Posted by
Julio Vazquez on
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Hi Nuno,
I have some thoughts (but not any useful answer, I'm afraid)...
1. Do you want to track cells in x,y too, or only in z? In addition to the Nikon implementation already mentioned, on the Zeiss 510 one can "autofocus" on the inner surface of the coverslip, and then move the focal plane to a specified distance from that surface. This will correct for drift of the focus drive. On the DeltaVision, the autofocus function takes a series of images around the previous z location, finds the plane of highest intensity, and then recenters the stack at the new z position. In both cases, the systems look for the most intense image and use that as the new reference. By using a combination of both approaches, in iteration, one could compensate both for mechanical drift, as well as for drift of the cell inside the sample. The problem is that the most intense plane is not necessarily the one you want, and/or may change within the sample over time. In photography, autofocus is achieved by looking for maximum contrast, rather than max intensity, and I could imagine something like this being implemented in microscopy. I think these approaches will work fine if you have a single cell in your field of view, but once you have many, how does one teach the software to pay attention to one specific cell and ignore the others, so that focus doesn't keep jumping between cells? Some image analysis software have tools for object tracking, where individual objects (cells) are identified based on total intensity, and possibly morphological parameters. I guess one could use such an approach to force the microscope to stay on one specific object and track it in x,y,z over time, but complex samples where there are many cells changing shape and intensity over time would be very difficult for the software to track... We have experienced this when trying to track objects for analysis purposes... it works OK with good images and few objects, but gets messy rather quickly otherwise
2. Regarding the autoexposure issue, again DeltaVision has a function where a brief series of short exposures is taken and then exposure time is set a value that gives a preset max intensity. This is probably how most autoexposure routines work with microscopy acquisition software, and while it is true that some implementations use quite heavy doses of exposure, the DeltaVision implementation generally uses only a small fraction of the exposure time you would use for normal acquisition. Bleaching is certainly increased, but not outrageously.
What you suggest is a system where images are collected, and based on post-acquisition analysis of one given image, exposure would be adjusted for the next time point, therefore avoiding the need for extra exposure required by a conventional autoexposure routine. The major problem I see with this is that if one time point is grossly overexposed (saturated), how does the software calculate the correction factor for the next time point? In addition, such a system is clearly most important in cases where the intensity of the sample is changing significantly. But then, how can the software predict the rate of change? It might work if the rate is linear, but even so, one has to wait for an image to deviate from the desired exposure level to implement a change for to bring the next exposure to a desired value... we would still end up with stacks of varying intensities cycling around an optimal value... Finally, one problem we have seen with systems based on feedback from average image intensities, is that the object you are interested in may be a minor contributor to the total image intensity, and therefore your autofocus, or autoexposure, may be responding to extraneous things that are irrelevant for your experiment. For instance, if you base your autoexposure on total image intensity, and your cell is quite small, the autoexposure may be following the changes in background fluorescence, and not the changes in your cell. On the other hand, if it is adjusting to the max intensity, then you have to find a field where the cell of interest is also the brightest object...
3. Most tracking system I can think of use some sort of live feed back: the autofocus on your photo camera estimates the distance to the object just before you click the shutter, or, if in "continuous" mode, keeps measuring and estimating the distance, so that when you click the shutter, the camera will focus where it thinks the object will be. You still need to tell the camera which object to focus on (by keeping it in the crosshair), or use some fancy algorithm that makes assumptions as to what the object of interest looks like. I suppose a missile tracking system would also rely on continuous feed back in real time to anticipate the next location of the missile. I think such a system will most likely fail if the time delay between measurement and action increases, if the object has a highly non-linear trajectory (changes of direction and velocity), and if there is crowding (1 missile to track among 100 identical decoys). Unfortunately, most of these caveats seem to apply to some extent in real-life microscopy, and that is perhaps why an autoexpose/autofocus function just before acquisition might be the most reliable... On the other hand, if you can implement a system such as the one you describe, I would love to invest in your business (although I don't have that much to invest, unfortunately)! We actually had a user who wanted to follow yeast cells as they underwent mitosis, and those guys do jump around like crazy. Eventually, she did what you suggest, except that she was part of the feed back loop: she just kept looking at the images on the monitor as they were being acquired and manually refocusing the microscope. Couldn't find any software that would do that better than she did...
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Julio Vazquez
Fred Hutchinson Cancer Research Center
Seattle, WA 98109-1024
On Dec 7, 2007, at 8:41 AM, Nuno Moreno wrote:
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Autoexpose will bleach everything, right?
Regarding the adaptative focus that I mentioned before, there are commercial system that with minimum light and before an acquisition "measure" the cell position and adapt the focus. But this is an half adaptation. It could be that it does not need to readjust the focus.
What I was counting with would be after the acquisition, if it is out of focus, it make the adjustment base in some kind of sensitivity parameter. This could be after 10 time points but it might be that it would never need such adjustment.
About the intensity variations I'm not talking about post processing adjustments. If it gets saturated there are no post processing that can help you.
Regards,
NM
Shalin Mehta wrote:
Wouldn't auto-exposure on cameras suffice for maintaining constant intensity?
Apparently most of the commercial adaptive optics systems are geared towards astronomy. Perhaps you have known this already:
http://cfao.ucolick.org/
Interesting to note that James Webb space telescope will have hardware and intelligence for adaptive optics evolved from algorithms developed for correcting aberrations for hubble telescope.
Regards,
Shalin
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Does anyone knows any commercial widefield SYSTEM that makes an
adaptative focus. And I mean adaptative (follows the cell.
The other feature is a commercial system that keeps intensities, i.e.,
if you have something with different protein expression levels over
time, the system will correct the exposure time so that at the end the
intensities are constant.
Many thanks,
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Nuno Moreno
Cell Imaging Unit
Instituto Gulbenkian de Ciência
phone +351 214464606
fax +351 214407970
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~~~~~~~~~~~~~~~~~~~~~~~~~
Shalin Mehta
Graduate Student in Bioengineering, NUS
mobile: +65-90694182
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Nuno Moreno
Cell Imaging Unit
Instituto Gulbenkian de Ciência
phone +351 214464606
fax +351 214407970