not a confocal question - features of a widefield
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Nuno Moreno |
not a confocal question - features of a widefield
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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://www.igc.gulbekian.pt phone +351 214464606 fax +351 214407970 |
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Shalin Mehta |
Re: not a confocal question - features of a widefield
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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]> wrote: Search the CONFOCAL archive at -- ~~~~~~~~~~~~~~~~~~~~~~~~~ Shalin Mehta Graduate Student in Bioengineering, NUS mobile: +65-90694182 blog: shalin.wordpress.com ~~~~~~~~~~~~~~~~~~~~~~~~~~ |
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Nuno Moreno |
Re: not a confocal question - features of a widefield
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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 |
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
I've been thinking about building a system like that ever since one of our users started doing overnight recordings and his samples sometimes drift vertically and thus would need an autofocus system to correct for that. If you could tap into the Z-controller circuit of your system, you would be able to drive that with a signal that's proportional to the vertical drift. The drift could be scaled with
e.g. calculating average intensity during a Z-stack from a certain area of the image that's fairly thin. Similar logic would work for the autoexposure as well. Want to try it?
Zoltan
On Dec 7, 2007 4:41 PM, Nuno Moreno <[hidden email]> wrote: Autoexpose will bleach everything, right? -- -- Zoltan Cseresnyes Facility manager, Imaging Suite Dept. of Zoology University of Cambridge Downing Street, Cambridge CB2 3EJ UK Tel.: (++44) (0)1223 769282 Fax.: (++44) (0)1223 336676 |
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Engstrom, Lars |
Re: not a confocal question - features of a widefield
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In reply to this post by Nuno Moreno
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal I just re-read your message and realize you are looking for cell tracking ability. I know MediaCybernetics acquisition software QED has a cell tracking function. I never used it and don't think it included an autofocus but I really liked their user interface and many of their features. http://www.mediacy.com/index.aspx?page=InVivo No commercial interest. -Lars -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Nuno Moreno Sent: Friday, December 07, 2007 6:43 AM To: [hidden email] Subject: not a confocal question - features of a widefield 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://www.igc.gulbekian.pt phone +351 214464606 fax +351 214407970 |
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Engstrom, Lars |
Re: not a confocal question - features of a widefield
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In reply to this post by Zoltan
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Nikon has a Perfect Focus System I believe for their TE2000
series. It uses an LED (770nm) to focus on the coverslip and adjust Z prior to
image acquisition.
I haven't tried it but sounds similar to what you are
looking for and could use their system as an example for building your
own.
-Lars From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Zoltan Cseresnyes Sent: Friday, December 07, 2007 9:50 AM To: [hidden email] Subject: Re: not a confocal question - features of a widefield I've been thinking about building a system like that ever since one of our
users started doing overnight recordings and his samples sometimes drift
vertically and thus would need an autofocus system to correct for that. If
you could tap into the Z-controller circuit of your system, you would be able to
drive that with a signal that's proportional to the vertical drift. The
drift could be scaled with e.g. calculating average intensity during a
Z-stack from a certain area of the image that's fairly thin. Similar logic
would work for the autoexposure as well. Want to try it?
