Re: CONFOCALMICROSCOPY Digest - 23 Mar 2009 to 24 Mar 2009 (#2009-55)
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Tim Moran |
Re: CONFOCALMICROSCOPY Digest - 23 Mar 2009 to 24 Mar 2009 (#2009-55)
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W From: Confocal Microscopy List To: [hidden email] Sent: Wed Mar 25 01:02:55 2009 Subject: CONFOCALMICROSCOPY Digest - 23 Mar 2009 to 24 Mar 2009 (#2009-55)
I've been getting this error when i shoot larger z stacks and when i shoot longer time series Serious Error: NO file could be found matching the specification 'C:\Temp\untitled\untitled1_raw*.pic' then i either click ok or close the dialog box and lasersharp 2000 closes/crashes but when i shoot smaller stacks this does NOT happen any help would be greatly appreciated ive tried deleting the C:\Temp folder and this does nothing The reason i need this to finish is so that i can then export it to my desired location on a different server. Thank You --- Matiar Jafari I've been getting this error when i shoot larger z stacks and when i shoot longer time series Serious Error: NO file could be found matching the specification 'C:\Temp\untitled\untitled1_raw*.pic' then i either click ok or close the dialog box and lasersharp 2000 closes/crashes but when i shoot smaller stacks this does NOT happen any help would be greatly appreciated ive tried deleting the C:\Temp folder and this does nothing The reason i need this to finish is so that i can then export it to my desired location on a different server. Thanks in advance At 02:50 24.03.2009, you wrote: >Hi All, > >Thanks for all the responses. Looks like it is >time to lube up the objective and...... > >I have got hold of some Ultrasound gel from the >hospital here. The stuff i have is a pale blue >colour. Is this like the stuff you have Steffen? Nope. Our hospital has two kinds, the "normal" one to which my previous post applies is colorless. They also have another one, extra-viscous, which is blue. But when I saw on the label of the blue one that it contains 80% Glycerol, I didn't even try that. No idea whether this is some kind of international color code or whether it changes from one block to the next. Alternatively, you may be able to mix it yourself: Order 1,2 Propylenglycol and extract the superabsorber from a disposable diaper (unused).... BTW, apparently KY-Jelly is a brand better known in some countries than in others, anyway I had to look it up. The respective Wikipedia article says "K-Y Jelly's original stated purpose was as a <http://en.wikipedia.org/wiki/Surgical_lubricant>surgical lubricant, and it was often chosen by doctors because of its natural base. " I didn't even know that surgical lubricants exist, but if such a thing is still in use today, it might also be an alternative for immersion. 'Ask your local doctor or pharmacist if you have any questions or concerns about using this medicine...' Happy lubricating Steffen >Cheers > > >Cam -- --------------------------------------------------------------------------------------------------- Steffen Dietzel, PD Dr. rer. nat Ludwig-Maximilians-Universität München Walter-Brendel-Zentrum für experimentelle Medizin (WBex) Head of light microscopy Mail room (for letters etc.): Marchioninistr. 15, D-81377 München Building location and address for courier, parcel services etc: Marchioninistr. 27, D-81377 München (Großhadern) Phone: +49/89/2180-76509 Fax-to-email: +49/89/2180-9976509 KY jelly is used mostly as a sexual rather than surgical lubricant, but its function is the same in both cases. I'm sure an equivalent product can be found in any pharmacy. It'll be next to the condoms. Guy Optical Imaging Techniques in Cell Biology by Guy Cox CRC Press / Taylor & Francis http://www.guycox.com/optical.htm ______________________________________________ Associate Professor Guy Cox, MA, DPhil(Oxon) Electron Microscope Unit, Madsen Building F09, University of Sydney, NSW 2006 ______________________________________________ Phone +61 2 9351 3176 Fax +61 2 9351 7682 Mobile 0413 281 861 ______________________________________________ http://www.guycox.net -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Steffen Dietzel Sent: Tuesday, 24 March 2009 8:39 PM To: [hidden email] Subject: Re: Optical Coupling Gels At 02:50 24.03.2009, you wrote: >Hi All, > >Thanks for all the responses. Looks like it is time to lube up the >objective and...... > >I have got hold of some Ultrasound gel from the hospital here. The >stuff i have is a pale blue colour. Is this like the stuff you have >Steffen? Nope. Our hospital has two kinds, the "normal" one to which my previous post applies is colorless. They also have another one, extra-viscous, which is blue. But when I saw on the label of the blue one that it contains 80% Glycerol, I didn't even try that. No idea whether this is some kind of international color code or whether it changes from one block to the next. Alternatively, you may be able to mix it yourself: Order 1,2 Propylenglycol and extract the superabsorber from a disposable diaper (unused).... BTW, apparently KY-Jelly is a brand better known in some countries than in others, anyway I had to look it up. The respective Wikipedia article says "K-Y Jelly's original stated purpose was as a <http://en.wikipedia.org/wiki/Surgical_lubricant>surgical lubricant, and it was often chosen by doctors because of its natural base. " I didn't even know that surgical lubricants exist, but if such a thing is still in use today, it might also be an alternative for immersion. 