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I'm not sure what you're looking for, but you can have a look at this method for live 3D tracking of cells that allow the microscope to "follow" the cells :
J Microsc. 2004 Nov;216(Pt 2):131-7.
Automatic real-time three-dimensional cell tracking by fluorescence microscopy.
Rabut G, Ellenberg J.
Live cell imaging has become an indispensable technique for cell
biologists. However, when grown on coverslip glass used for live cell
imaging many cultured cells move even during relatively short
observation times and focus can drift as a result of mechanical
instabilities and/or temperature fluctuations. Time-lapse imaging
therefore requires constant adjustment of the imaging field and focus
position to keep the cell of interest centred in the imaged volume. We
show here that this limitation can be overcome by tracking cells in a
fully automated way using the mass centre of cellular fluorescence.
Combined with automated multiple location revisiting, this method
dramatically increases the throughput of high-resolution live cell
imaging experiments.
The article is freely available on the blackwell/synergy website.
> 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...
>
>
>
>
> --
> Julio Vazquez
> Fred Hutchinson Cancer Research Center
> Seattle, WA 98109-1024
>
>
>
http://www.fhcrc.org/>
>
>
>
>
>
> On Dec 7, 2007, at 8:41 AM, Nuno Moreno wrote:
>
> Search the CONFOCAL archive at
>
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal>
> 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:
> Search the CONFOCAL archive at
>
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear Nuno,
> 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
> On Dec 7, 2007 10:43 PM, Nuno Moreno <
[hidden email]> <mailto:
[hidden email]>> wrote:
> Search the CONFOCAL archive at
>
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> 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,
> --
> Nuno Moreno
> Cell Imaging Unit
> Instituto Gulbenkian de Ciência
>
http://uic.igc.gulbekian.pt <
http://uic.igc.gulbekian.pt>
>
http://www.igc.gulbekian.pt> phone +351 214464606
> fax +351 214407970
> --
> ~~~~~~~~~~~~~~~~~~~~~~~~~
> Shalin Mehta
> Graduate Student in Bioengineering, NUS
> mobile: +65-90694182
> blog:
shalin.wordpress.com <
http://shalin.wordpress.com
>
> ~~~~~~~~~~~~~~~~~~~~~~~~~~
>
> --
> Nuno Moreno
> Cell Imaging Unit
> Instituto Gulbenkian de Ciência
>
http://uic.igc.gulbekian.pt
>
http://www.igc.gulbekian.pt> phone +351 214464606
> fax +351 214407970
>