Zoltan
On Dec 7, 2007 4:41 PM, Nuno Moreno <[hidden email]>
wrote: Autoexpose will bleach everything, right? -- -- Zoltan Cseresnyes Facility manager, Imaging Suite Dept. of Zoology University of Cambridge Downing Street, Cambridge CB2 3EJ UK Tel.: (++44) (0)1223 769282 Fax.: (++44) (0)1223 336676 |
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Nuno Moreno |
Re: not a confocal question - features of a widefield
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In reply to this post by Zoltan
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Yes. In fact I just did it and I was wondering if there was some kind of commercial solution to compare. Even Deltavison what they do is having a switchable auto-focus that exposes with low light before taking the "real" picture, which is not adaptative. It might work better than mine because thinks are better integrated but it is not more elegant :) The adaptative exposure its so stupidly simple that I don't know why it is not integrated the most of the well know software. Not IPP, metamorph , Leica, Zeiss or Deltavision...as far has I know. Maybe I have to sell it ;). Regards, NM Zoltan Cseresnyes wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > I've been thinking about building a system like that ever since one of > our users started doing overnight recordings and his samples sometimes > drift vertically and thus would need an autofocus system to correct for > that. If you could tap into the Z-controller circuit of your system, > you would be able to drive that with a signal that's proportional to the > vertical drift. The drift could be scaled with e.g. calculating average > intensity during a Z-stack from a certain area of the image that's > fairly thin. Similar logic would work for the autoexposure as well. > Want to try it? > > Zoltan > > On Dec 7, 2007 4:41 PM, Nuno Moreno <[hidden email] > <mailto:[hidden email]>> 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 > <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]> > > <mailto:[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://uic.igc.gulbekian.pt <http://uic.igc.gulbekian.pt/>> > > http://www.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/> > <http://shalin.wordpress.com <http://shalin.wordpress.com/>> > > ~~~~~~~~~~~~~~~~~~~~~~~~~~ > > -- > Nuno Moreno > Cell Imaging Unit > Instituto Gulbenkian de Ciência > http://uic.igc.gulbekian.pt <http://uic.igc.gulbekian.pt/> > http://www.igc.gulbekian.pt <http://www.igc.gulbekian.pt/> > phone +351 214464606 > fax +351 214407970 > > > > > -- > -- > Zoltan Cseresnyes > Facility manager, Imaging Suite > Dept. of Zoology University of Cambridge > Downing Street, Cambridge > CB2 3EJ UK > > Tel.: (++44) (0)1223 769282 > Fax.: (++44) (0)1223 336676 -- 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 |
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Nuno Moreno |
Re: not a confocal question - features of a widefield
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In reply to this post by Engstrom, Lars
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal This does not track cells that are moving in Z, right? Engstrom, Lars wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > Nikon has a Perfect Focus System I believe for their TE2000 series. It > uses an LED (770nm) to focus on the coverslip and adjust Z prior to > image acquisition. > http://nikonusa.com/fileuploads/pdfs/Instruments/TE2000_PFS.pdf > > I haven't tried it but sounds similar to what you are looking for and > could use their system as an example for building your own. > > -Lars > > ------------------------------------------------------------------------ > *From:* Confocal Microscopy List [mailto:[hidden email]] > *On Behalf Of *Zoltan Cseresnyes > *Sent:* Friday, December 07, 2007 9:50 AM > *To:* [hidden email] > *Subject:* Re: not a confocal question - features of a widefield > > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > I've been thinking about building a system like that ever since one of > our users started doing overnight recordings and his samples sometimes > drift vertically and thus would need an autofocus system to correct for > that. If you could tap into the Z-controller circuit of your system, > you would be able to drive that with a signal that's proportional to the > vertical drift. The drift could be scaled with e.g. calculating average > intensity during a Z-stack from a certain area of the image that's > fairly thin. Similar logic would work for the autoexposure as well. > Want to try it? > > Zoltan > > On Dec 7, 2007 4:41 PM, Nuno Moreno <[hidden email] > <mailto:[hidden email]>> 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 > <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]> > > <mailto:[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://uic.igc.gulbekian.pt <http://uic.igc.gulbekian.pt/>> > > http://www.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/><http://shalin.wordpress.com > <http://shalin.wordpress.com/>> > > ~~~~~~~~~~~~~~~~~~~~~~~~~~ > > -- > Nuno Moreno > Cell Imaging Unit > Instituto Gulbenkian de Ciência > http://uic.igc.gulbekian.pt <http://uic.igc.gulbekian.pt/> > http://www.igc.gulbekian.pt <http://www.igc.gulbekian.pt/> > phone +351 214464606 > fax +351 214407970 > > > > > -- > -- > Zoltan Cseresnyes > Facility manager, Imaging Suite > Dept. of Zoology University of Cambridge > Downing Street, Cambridge > CB2 3EJ UK > > Tel.: (++44) (0)1223 769282 > Fax.: (++44) (0)1223 336676 -- 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 |
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Nuno Moreno |
Re: not a confocal question - features of a widefield
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In reply to this post by Engstrom, Lars
