- Put all lenses off. Modulate the positions and angles of the
lasers and mirrors to make the laser light pass through the
centers of the respective diaphragms. Then the laser light
goes horizontally at the height of the optical port of the
microscope. - Put the expanders and relay lenses into the laser lines and
optimize the line in the same manner as done before putting
the lenses. - Tune on the cameras and modulate the laser position to be the
center of the field of view.
3.3.2 Observation 1. Turn on the excitation lasers.
- Place appropriate neutral density filters in respective laser lines.
- Modulate the full mirror angle to have the laser light pass
vertically from the objective. - Modulate the distance between the beam expander lenses to
make the laser light be a parallel beam. - Place a sample glass-bottom dish on the microscope stage.
- Make the incident angle of the laser light be large to satisfy the
TIRF condition. - Focus at the upper surface of the glass bottom. Small particles
of fluorescent substances adsorbing on the surface of the glass
will be detected. - Modulate the incident angle of the excitation laser light to be
slightly smaller to make oblique illumination (seeNote4). - Optimize the focus position roughly to observe intracellular
RNAs. Find a cell expressing the probes at an appropriate level.
Fig. 3Layout of optical setup in the microscope system to monitor the dynamics of TERRA, telomeres, and
hnRNPA1 in living cells
344 Hideaki Yoshimura and Takeaki Ozawa