off the first mirror behind the scope,
potentially even off the second mirror,
and then making its way back toward the laser.
I like to use this as my opportunity
to reposition any mirrors necessary
by aligning the beam backward toward the imaging laser.
This will get my imaging beam closer to the target
when we switch over to the imaging laser
and work it back through the microscope.
Step Eight
Remove the alignment laser.
Once your imaging laser is on and warmed up,
open the shutter to begin using the imaging beam.
Always wear safety glasses
when working with the powerful IR imaging beam.
An IR viewing card will assist in safely viewing the beam.
If your laser has an alignment mode, use it.
An alignment beam is generally a much lower power beam
that is safer to work with, and it is in the visible red
so it is easier to see than the IR beam.
If your laser does not have an alignment beam,
tune it to its shortest possible wavelength
at which it will stably operate.
Use your Pockels cell
or half wave plate and polarizer combination
to reduce the output power of the beam to a very safe level.
Step Nine
Use the last two mirrors before the periscope of the microscope
to adjust the beam into and through the two table pinholes
you just fixed to the table.
Once the imaging laser is through the center
of those two pinholes, you are very close
to a perfectly aligned system.
Go ahead and open your pinholes once the beam is centered.
The final step is to remove the alignment pinholes over the
tube lens and scan lens on the back of the microscope,
and view the imaging beam on the back aperture of the objective.
This is easily done by sliding a piece of business card
or paper in the narrow space directly over the objective.
This will allow you to view the shape and position of the beam.
It should either be round,
or you will perhaps see some of the edges
of your scanner mirrors if the beam is expanded
to fully fill them.
The last adjustment you need to make is to tweak the last mirror
before the periscope tube
to center the beam through the objective.
I generally do this by looking both at a piece of paper
above the objective, as well as putting a piece of paper
underneath the objective to make sure that the light throughput
of the objective is round and even.
Now your Sutter two-photon microscope
should be optimized for imaging.
This completes beam alignment of your two-photon
laser scanning system the Sutter Moveable Objective Microscope.
Leaving the two pinholes on the table will allow
for quick checks and potentially easy corrections
of the beam alignment of your system.
However, if there are any unexpected variations,
it is recommended to start the alignment procedure
from the beginning as we have shown here.
Happy imaging!
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