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High Speed Synchronization

01:00:12.01 So, if you just happened to watch
01:00:15.18 the section on software control of microscopes,
01:00:17.15 you may have noticed that it took quite a bit of time
01:00:19.21 to acquire that two-channel z-stack,
01:00:22.20 and in part that is because the different components
01:00:25.03 need to move in place
01:00:26.17 — the dichroic needs to move,
01:00:28.08 the z motor needs to move —
01:00:32.05 but it’s also cumbersome for the software
01:00:34.14 to give a command to the camera, read out the image,
01:00:37.25 wait for that to be done,
01:00:39.25 and then do the next, etc, etc.
01:00:43.10 So, there are ways to speed this process up,
01:00:45.09 and one of them is to use the camera, now,
01:00:49.25 as a video camera,
01:00:51.29 and then use equipment that is moving,
01:00:55.29 that is so fast that it can move
01:00:59.11 in between the exposures that this video camera is taking.
01:01:04.15 Now, on top of that, we need to synchronize these components,
01:01:07.28 and the synchronization is now not anymore through software,
01:01:11.16 but it’s through electrical signals
01:01:14.18 coming from the cameras.
01:01:17.17 So, many of these scientific-grade cameras
01:01:20.09 have signals that indicate that they’re exposing.
01:01:23.24 So, the signal will… the electrical signal will go high
01:01:26.23 when the exposure starts,
01:01:28.05 and then when the exposure stops
01:01:29.24 it will drop down,
01:01:31.15 readout happens or something happens,
01:01:33.04 and then it goes up again.
01:01:35.08 And so, that signal we can use
01:01:38.16 to now trigger events in other hardware,
01:01:40.09 and I’ll show you how that is implemented here.
01:01:44.00 One other aspect is now…
01:01:45.13 okay, we get a video stream of images into the software.
01:01:49.04 The software needs to understand what is what.
01:01:52.29 So, it needs to still communicate with all this equipment
01:01:56.25 and to present it to you in a way
01:01:58.23 that you can work with it.
01:02:01.10 So, we’ll now go through these different components
01:02:03.27 and show how they are linked to each other
01:02:06.06 through electrical signals.
01:02:09.21 So, first we go to the camera.
01:02:12.14 The camera has a controller.
01:02:14.22 On the back of that controller is a BNC cable.
01:02:18.29 The BNC cable runs to this box here.
01:02:24.04 This device has a little microcontroller in it
01:02:26.21 that is reading out the state of this input continuously,
01:02:31.27 and you can load up a program,
01:02:34.06 a sequence of whatever this thing must do,
01:02:37.10 when the state of the input changes.
01:02:41.27 So, the software will load up the sequence
01:02:45.06 into this microcontroller device.
01:02:47.28 This one controls the AOTF,
01:02:50.24 an acousto-optical tunable filter,
01:02:53.20 and that basically is a very fast wavelength changer.
01:03:00.04 So, that AOTF is sitting there in front of the lasers, in the back,
01:03:04.27 and that will select, in this case,
01:03:07.16 either the blue or the yellow line coming through,
01:03:11.02 and can do that at microsecond timescales.
01:03:15.10 The electrical signal travels on,
01:03:17.26 and it travels to the controller for our z-drive.
01:03:21.25 And in this microscope, we have a piezo z-drive,
01:03:26.25 and that piezo z can move, also,
01:03:29.04 at very high speeds,
01:03:31.05 although it takes, still, quite a few milliseconds
01:03:34.03 for it to move to another z position.
01:03:37.29 So, also, that controller of the z drive
01:03:40.02 can be programmed to,
01:03:43.03 for instance, every two signals…
01:03:46.11 after two TTL signals to change the z position.
01:03:52.17 So now, putting this all together,
01:03:54.13 we can run a very fast multichannel z-stack.
01:03:59.05 If we now put this all together in software, then…
01:04:04.23 in this case, the user interface stays exactly the same.
01:04:08.29 So, in software, we can again say
01:04:11.06 that we want a z-stack,
01:04:14.17 we want multiple channels.
01:04:17.21 The software will figure out…
01:04:19.28 hey, I can use this electrical synchronization
01:04:23.14 and I can run the camera in video mode.
01:04:26.29 A couple things that are important are that the exposure time
01:04:30.06 for the channels is the same.
01:04:32.08 We do not want to be moving things like filter wheels
01:04:34.13 that are slow at the same time.
01:04:37.03 So, I use here configurations
01:04:39.17 that don’t do any of that.
01:04:41.11 And then, when I now press acquire,
01:04:45.00 and I’m asking here for steps of 0.2 microns
01:04:50.05 from -2 to +2
01:04:52.11 -- so, this is a total of 21 slices in 2 channels —
01:04:56.14 then we’ll see that that is being acquired
01:05:02.05 in, like, seconds… 2 seconds or so.
01:05:05.28 So, we get, like, a very fast acquisition of this data set,
01:05:09.23 only limited by the exposure time of the camera.
01:05:18.18 And again, we can go, here, through this dataset,
01:05:20.27 and you see that we have a nice focus somewhere here in the middle.

This Talk
Speaker: Nico Stuurman
Audience:
  • Researcher
Recorded: April 2012
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Talk Overview

When executing complex microscopy experiments, it is important to tightly synchronize the operation of camera(s), shutters, stages, etc.. This is usually done in software, however, current computer software has limits in its ability to respond quickly. This video shows how hardware synchronization, using TTL signals running through BNC cables, can dramatically speed up microscope image acquisition and reduce light exposure to biological samples.

Speaker Bio

Nico Stuurman

Nico Stuurman

Nico Stuurman is a Research Specialist at the University of California, San Francisco, in the lab of Ron Vale. Nico combines his expertise in computer programming and microscopy to advance many projects including the Open Source software, Micro-Manager. Continue Reading

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