FCS/FCCS advice
Locked
|
*****
To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hello, Apologies for the rather naive questions, but I have no experience with this. Someone here is keen to try FCS/FCCS but I'm not sure what we need (both in terms of hardware and software) to get this up and running. Can the FCS/FCCS data be acquired using a standard confocal microscope (we have Zeiss 510, Olympus FV1000 and Nikon A1R confocals with standard PMTs)? If so, what tools are available for the data analysis? If it is necessary to have some kind of special adaptation (different detectors/hardware correlation) what is the most cost effective solution for this? The person wanting to do these exps has suggested using AmCyan and Katushka for FCCS - can anyone forsee any problems with this combo? Thanks in advance for any help.. Simon |
|
|
*****
To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi Simon, FCS/FCCS does require special hardware and special software. On the detection side you need more sensitive detectors than conventional PMT offers. When I did FCS some time ago APD were the detectors of choice. Currently it might be also possible to use GaASP detectors. But I do not know whether one of the vendors is already using them. In order to do FCS/FCCS you also need special electronics and software. But this is usually already included if you are buying an upgrade kit. Picoquant is ONE of the vendors who is offering upgrade kits for various confocal microscopes (no commercial interest). http://www.picoquant.com/products.htm Maybe you can get in contact with them in order to discuss more details. IN general FCS/FCCS is not extremely difficult to do (once you have a set-up) but the interpretation of the data is not always straight forward. And it doesn't hurt to have experience with that if one really want to obtain reliable data. So just upgrading an instrument is probably not the method of choice. Unfortunately I have no clue about the mentioned fluorophores. I hope that helps. Cheers Arne --------------------------------------------------------------- Arne Seitz Head of Bioimaging and Optics Platform (PT-BIOP) Ecole Polytechnique Fédérale de Lausanne (EPFL) Faculty of Life Sciences Station 15, AI 0241 CH-1015 Lausanne Phone: +41 21 693 9618 Fax: +41 21 693 9585 http://biop.epfl.ch/ --------------------------------------------------------------- > -----Original Message----- > From: Confocal Microscopy List > [mailto:[hidden email]] On Behalf Of Simon > Walker > Sent: jeudi 19 avril 2012 12:40 > To: [hidden email] > Subject: FCS/FCCS advice > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Hello, > Apologies for the rather naive questions, but I have no experience with this. > Someone here is keen to try FCS/FCCS but I'm not sure what we need (both > in terms of hardware and software) to get this up and running. Can the > FCS/FCCS data be acquired using a standard confocal microscope (we have > Zeiss 510, Olympus FV1000 and Nikon A1R confocals with standard PMTs)? If > so, what tools are available for the data analysis? If it is necessary to have > some kind of special adaptation (different detectors/hardware correlation) > what is the most cost effective solution for this? > The person wanting to do these exps has suggested using AmCyan and > Katushka for FCCS - can anyone forsee any problems with this combo? > Thanks in advance for any help.. > Simon |
|
|
*****
To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** I would second what Arne said: both Becker & Hickl and Picoquant offer upgrade kits for commercial confocal systems. These should be by far the easiest solutions -- not necessarily the cheapest, though. When it comes to detectors, my impression is that Picoquant tend to prefer people using SPADs, whereas B&H also have GaAsP modules available. Having said that: nothing would prevent you from getting an upgrade kit without the detectors and just buying a GaAsP module from Hamamatsu or others. I have used both of these systems for FLIM, not FCS, so I can't say much to the specific functionality of the software. For the FLIM side my impression is that Picoquant is much more turn-key oriented, whereas B&H always had a "technical feel" to it. Depending on what you prefer, both have their upsides. I hope this helps at least a bit. Best, Christian |
|
|
In reply to this post by simon walker (BI)-2
*****
To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Simon, We have a good review/protocol on how to do FCS in vivo: Slaughter, B. D.; Unruh, J. R.; Li, R. Fluorescence fluctuation spectroscopy and imaging methods for examination of dynamic protein interactions in yeast. In Methods in Molecular Biology: Yeast Systems Biology. J.I. Castrillo and S.G. Oliver, Eds. (Springer, New York, 2011). Vol. 759, pp. 283-306. Of course there are others, for example, the technical one by Petra Schwille: www.biophysics.org/Portals/1/PDFs/Education/schwille.pdf As was already mentioned, you need a system with APD's. If you are going to do in vivo work the system also needs to be able to position the confocal spot inside the cell/organism with relatively high precision. We have had good luck with the Zeiss confocor3 system which is an add-on to the Lsm 510, but is rather expensive. ISS out of Urbana, Illinois provides add on systems in addition to the systems mentioned by previous posts. I would highly recommend demoing these systems before use--especially for in vivo measurements as there is a dramatic need for user-friendliness for these applications. No one wants to spend all day getting the microscope in the right spot on the right cell--it makes for unproductive and unreliable measurements. As far as fluorescent proteins are concerned, GFP-mCherry has been the best pair in our hands despite the fact that mCherry is misfolded half the time. We have not had luck with the cyan proteins in yeast, mostly because of the high autofluorescence in yeast at these wavelengths and the weakness of the cyan proteins. Joachim Mueller has had more luck with these in mammalian cells. Regardless of your fluorophore pair, you will have to correct for bleedthrough of the green into the red. Some of the manufacturers offer interlaced excitation that eliminates this issue (I know picoquant offers this with pulsed lasers but I'm not sure which others have it). This can be crucial for looking at typical in vivo interactions which often compete with endogenous protein interactions and can be quite low. Even Zeiss has an experimental interleaving option that we documented in this paper: Wood, C.; Huff, J.; Marshall, W.; Quingfeng Yu, E.; Unruh, J.; Slaughter, B.; Wiegraebe, W. Fluorescence correlation spectroscopy as tool for high-content-screening in yeast (HCS-FCS). Proc. of SPIE. 2011. 