Three-Dimensional Optical-Resolution Photoacoustic Microscopy - Biomedical Optical Imaging Technologies: Design and Applications - page 60

2.4 System Alignment

Among the three general design guidelines, the first two are related to component
selection,

while the third one is about system alignment. The following

acoustic-optical confocal alignment in OR-PAM is essential for high-quality

imaging:

1. Use pulse-echo ultrasound and an ultrasonic reflector to determine the position
of the acoustic focal plane, i.e., the time delay from the trigger signal to the
maximum pulse-echo ultrasonic signal.
2. Maximize the optical power passing through the pinhole by adjusting the pinhole
position within the plane perpendicular to the optical axis.
3. Check the optical diffraction pattern after the pinhole. If the beam pattern is not a
clear Airy's disc surrounded by gradually fading concentric circular rings, adjust
the pinhole position along the optical axis (i.e., away or towards the condenser
lens) until you see the typical circular aperture diffraction pattern.
4. If the circular rings are not concentric, fine-tune the pinhole position within the
plane perpendicular to the optical axis.
5. Place an optically absorbing flat target (e.g., a piece of black tape) in the acoustic
focal plane.
6. Adjust the vertical position (i.e., z position) of the microscope objective to
maximize the amplitude of the photoacoustic signal generated from the flat
target. At the maximum, the optical focus is aligned with the acoustic focus
in the vertical direction. Alternatively, users can place a 6-m carbon fiber in
the acoustic focal plane for the confocal alignment in the z direction. Repeat
B-scans across the carbon fiber while adjusting the vertical position of the
objective to minimize the cross-sectional diameter of the carbon fiber in the
image. At the minimum, the carbon fiber is in the optical focus. Using the 6-m
carbon fiber may lead to more accurate acoustic-optical confocal alignment in the
z direction.
7. With the same flat target, adjust the horizontal positions (i.e., x and y positions)
of the microscope objective until the photoacoustic signal generated from
the target shows a symmetric pattern as shown in Fig. 2.3 . At the symmetry
point, the optical focus is aligned with the acoustic focus in the horizontal
direction.
8. Repeat steps 6 and 7 until the photoacoustic signal is optimized in both shape
and amplitude.
9. Fix the positions of the pinhole and the microscope objective.

Note that only steps 4, 5, and 7 are required to be performed before experiments
to make sure that the confocal configuration is optimized. However, if the system is
found to be misaligned, users need to follow the entire procedure.