http://confocal-microscopy-list.275.s1.nabble.com/not-a-confocal-question-features-of-a-widefield-tp591195p591210.html
pixel via a feed-back loop while acquiring. But with this technique we are
idea to go for a C1. Still, cell movement has to be compensated by
acquiring several Z-stacks. However, if you are in a focal plane without
signal, the CLEM feature should avoid useless exposure. I have never seen
such a system in action, but it sounds nice. Maybe some customers can
> Search the CONFOCAL archive at
>
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal>
> -
> 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...
>
>
>
> --
> 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