Alignment of the GRIN lens

For in vitro and in vivo experiments, the sample was mounted on the rotational stage MAG-2 and further fixed to a three-axis translation stage, which enabled precise adjustment of the angle and position of the GRIN lens. A visible laser diode was introduced to the endomicroscopy system to assist the alignment. The laser beam was first aligned with the optical axis of the objective. Then, the objective was removed, and the tilt/tip of the GRIN lens was adjusted so that the laser was normally incident on the upper surface of the GRIN lens. This guaranteed that the optical axes of the GRIN lens and the objective were parallel to each other. Next, we looked at the imaging plane of the objective through the eyepiece and translated the GRIN lens until its upper surface came into focus. Then, the objective was lifted 100 mm up so that the GRIN lens can operate at its designed working distance. Last, we switched to the two-photon imaging mode and translated the GRIN lens sideways until the fluorescence image appeared at the center of the FOV.

The details for each step are listed below: 

1. Implant the GRIN lens into the imaging sample and mount them on a rotational stage such as MAG-2, NARISHIGE).
2. Introduce a visible laser diode to assist the alignment of the GRIN lens. 
3. Align the laser beam to be parallel with the optical axis of the 10X objective. This could be achieved by putting a flat mirror at the back pupil plane of the objective and adjusting the angle of the incident laser such that the reflected light follows the incident path.
4. Roughly place the GRIN lens at the focal plane of the objective.
5. Remove the objective. 
6. Adjust the tip and tilt angles of the rotational stage of the GRIN lens such that the laser beam is normally incident on the surface of the GRIN lens. This could be achieved by ensuring that the reflected laser from the upper surface of the GRIN lens follows the incident path. This guarantees that the optical axes of the GRIN lens and the objective are parallel to each other.
7. Put the objective back on the objective holder.
8. Look at the imaging plane of the objective through the eyepiece and translate the GRIN lens until its upper surface comes into focus.
9. Lift the objective 100 mm up so that the GRIN lens operates at its designed working distance.
10. Switch to the two-photon imaging mode and translate the GRIN lens sideways using a translational stage until the fluorescence image appears at the center of the imaging FOV.