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Yes, but again autofocus is a concept that I never liked. This means exposing your cells to more light...and it might be that cells are just at the same place! Regards, NM Engstrom, Lars wrote: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > I just re-read your message and realize you are looking for cell tracking ability. I know MediaCybernetics acquisition software QED has a cell tracking function. I never used it and don't think it included an autofocus but I really liked their user interface and many of their features. > http://www.mediacy.com/index.aspx?page=InVivo > No commercial interest. > > -Lars > > -----Original Message----- > From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Nuno Moreno > Sent: Friday, December 07, 2007 6:43 AM > To: [hidden email] > Subject: not a confocal question - features of a widefield > > 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://www.igc.gulbekian.pt phone +351 214464606 fax +351 214407970 |
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Bill Miller-3 |
Re: not a confocal question - features of a widefield
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In reply to this post by Nuno Moreno
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal see http://www.pnas.org/cgi/content/full/103/46/17137 At 06:17 PM 12/7/2007 +0000, Nuno Moreno wrote: >Search the CONFOCAL archive at >http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > >Yes. In fact I just did it and I was wondering >if there was some kind of commercial solution to compare. > >Even Deltavison what they do is having a >switchable auto-focus that exposes with low >light before taking the "real" picture, which is >not adaptative. It might work better than mine >because thinks are better integrated but it is not more elegant :) > >The adaptative exposure its so stupidly simple >that I don't know why it is not integrated the >most of the well know software. Not IPP, >metamorph , Leica, Zeiss or Deltavision...as far has I know. > >Maybe I have to sell it ;). > >Regards, >NM > > > >Zoltan Cseresnyes wrote: >>Search the CONFOCAL archive at >>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >>I've been thinking about building a system like >>that ever since one of our users started doing >>overnight recordings and his samples sometimes >>drift vertically and thus would need an >>autofocus system to correct for that. If you >>could tap into the Z-controller circuit of your >>system, you would be able to drive that with a >>signal that's proportional to the vertical >>drift. The drift could be scaled with e.g. >>calculating average intensity during a Z-stack >>from a certain area of the image that's fairly >>thin. Similar logic would work for the autoexposure as well. >>Want to try it? Zoltan >>On Dec 7, 2007 4:41 PM, Nuno Moreno >><[hidden email] <mailto:[hidden email]>> 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 >> <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]> >> > >> <mailto:[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://uic.igc.gulbekian.pt <http://uic.igc.gulbekian.pt/>> >> > http://www.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/> >> <http://shalin.wordpress.com <http://shalin.wordpress.com/>> >> > ~~~~~~~~~~~~~~~~~~~~~~~~~~ >> -- >> Nuno Moreno >> Cell Imaging Unit >> Instituto Gulbenkian de Ciência >> http://uic.igc.gulbekian.pt <http://uic.igc.gulbekian.pt/> >> http://www.igc.gulbekian.pt <http://www.igc.gulbekian.pt/> >> phone +351 214464606 >> fax +351 214407970 >> >> >>-- >>-- >>Zoltan Cseresnyes >>Facility manager, Imaging Suite >>Dept. of Zoology University of Cambridge >>Downing Street, Cambridge >>CB2 3EJ UK >>Tel.: (++44) (0)1223 769282 >>Fax.: (++44) (0)1223 336676 > >-- >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 |
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Cathcart, Chris |
Re: not a confocal question - features of a widefield
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In reply to this post by Nuno Moreno
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Been following this string . If this is such a big problem, everyone should be on the TiPFS system. New system is absolutely perfect. BTW, how come you'r not down in the islands? I need a place to go to soon. Visiting friends in NC this weekend after the ASCB meeting, saw Barbara . Chris Cathcart Nikon Confocal Imaging 416-554-2512 Visit www.nikonconfocal.com -----Original Message----- From: Bill Miller [mailto:[hidden email]] Sent: Friday, December 07, 2007 1:35 PM To: [hidden email] Subject: Re: not a confocal question - features of a widefield Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal see http://www.pnas.org/cgi/content/full/103/46/17137 At 06:17 PM 12/7/2007 +0000, Nuno Moreno wrote: >Search the CONFOCAL archive at >http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > >Yes. In fact I just did it and I was wondering if there was some kind >of commercial solution to compare. > >Even Deltavison what they do is having a switchable auto-focus that >exposes with low light before taking the "real" picture, which is not >adaptative. It might work better than mine because thinks are better >integrated but it is not more elegant :) > >The adaptative exposure its so stupidly simple that I don't know why it >is not integrated the most of the well know software. Not IPP, >metamorph , Leica, Zeiss or Deltavision...as far has I know. > >Maybe I have to sell it ;). > >Regards, >NM > > > >Zoltan Cseresnyes wrote: >>Search the CONFOCAL archive at >>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >>I've been thinking about building a system like that ever since one of >>our users started doing overnight recordings and his samples sometimes >>drift vertically and thus would need an autofocus system to correct >>for that. If you could tap into the Z-controller circuit of your >>system, you would be able to drive that with a signal that's >>proportional to the vertical drift. The drift could be scaled with >>e.g. >>calculating average intensity during a Z-stack from a certain area of >>the image that's fairly thin. Similar logic would work for the >>autoexposure as well. >>Want to try it? Zoltan >>On Dec 7, 2007 4:41 PM, Nuno Moreno >><[hidden email] <mailto:[hidden email]>> 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 >> <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]> >> > >> <mailto:[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://uic.igc.gulbekian.pt <http://uic.igc.gulbekian.pt/>> >> > http://www.