Biology Reference
In-Depth Information
mounting of the kineFLEX 4-axis adjuster. 45 -orientated lasers, such as the
50 mW, 532 nmDPSS laser fromCrystaLasers, require a custommachined holder.
￿ Use the kineFLEX coarse alignment tool in two orientations to adjust the 4-axis
alignment screws and find the optical axis of the laser beam.
￿ Replace the alignment tool with the kineFLEX collimator of the fiber matching
the laser wavelength.
￿ Monitor the optical power on the other end of this fiber and perform a fine
adjustment of the 4-axis alignment screws to maximize the power throughput.
Typically, we obtain better than 50% power throughput.
￿ Connect the fiber to the correct input port of the iFLEX-Adder unit. Measure the
output power at the end of the output fiber (OF6). The power throughput should
be better than 25% of the free space laser power.
￿ Turn off the first laser and perform the same steps for the other lasers.
￿ The alignment of the laser subsystem is now complete. The system has
exceptional long-term stability and typically does not require realignment, unless
the configuration has changed.
15.8.3 Step 3: alignment of DualView and QuadView simultaneous
imaging systems
￿ Place the test glass slide with precision etched chromiummask (Photometrics) on
the sample stage. Focus on the sample with a 20
air objective.
￿ Switch the emission light path to the side port connected to the QuadView optic.
￿ Adjust the exposure time of the eVolve-512 camera and observe the image in live
view.
￿ Set the QuadView to Bypass mode and replace its filter cube by the Bypass cube.
￿ Adjust the four Entrance Aperture slits so that they are just at the edges of the
camera image. If necessary, change the rotation of the camera relative to the
QuadView to ensure that the slits are parallel to the image edges.
￿ Set the QuadView to QuadView mode.
￿ Adjust the second set of slits so that the visible image is in the center and exactly
50% of the size of the full image.
￿ Replace the filter cube with the fluorescence cube.
￿ Four subimageswithdifferent intensity appear insteadof the single small (50%) image.
￿ Adjust for each subimage the position (up-down and left-right) so that they
are located in their respective quadrants of the camera image and are
nonoverlapping with each other. The grid lines of the test specimen facilitate the
fine adjustment of the channels relative to each other.
￿ The alignment of the QuadView unit is now complete.
15.8.4 Step 4: calibration of the multiwell plate geometry
￿ Place the microplate on top of the stage insert.
￿ Select a low magnification (10
) air objective and focus on the well bottom.
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