Yokogawa head
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Hi, Can someone give me a ballpark figure for the cost to add a
yokogawa head and Krypton/argon mixed gas laser to an existing imaging station?
I need a number for planning purposes. Vendors can feel free to contact me off
of the list. Thanks Chris Christopher
Navara Ph.D. |
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Dear list,
actually we are somewhat interested in a similar issue; we have a Nikon TE2000E microscope with widefield laser excitation and one EMCCD camera and we'd like to hear other opinions on the possibilities to upgrade this system to a semi-confocal imaging system with a spinning disc module, but maintain the existing light sources and detection. The application is to look at intracellular transport (to acquire and track dim fluorescent particles or endosomes in living cells; about 10 fps should be OK). Or do you think that investing in a piezo-driven stage and 3D deconvolution will do the trick? And if it is still possible to image then at approximately 10fps? The thickness of the stack per frame should only be as thin as possible to be able to do deconvolution, so I'd guess 1µm (so according to Nycquist 6 to 7 frames per stack; wich makes a total of 60-70 fps). Is this achievable? Thanks a lot in advance for the info; it is very much appreciated! Dries. On 26/03/2008, Navara, Christopher S <[hidden email]> wrote: Search the CONFOCAL archive at <a href="http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal" target="_blank" onclick="return top.js.OpenExtLink(window,event,this)">http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal -- Dries Vercauteren, PhD student Master of Bioscience Engineering: Cell and Gene Biotechnology Ghent Research Group on Nanomedicines www.ugent.be/fw/en/research/biofys Faculty of pharmaceutical sciences, Ghent University Harelbekestraat 72, 9000 Ghent Belgium Phone: +329/264 80 49 Mobile: +32485/30 69 80 E-mail: [hidden email] [hidden email] |
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Dear Dries,
for the decon approach, this will be difficult if the
particles you are looking at are moving during the time required to take a
stack, because in case this will destort the PSF remarkably, which in my
experience results in filtering out fast moving particles. Also, for proper
decon 6 z-planes is far too little info for many algorithms, the signal from a
diff limited spot will typically spread over about 10microns, so the amount
of info you have to provide is rather something like 35-50
z-planes per timepoint.
60 fps is feasible depending on the number of pixels you
want to read and the amount of signal you exspect. Reading an emccd at more than
10Mhz is typically not possible, so with this type of camera you will
be restricted to very small fields if you want to oversample at high res.
I
would strongly recommend the spinning disk approach, you can surely use the
emccd, lasers are usually fiber coupled to the Yokogawwa unit. Your laser should
provide at least 15mW of power per line, because light is split to 1k beamlets
and transmission though the disk reduces the intensity
further.
Cheers, jens
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Dries Vercauteren Sent: Mittwoch, 26. März 2008 14:41 To: [hidden email] Subject: Re: Yokogawa head actually we are somewhat interested in a similar issue; we have a Nikon TE2000E microscope with widefield laser excitation and one EMCCD camera and we'd like to hear other opinions on the possibilities to upgrade this system to a semi-confocal imaging system with a spinning disc module, but maintain the existing light sources and detection. The application is to look at intracellular transport (to acquire and track dim fluorescent particles or endosomes in living cells; about 10 fps should be OK). Or do you think that investing in a piezo-driven stage and 3D deconvolution will do the trick? And if it is still possible to image then at approximately 10fps? The thickness of the stack per frame should only be as thin as possible to be able to do deconvolution, so I'd guess 1µm (so according to Nycquist 6 to 7 frames per stack; wich makes a total of 60-70 fps). Is this achievable? Thanks a lot in advance for the info; it is very much appreciated! Dries. On 26/03/2008, Navara,
Christopher S <[hidden email]> wrote:
Search the CONFOCAL archive at <A onclick="return top.js.OpenExtLink(window,event,this)" href="http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal" target=_blank>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal -- Dries Vercauteren, PhD student Master of Bioscience Engineering: Cell and Gene Biotechnology Ghent Research Group on Nanomedicines www.ugent.be/fw/en/research/biofys Faculty of pharmaceutical sciences, Ghent University Harelbekestraat 72, 9000 Ghent Belgium Phone: +329/264 80 49 Mobile: +32485/30 69 80 E-mail: [hidden email] [hidden email] |
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As a counterpoint to Jens, I would say that if your particles are moving around, you may need to collect multiple sections anyway, and there may not be a huge advantage to using a spinning disk... The main speed advantage of a spinning disk, in my opinion, is that you can get single confocal sections, as opposed to having to collect stacks for deconvolution. If you need to collect many z sections anyway, all other things being equal, a regular widefield may be more sensitive (because you avoid all the light loss due to the extra spinning disc), and you may be able to use shorter exposures. However, as Jens said, if your particles are moving fast, 3-D deconvolution will be tricky at best. I must say however that we sometimes got very good deconvolution results of single sections (for instance with Improvision's restoration package). So, at least, you may want to give it a try before you make a final decision, since there is a significant price difference between a deconvolution package and a spinning disk outfit... I would recommend you find someone with a deconvolution microscope and try it out with your samples (or even use your own, even if you don't have z-stack capability, and deconvolve the single sections to see if they are useable for your purpose). I would not personally spend 50K or more without having run a test to check whether this will work for me... -- Julio Vazquez, Fred Hutchinson Cancer Research Center Seattle, WA 98109-1024 == On Mar 28, 2008, at 2:33 PM, Rietdorf, Jens wrote: Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal |
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