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/> >> <http://shalin.wordpress.com <http://shalin.wordpress.com/>> >> > ~~~~~~~~~~~~~~~~~~~~~~~~~~ >> -- >> Nuno Moreno >> Cell Imaging Unit >> Instituto Gulbenkian de Ciência >> http://uic.igc.gulbekian.pt <http://uic.igc.gulbekian.pt/> >> http://www.igc.gulbekian.pt <http://www.igc.gulbekian.pt/> >> phone +351 214464606 >> fax +351 214407970 >> >> >>-- >>-- >>Zoltan Cseresnyes >>Facility manager, Imaging Suite >>Dept. of Zoology University of Cambridge Downing Street, Cambridge >>CB2 3EJ UK >>Tel.: (++44) (0)1223 769282 >>Fax.: (++44) (0)1223 336676 > >-- >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 IMPORTANT: The contents of this email and any attachments are confidential. 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Julio Vazquez |
Re: not a confocal question - features of a widefield
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In reply to this post by Nuno Moreno
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
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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... -- 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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Michael Weber-4 |
Re: not a confocal question - features of a widefield
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear all, the solution for the "auto exposure" function might be Controlled Light Exposure Microscopy (CLEM), which controlls the excitation time for every pixel via a feed-back loop while acquiring. But with this technique we are coming back to Confocals: http://www.nikoninstruments.eu/news.php?n=550 Since Nikon also offers the hardware autofocus system, it might be one 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 comment on that?! cheers, Michael > 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 |
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Nuno Moreno |
Re: not a confocal question - features of a widefield
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In reply to this post by Julio Vazquez
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal I'm very happy with all this replies. I would never guess that this was such an interesting/actual topic. Bellow you can find some answers to very important points. I think there is a new concept born here: ADAPTATIVE MICROSCOPY. The name looks nice and I hope in a near future all manufacturers will have a similar packaged. ;) Regards, NM Julio Vazquez wrote: > 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 "contrast normalization" but when you have to many cells it can be confused. It is for specific applications but its so simple that I was just wondering why it is still not implemented yet. > > 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. I absolutely agree! > > 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? It goes gradually to the target value. You will get the over exposed value but I don't see how it will get such a big difference. Here you can find and example of a time laplse of more than 13 hours where the exposure time of one channel decreased from ~6000 milliseconds to ~1500 milliseconds with no post processing. I must admit that without post processing shorter exposure times or higher time between frames you will get flickering. http://uic.igc.gulbenkian.pt/pics/adaptExpTimeXvid.avi 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... yes but you can correct those small changes by software afterwords. 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. Yes. If there is external factors like a bit of crap floating around your are done. 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... This night there is a timelapse with a malaria parasite and a cell stained with lisotraker. It will not work very well for the parasite because its vary small. The focus adaptation is made in the cell lysotracker channel. > > 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. on this case it will not work properly unless you are imaging a single or few yeasts. What you can give is a sensitivity value that will adjust focus more often but you might come up to a point where its always trying to get the best focus and will bleach more than it should. In this case the deltavision solution will probably work better. 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... Yes but if your timelapse is more than 10 hours... > > > > -- > 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 > -- 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 |
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lechristophe |
Re: not a confocal question - features of a widefield
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In reply to this post by Julio Vazquez
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
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. doi:10.1111/j.0022-2720.2004.01404.x Summary : 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. Christophe On Dec 7, 2007 8:36 PM, Julio Vazquez <[hidden email]> wrote: > 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 > |
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Julio Vazquez |
Re: not a confocal question - features of a widefield
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In reply to this post by Nuno Moreno
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
On Dec 7, 2007, at 2:03 PM, Nuno Moreno wrote:
Even so, it should be no problem... you can always find motivated grad students. Besides, a one year grad student stipend costs about the same as a 1-year maintenance agreement for some of the imaging software packages we own... Just (barely) kidding! Julio.