'Ask your local doctor or pharmacist if you have any questions or concerns about using this medicine...' Happy lubricating Steffen >Cheers > > >Cam -- --------------------------------------------------------------------------------------------------- Steffen Dietzel, PD Dr. rer. nat Ludwig-Maximilians-Universität München Walter-Brendel-Zentrum für experimentelle Medizin (WBex) Head of light microscopy Mail room (for letters etc.): Marchioninistr. 15, D-81377 München Building location and address for courier, parcel services etc: Marchioninistr. 27, D-81377 München (Großhadern) Phone: +49/89/2180-76509 Fax-to-email: +49/89/2180-9976509 No virus found in this incoming message. Checked by AVG. Version: 7.5.557 / Virus Database: 270.11.24/2018 - Release Date: 23/03/2009 6:52 AM No virus found in this outgoing message. Checked by AVG. Version: 7.5.557 / Virus Database: 270.11.24/2018 - Release Date: 23/03/2009 6:52 AM Another one for 1st thing in the morning Sally Dowling, PhD Technical sales BD Bioimaging 301-351-0524 www.bd.com/bioimaging ----- Original Message ----- From: Steffen Dietzel [[hidden email]] Sent: 03/24/2009 10:39 AM CET To: [hidden email] Subject: Re: Optical Coupling Gels At 02:50 24.03.2009, you wrote: >Hi All, > >Thanks for all the responses. Looks like it is >time to lube up the objective and...... > >I have got hold of some Ultrasound gel from the >hospital here. The stuff i have is a pale blue >colour. Is this like the stuff you have Steffen? Nope. Our hospital has two kinds, the "normal" one to which my previous post applies is colorless. They also have another one, extra-viscous, which is blue. But when I saw on the label of the blue one that it contains 80% Glycerol, I didn't even try that. No idea whether this is some kind of international color code or whether it changes from one block to the next. Alternatively, you may be able to mix it yourself: Order 1,2 Propylenglycol and extract the superabsorber from a disposable diaper (unused).... BTW, apparently KY-Jelly is a brand better known in some countries than in others, anyway I had to look it up. The respective Wikipedia article says "K-Y Jelly's original stated purpose was as a <http://en.wikipedia.org/wiki/Surgical_lubricant>surgical lubricant, and it was often chosen by doctors because of its natural base. " I didn't even know that surgical lubricants exist, but if such a thing is still in use today, it might also be an alternative for immersion. 'Ask your local doctor or pharmacist if you have any questions or concerns about using this medicine...' Happy lubricating Steffen >Cheers > > >Cam -- --------------------------------------------------------------------------------------------------- Steffen Dietzel, PD Dr. rer. nat Ludwig-Maximilians-Universität München Walter-Brendel-Zentrum für experimentelle Medizin (WBex) Head of light microscopy Mail room (for letters etc.): Marchioninistr. 15, D-81377 München Building location and address for courier, parcel services etc: Marchioninistr. 27, D-81377 München (Großhadern) Phone: +49/89/2180-76509 Fax-to-email: +49/89/2180-9976509 ----------------------------------------- ******************************************************************* IMPORTANT MESSAGE FOR RECIPIENTS IN THE U.S.A.: This message may constitute an advertisement of a BD group's products or services or a solicitation of interest in them. If this is such a message and you would like to opt out of receiving future advertisements or solicitations from this BD group, please forward this e-mail to [hidden email]. ******************************************************************* This message (which includes any attachments) is intended only for the designated recipient(s). It may contain confidential or proprietary information and may be subject to the attorney-client privilege or other confidentiality protections. If you are not a designated recipient, you may not review, use, copy or distribute this message. If you received this in error, please notify the sender by reply e-mail and delete this message. Thank you. ******************************************************************* Corporate Headquarters Mailing Address: BD (Becton, Dickinson and Company) 1 Becton Drive Franklin Lakes, NJ 07417 U.S.A. ******************************************************************* Is this to combine with Stephen Cody's "special" technique? It could raise some eyebrows with lab shelves stocked with these items.. Simon -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Guy Cox Sent: 24 March 2009 09:48 To: [hidden email] Subject: Re: Optical Coupling Gels KY jelly is used mostly as a sexual rather than surgical lubricant, but its function is the same in both cases. I'm sure an equivalent product can be found in any pharmacy. It'll be next to the condoms. Guy Optical Imaging Techniques in Cell Biology by Guy Cox CRC Press / Taylor & Francis http://www.guycox.com/optical.htm ______________________________________________ Associate Professor Guy Cox, MA, DPhil(Oxon) Electron Microscope Unit, Madsen Building F09, University of Sydney, NSW 2006 ______________________________________________ Phone +61 2 9351 3176 Fax +61 2 9351 7682 Mobile 0413 281 861 ______________________________________________ http://www.guycox.net -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Steffen Dietzel Sent: Tuesday, 24 March 2009 8:39 PM To: [hidden email] Subject: Re: Optical Coupling Gels At 02:50 24.03.2009, you wrote: >Hi All, > >Thanks for all the responses. Looks like it is time to lube up the >objective and...... > >I have got hold of some Ultrasound gel from the hospital here. The >stuff i have is a pale blue colour. Is this like the stuff you have >Steffen? Nope. Our hospital has two kinds, the "normal" one to which my previous post applies is colorless. They also have another one, extra-viscous, which is blue. But when I saw on the label of the blue one that it contains 80% Glycerol, I didn't even try that. No idea whether this is some kind of international color code or whether it changes from one block to the next. Alternatively, you may be able to mix it yourself: Order 1,2 Propylenglycol and extract the superabsorber from a disposable diaper (unused).... BTW, apparently KY-Jelly is a brand better known in some countries than in others, anyway I had to look it up. The respective Wikipedia article says "K-Y Jelly's original stated purpose was as a <http://en.wikipedia.org/wiki/Surgical_lubricant>surgical lubricant, and it was often chosen by doctors because of its natural base. " I didn't even know that surgical lubricants exist, but if such a thing is still in use today, it might also be an alternative for immersion. 'Ask your local doctor or pharmacist if you have any questions or concerns about using this medicine...' Happy lubricating Steffen >Cheers > > >Cam -- --------------------------------------------------------------------------------------------------- Steffen Dietzel, PD Dr. rer. nat Ludwig-Maximilians-Universität München Walter-Brendel-Zentrum für experimentelle Medizin (WBex) Head of light microscopy Mail room (for letters etc.): Marchioninistr. 15, D-81377 München Building location and address for courier, parcel services etc: Marchioninistr. 27, D-81377 München (Großhadern) Phone: +49/89/2180-76509 Fax-to-email: +49/89/2180-9976509 No virus found in this incoming message. Checked by AVG. Version: 7.5.557 / Virus Database: 270.11.24/2018 - Release Date: 23/03/2009 6:52 AM No virus found in this outgoing message. Checked by AVG. Version: 7.5.557 / Virus Database: 270.11.24/2018 - Release Date: 23/03/2009 6:52 AM Sorry, didn't mean to reply to group, too early for me to get a good chuckle from the banter, perhaps! Apologies, sally Sally Dowling, PhD Technical sales BD Bioimaging 301-351-0524 www.bd.com/bioimaging ----- Original Message ----- From: Sally_Dowling Sent: 03/24/2009 12:22 PM CET To: [hidden email] Subject: Re: Optical Coupling Gels Another one for 1st thing in the morning Sally Dowling, PhD Technical sales BD Bioimaging 301-351-0524 www.bd.com/bioimaging ----- Original Message ----- From: Steffen Dietzel [[hidden email]] Sent: 03/24/2009 10:39 AM CET To: [hidden email] Subject: Re: Optical Coupling Gels At 02:50 24.03.2009, you wrote: >Hi All, > >Thanks for all the responses. Looks like it is >time to lube up the objective and...... > >I have got hold of some Ultrasound gel from the >hospital here. The stuff i have is a pale blue >colour. Is this like the stuff you have Steffen? Nope. Our hospital has two kinds, the "normal" one to which my previous post applies is colorless. They also have another one, extra-viscous, which is blue. But when I saw on the label of the blue one that it contains 80% Glycerol, I didn't even try that. No idea whether this is some kind of international color code or whether it changes from one block to the next. Alternatively, you may be able to mix it yourself: Order 1,2 Propylenglycol and extract the superabsorber from a disposable diaper (unused).... BTW, apparently KY-Jelly is a brand better known in some countries than in others, anyway I had to look it up. The respective Wikipedia article says "K-Y Jelly's original stated purpose was as a <http://en.wikipedia.org/wiki/Surgical_lubricant>surgical lubricant, and it was often chosen by doctors because of its natural base. " I didn't even know that surgical lubricants exist, but if such a thing is still in use today, it might also be an alternative for immersion. 