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.
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).
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).
This week I finally went live, i.e. started live imaging.
The first session, on Tuesday, was ok.
I came early to start up the systems. The most important thing is the heating chamber. Heating is important since the temperature can affect the quality of the imaging. How? The metal tray (where you place your sample) expands when heated. The lenses in the objective also. The immersion oil changes viscosity. So does the cell media. So when imaging mammalian cells at 37C, it takes time for all components to equalize their temp on 37C.
3 hours later, I was ready to image. I started the camera, the computer, the lasers. The only thing left to do before starting is align the laser so it will go directly up through the objective (at 90 degree to the sample). The laser sometimes drifts, or users change it and so one has to check before starting.
The laser was waaayyy off the target. I tried my best. Then, over the next 4-5 hours(!), three other people tried until our physicists succeeded, in a way (it’s centered but the laser looked dispersed, not sharp).
Problem is, the temp in the chamber isn’t equalized now (since we kept opening the chamber, switching objectives, removed the metal plate holder etc…). I tried imaging without waiting, but the z-axis kept drifting. So now I have to wait another half-hour or hour to let everything heat up properly and it’s already 6pm.
Guess I won’t be doing lots of imaging today…
The other day I started imaging, when all of a sudden I could not see any image on the screen; nor through the ocular.
Immediately I noticed that clicking the shutter button does not make the unique sound of the shutter opening.
I called for help and here is what I learned:
The shutter is controlled by an electronic box sitting on the shelf. The LED light indicating shutter opening was on. Changing the port of the fluorescent shutter to that of the transmitted light shutter did not help. Crossing the cables of the two shutters indicated that the problem is not the electronics or the cable since the shutter for the transmitted light worked fine with the port & cable of the other shutter.
So, the problem was the shutter itself. We dismantled it and indeed, the flaps of the shutter were stuck. No way to fix that easily. What we did was to replace the broken shutter with that of the transmitted light. So we can’t use transmitted light, but can still have fluorescent imaging.
A few points about our shutter:
It was costume built so that on the back side (the one pointing towards the light source) is coated with zinc. This was done in order to reflect the light when the shutter is closed, to prevent over- heating.
On top of that, there is a gap of ~half centimeter in the shutter housing, to allow air flow, again to prevent over-heating.
I found the experience annoying yet educating.