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In reply to this post by Nuno Moreno
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi all, I would like suggestions for acquisition of the cheapest useful confocal for live cell imaging (laser or disk-based). We have an old Biorad 1024 (Nikon based) but its lifetime is approaching. Thanks -- Dr Pedro J Camello Dpt Physiology Faculty of Veterinary Sciences University of Extremadura 10071 Caceres Spain Ph: 927257100 Extension 1321 Fax:927257110 |
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F Javier Díez Guerra |
Re: Advice for a cheap confocal
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Pedro, Recientemente, tras el traslado al nuevo edificio, hemos renovado un sistema de microscopia confocal Radiance 2000 de BioRad. Se trata de un microscopio Zeiss Axiovert 100 (invertido) con optica 25x, 40x y 63x (DIC), y tres laseres (488, 543, 633). Habla con Ramón Monteagudo o Pepe Sanz (Grupo Taper) para más información sobre componentes y coste. Un saludo, At 17:44 09/12/2007, you wrote: >Search the CONFOCAL archive at >http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > >Hi all, > >I would like suggestions for acquisition of the cheapest useful confocal >for live cell imaging (laser or disk-based). We have an old Biorad 1024 >(Nikon based) but its lifetime is approaching. > >Thanks > >-- >Dr Pedro J Camello >Dpt Physiology >Faculty of Veterinary Sciences >University of Extremadura >10071 Caceres >Spain >Ph: 927257100 Extension 1321 >Fax:927257110 F Javier Diez-Guerra, PhD Profesor Titular Centro de Biologia Molecular Severo Ochoa C/ Nicolás Cabrera, 1 Universidad Autónoma Ctra Colmenar Viejo Km 15 Cantoblanco, 28049 Madrid SPAIN phone: +34 91 196 4612 e-mail: [hidden email] |
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In reply to this post by Pedro Camello
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear Dr Camello, I saw your request for a low cost confocal on the confocal listserver. VisiTech International are manufacturers of confocal imaging systems. We are able to offer a variety of confocal technologies including spinning disc systems from Yokogawa, an advanced multi-point confocal scanner of our own design which eliminates the artefacts normally associated with spinning disc systems, and a high speed single point confocal system using Acousto Optical Deflection technology. I would like to discuss your requirements to see how best we can help you to obtain a confocal system within your budget. When would be a good time to call you? Best regards Ken Ken Bell Product Specialist VisiTech International Ltd Unit 92, Silverbriar Sunderland Enterprise Park (East) Sunderland SR5 2TQ, UK Tel +44 191 516 6255 Fax +44 191 516 6258 email: [hidden email] web: www.visitch.co.uk -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Pedro J Camello Sent: 09 December 2007 16:45 To: [hidden email] Subject: Advice for a cheap confocal Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi all, I would like suggestions for acquisition of the cheapest useful confocal for live cell imaging (laser or disk-based). We have an old Biorad 1024 (Nikon based) but its lifetime is approaching. Thanks -- Dr Pedro J Camello Dpt Physiology Faculty of Veterinary Sciences University of Extremadura 10071 Caceres Spain Ph: 927257100 Extension 1321 Fax:927257110 |
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