7905, 79050H. Hope this helps! Jay -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Simon Walker Sent: Thursday, April 19, 2012 5:40 AM To: [hidden email] Subject: FCS/FCCS advice ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hello, Apologies for the rather naive questions, but I have no experience with this. Someone here is keen to try FCS/FCCS but I'm not sure what we need (both in terms of hardware and software) to get this up and running. Can the FCS/FCCS data be acquired using a standard confocal microscope (we have Zeiss 510, Olympus FV1000 and Nikon A1R confocals with standard PMTs)? If so, what tools are available for the data analysis? If it is necessary to have some kind of special adaptation (different detectors/hardware correlation) what is the most cost effective solution for this? The person wanting to do these exps has suggested using AmCyan and Katushka for FCCS - can anyone forsee any problems with this combo? Thanks in advance for any help.. Simon |
|
|
In reply to this post by simon walker (BI)-2
*****
To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi, Not straightforward FCS but RICS, Enrico Graton has several papers with it in conventional confocals. http://www.lfd.uci.edu/~gratton/ also this one for adapting a confocal http://rsi.aip.org/resource/1/rsinak/v76/i3/p033106_s1 On my PhD I've used Confocor1..with PMTs as far as I remember. Of course detection efficienty is critical. Also, in theory, if you could park the beam and make the image you should be able to do it, I would say. We are know asking Leica to be able to control the galvos for trying with the new hyd detectors. I must say that I'm not an specialist in the subject, I might be simplistic. Regarding software I've used a commercial package www.sstcenter.com. I just found this but never test it. http://research.stowers.org/imagejplugins/ Looks interesting though...its free and I'm cheap, Nuno Moreno CEO @ www.cirklo.org CTO @ www.igc.pt Regarding software I'm not a specialist but I've used for my PhD thesis On Apr 19, 2012, at 11:39 AM, Simon Walker wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Hello, > Apologies for the rather naive questions, but I have no experience with this. > Someone here is keen to try FCS/FCCS but I'm not sure what we need (both > in terms of hardware and software) to get this up and running. Can the > FCS/FCCS data be acquired using a standard confocal microscope (we have > Zeiss 510, Olympus FV1000 and Nikon A1R confocals with standard PMTs)? If > so, what tools are available for the data analysis? If it is necessary to have > some kind of special adaptation (different detectors/hardware correlation) > what is the most cost effective solution for this? > The person wanting to do these exps has suggested using AmCyan and > Katushka for FCCS - can anyone forsee any problems with this combo? > Thanks in advance for any help.. > Simon |
|
|
In reply to this post by simon walker (BI)-2
*****
To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** One more note: AmCyan is a tetramer and Katushka is a dimer. This could cause unwanted interactions of your target proteins or disruption of other interactions. I would recommend sticking with monomeric tags if possible. Jay -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Simon Walker Sent: Thursday, April 19, 2012 5:40 AM To: [hidden email] Subject: FCS/FCCS advice ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hello, Apologies for the rather naive questions, but I have no experience with this. Someone here is keen to try FCS/FCCS but I'm not sure what we need (both in terms of hardware and software) to get this up and running. Can the FCS/FCCS data be acquired using a standard confocal microscope (we have Zeiss 510, Olympus FV1000 and Nikon A1R confocals with standard PMTs)? If so, what tools are available for the data analysis? If it is necessary to have some kind of special adaptation (different detectors/hardware correlation) what is the most cost effective solution for this? The person wanting to do these exps has suggested using AmCyan and Katushka for FCCS - can anyone forsee any problems with this combo? Thanks in advance for any help.. Simon |
|
|
*****
To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Thanks so much to everyone for the advice - exactly what I was hoping for and really helpful. Looks like we'll have to save up for some new detectors then! -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Unruh, Jay Sent: 19 April 2012 16:09 To: [hidden email] Subject: Re: FCS/FCCS advice ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** One more note: AmCyan is a tetramer and Katushka is a dimer. This could cause unwanted interactions of your target proteins or disruption of other interactions. I would recommend sticking with monomeric tags if possible. Jay -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Simon Walker Sent: Thursday, April 19, 2012 5:40 AM To: [hidden email] Subject: FCS/FCCS advice ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hello, Apologies for the rather naive questions, but I have no experience with this. Someone here is keen to try FCS/FCCS but I'm not sure