The stage and the objective

I was happily imaging today when suddenly, the stage control went a little crazy. But then, all the excitation lights were dimmer. Moreover, I could not see any fluorescent signal anymore, not even the DAPI signal which is usually very strong. Initially I thought that the filter cube or dichroic mirror were misaligned, or maybe the shutter were not functioning properly.  But the solution turned out to be very simple.

What happened?

Well, when the motorized stage control went crazy, it sent the objective all the way to the left on the X axis. Our stage is built in such a way that the slide holder is lower than the entire stage (see image).

stage & objective

So, when the objective touched the elevated part of the stage, it caused it to rotate by a few millimeters out of its place. Since the objective wasn’t perfectly aligned (i.e. snapped into position) only part of the excitation light was able to go through, but very little (if any) of the emission light got back to the ocular/camera.

So eventually all I had to do was snap the objective back into place.

(Thanks to Caro from our lab who helped me figure it out).

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