'Ask your local doctor or pharmacist if you have any questions or concerns about using this medicine...' Happy lubricating Steffen >Cheers > > >Cam -- --------------------------------------------------------------------------------------------------- Steffen Dietzel, PD Dr. rer. nat Ludwig-Maximilians-Universität München Walter-Brendel-Zentrum für experimentelle Medizin (WBex) Head of light microscopy Mail room (for letters etc.): Marchioninistr. 15, D-81377 München Building location and address for courier, parcel services etc: Marchioninistr. 27, D-81377 München (Großhadern) Phone: +49/89/2180-76509 Fax-to-email: +49/89/2180-9976509 ----------------------------------------- ******************************************************************* IMPORTANT MESSAGE FOR RECIPIENTS IN THE U.S.A.: This message may constitute an advertisement of a BD group's products or services or a solicitation of interest in them. If this is such a message and you would like to opt out of receiving future advertisements or solicitations from this BD group, please forward this e-mail to [hidden email]. ******************************************************************* This message (which includes any attachments) is intended only for the designated recipient(s). It may contain confidential or proprietary information and may be subject to the attorney-client privilege or other confidentiality protections. If you are not a designated recipient, you may not review, use, copy or distribute this message. If you received this in error, please notify the sender by reply e-mail and delete this message. Thank you. ******************************************************************* Corporate Headquarters Mailing Address: BD (Becton, Dickinson and Company) 1 Becton Drive Franklin Lakes, NJ 07417 U.S.A. ******************************************************************* Actually I've never used Stephen's condom technique, but we use KY jelly all the time in plant physiology. You can seal a chamber to a leaf with it to measure gas exchange, for example, and just rinse it off afterwards. But a bottle lasts a long time (much longer than one used as intended!) so there's not much embarrassment factor involved. I'd never thought to check its optical properties. As I said before, since it's intended for use in body cavities I'd have thought it should be close to isotonic, but I don't know. Guy Optical Imaging Techniques in Cell Biology by Guy Cox CRC Press / Taylor & Francis http://www.guycox.com/optical.htm ______________________________________________ Associate Professor Guy Cox, MA, DPhil(Oxon) Electron Microscope Unit, Madsen Building F09, University of Sydney, NSW 2006 ______________________________________________ Phone +61 2 9351 3176 Fax +61 2 9351 7682 Mobile 0413 281 861 ______________________________________________ http://www.guycox.net -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of simon walker (BI) Sent: Tuesday, 24 March 2009 10:37 PM To: [hidden email] Subject: Re: Optical Coupling Gels Is this to combine with Stephen Cody's "special" technique? It could raise some eyebrows with lab shelves stocked with these items.. Simon -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Guy Cox Sent: 24 March 2009 09:48 To: [hidden email] Subject: Re: Optical Coupling Gels KY jelly is used mostly as a sexual rather than surgical lubricant, but its function is the same in both cases. I'm sure an equivalent product can be found in any pharmacy. It'll be next to the condoms. Guy Optical Imaging Techniques in Cell Biology by Guy Cox CRC Press / Taylor & Francis http://www.guycox.com/optical.htm ______________________________________________ Associate Professor Guy Cox, MA, DPhil(Oxon) Electron Microscope Unit, Madsen Building F09, University of Sydney, NSW 2006 ______________________________________________ Phone +61 2 9351 3176 Fax +61 2 9351 7682 Mobile 0413 281 861 ______________________________________________ http://www.guycox.net -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Steffen Dietzel Sent: Tuesday, 24 March 2009 8:39 PM To: [hidden email] Subject: Re: Optical Coupling Gels At 02:50 24.03.2009, you wrote: >Hi All, > >Thanks for all the responses. Looks like it is time to lube up the >objective and...... > >I have got hold of some Ultrasound gel from the hospital here. The >stuff i have is a pale blue colour. Is this like the stuff you have >Steffen? Nope. Our hospital has two kinds, the "normal" one to which my previous post applies is colorless. They also have another one, extra-viscous, which is blue. But when I saw on the label of the blue one that it contains 80% Glycerol, I didn't even try that. No idea whether this is some kind of international color code or whether it changes from one block to the next. Alternatively, you may be able to mix it yourself: Order 1,2 Propylenglycol and extract the superabsorber from a disposable diaper (unused).... BTW, apparently KY-Jelly is a brand better known in some countries than in others, anyway I had to look it up. The respective Wikipedia article says "K-Y Jelly's original stated purpose was as a <http://en.wikipedia.org/wiki/Surgical_lubricant>surgical lubricant, and it was often chosen by doctors because of its natural base. " I didn't even know that surgical lubricants exist, but if such a thing is still in use today, it might also be an alternative for immersion. 