what we need (both in terms of hardware and software) to get this up and running. Can the FCS/FCCS data be acquired using a standard confocal microscope (we have Zeiss 510, Olympus FV1000 and Nikon A1R confocals with standard PMTs)? If so, what tools are available for the data analysis? If it is necessary to have some kind of special adaptation (different detectors/hardware correlation) what is the most cost effective solution for this? The person wanting to do these exps has suggested using AmCyan and Katushka for FCCS - can anyone forsee any problems with this combo? Thanks in advance for any help.. Simon The Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT Registered Charity No. 1053902. The information transmitted in this email is directed only to the addressee. If you received this in error, please contact the sender and delete this email from your system. The contents of this e-mail are the views of the sender and do not necessarily represent the views of the Babraham Institute. Full conditions at: www.babraham.ac.uk<http://www.babraham.ac.uk/email_disclaimer.html> |
|
|
*****
To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hello, FCS and FCCS can be done with the Olympus FV1000, the Zeiss LSM710 or 780 and the Nikon C1 microscope straightforward without any additional hardware. You do need the softweare for data analyis though. You can also perform Raster Image Correlation Spectroscopy (RICS also including ICS, tICS, and STICS) as noted earlier by Nuno as well as the Number and Molecular Brightness Analysis (N&B) for determining aggregation of proteins spatially in an image. For the FCS part I have up loaded a tutorial for the FV1000. Just copy and paste this link to access the file: https://webfiles.uci.edu/xythoswfs/webui/_xy-8018478_1-t_DWD3mp7i We have many tutorial on line if you want to know more at: http://www.lfd.uci.edu/globals/tutorials/ I hope this helps! Michelle > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Thanks so much to everyone for the advice - exactly what I was hoping for > and really helpful. > Looks like we'll have to save up for some new detectors then! > > -----Original Message----- > From: Confocal Microscopy List [mailto:[hidden email]] > On Behalf Of Unruh, Jay > Sent: 19 April 2012 16:09 > To: [hidden email] > Subject: Re: FCS/FCCS advice > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > One more note: AmCyan is a tetramer and Katushka is a dimer. This could > cause unwanted interactions of your target proteins or disruption of other > interactions. I would recommend sticking with monomeric tags if possible. > > Jay > > -----Original Message----- > From: Confocal Microscopy List [mailto:[hidden email]] > On Behalf Of Simon Walker > Sent: Thursday, April 19, 2012 5:40 AM > To: [hidden email] > Subject: FCS/FCCS advice > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Hello, > Apologies for the rather naive questions, but I have no experience with > this. > Someone here is keen to try FCS/FCCS but I'm not sure what we need (both > in terms of hardware and software) to get this up and running. Can the > FCS/FCCS data be acquired using a standard confocal microscope (we have > Zeiss 510, Olympus FV1000 and Nikon A1R confocals with standard PMTs)? If > so, what tools are available for the data analysis? If it is necessary to > have some kind of special adaptation (different detectors/hardware > correlation) what is the most cost effective solution for this? > The person wanting to do these exps has suggested using AmCyan and > Katushka for FCCS - can anyone forsee any problems with this combo? > Thanks in advance for any help.. > Simon > The Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT > Registered Charity No. 1053902. > The information transmitted in this email is directed only to the > addressee. If you received this in error, please contact the sender and > delete this email from your system. The contents of this e-mail are the > views of the sender and do not necessarily represent the views of the > Babraham Institute. Full conditions at: > www.babraham.ac.uk<http://www.babraham.ac.uk/email_disclaimer.html> > > -- Michelle Digman, Ph.D. University of California, Irvine Laboratory for Fluorescence Dynamics 3204 Natural Sciences II Office:(949) 824-3255 http://www.lfd.uci.edu/ |
|
|
In reply to this post by simon walker (BI)-2
*****
To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hallo, what Michelle said. Our facility has a FV1000 confocal, one of our users was contemplating building a FCS hardware box, in the end went ahead and tried the Raster Image Correlation Spectroscopy on the FV1000 (and published a paper). You can download a fully functional 1-month evaluation copy of the RICS/FCS software from Laboratory for Fluorescence Dynamics, so I think it is definitely worth a try - no money, just time invested. Besides RICS, at least on the FV1000 you can park the beam and do spot scanning, so I think standard-type FCS should be possible (but I have not tried that myself. I agree that detector efficiency is crucial. Zeiss, Olympus and Leica all have their new detectors (GaAsP PMTs or Hybrid detectors) as an upgrade option - I have not talked to Nikon lately, so I do not know if they have one or not. It is my understanding that RICS or FCS analyses are more straightforward if the detection is in photon counting mode. Nikon does not have photon counting (yet?). During our recent Leica STED/confocal demo I used the HyD in photon counting regime and compared to the standard PMT. It is a huge improvement in sensitivity; I have not tried the Zeiss and Olympus GaAsP detectors yet, so I cannot comment on their suitability for photon counting - sometimes increased quantum efficiency comes with increased dark noise. I would love to hear from users who tested them. Stan Vitha Microscopy and Imaging Center Texas A&M University |
«
Return to Confocal Microscopy List
|
1 view|%1 views
| Free forum by Nabble | Edit this page |