'Ask your local doctor or pharmacist if you have any questions or concerns about using this medicine...' Happy lubricating Steffen >Cheers > > >Cam -- --------------------------------------------------------------------------------------------------- Steffen Dietzel, PD Dr. rer. nat Ludwig-Maximilians-Universität München Walter-Brendel-Zentrum für experimentelle Medizin (WBex) Head of light microscopy Mail room (for letters etc.): Marchioninistr. 15, D-81377 München Building location and address for courier, parcel services etc: Marchioninistr. 27, D-81377 München (Großhadern) Phone: +49/89/2180-76509 Fax-to-email: +49/89/2180-9976509 No virus found in this incoming message. Checked by AVG. Version: 7.5.557 / Virus Database: 270.11.24/2018 - Release Date: 23/03/2009 6:52 AM No virus found in this outgoing message. Checked by AVG. Version: 7.5.557 / Virus Database: 270.11.24/2018 - Release Date: 23/03/2009 6:52 AM No virus found in this incoming message. Checked by AVG. Version: 7.5.557 / Virus Database: 270.11.24/2018 - Release Date: 23/03/2009 6:52 AM No virus found in this outgoing message. Checked by AVG. Version: 7.5.557 / Virus Database: 270.11.24/2018 - Release Date: 23/03/2009 6:52 AM Folks, As many of you know, Quantitative Fluorescence
Microscopy is a course run at the Mount Desert Island Bio Labs annually. This
is an intensive lab/lecture course, focusing on fluorescence microscopy in all
its forms, including widefield, Confocal, Multiphoton, Decon, TIRF, FRET etc,
though with an emphasis on live cell methods. We also encourage students
to provide their own specimens such that the hands on components of the course
are as enriching as possible. While we have had a healthy applicant pool, we
have also had a surprising number of dropouts/cancellations,(we are guessing
that the economy isnt exactly helping).... Right now we need to fill 3 of the
32 slots, so if you or anyone you work with/for might be interested please
encourage them to apply. The dates for course are from Saturday May
30th 2009 to the following Friday (June 5th) . The url for the course is http://www.cbi.pitt.edu/qfm/index.html Thanks Simon Simon C.
Watkins Ph.D, FRCPath Professor and Vice Chair, Cell Biology and Physiology Professor, Immunology Director, Center for Biologic Imaging BSTS 225, University of Pittsburgh 3500 Terrace St. Pittsburgh PA 15261 Tel: 412-352-2277 Fax:412-648-2797 URL: http://www.cbi.pitt.edu Try increasing the memory allocated to image acquisition. In Tools select System Config and set memory to size smaller than Computer RAM. Also make sure your computer hard drive has plenty of space. You cannot collect images/stacks greater than the number set in System Config. good luck, michael On Mar 24, 2009, at 2:57 AM, Matiar Jafari wrote: > I've been getting this error when i shoot larger z stacks and when i > shoot longer time series > > Serious Error: NO file could be found matching the specification > 'C:\Temp\untitled\untitled1_raw*.pic' > > then i either click ok or close the dialog box and lasersharp 2000 > closes/crashes > > but when i shoot smaller stacks this does NOT happen any help would be > greatly appreciated > > ive tried deleting the C:\Temp folder and this does nothing > > The reason i need this to finish is so that i can then export it to my > desired location on a different server. > > Thank You > --- > Matiar Jafari [hidden email] Won't this hysteresis play havoc with software-based autofocus routines? Any ideas on compatibility with such techniques? Saludos Chris IBt/UNAM On Mon, 23 Mar 2009 08:56:00 -0400, John Oreopoulos <[hidden email]> wrote: >We purchased a bottle of this product a few months back and I've used >it a few times when doing live-cell imaging. One thing I noticed that >was different when using this compared to regular immersion oil for >fluorescence was that there was a short lag time in terms of >adjusting the focus of the microscope and the image coming into focus >on my preview computer screen (and in the eyepieces). I guess this >has to do with the viscosity of the oil. It took a while to get >adjusted to this because I would keep overshooting the fine focus, >but other than that it works great (no commercial interest). > >John Oreopoulos > > >On 23-Mar-09, at 6:42 AM, Christophe Leterrier wrote: > >> I would like to point out that Cargille has a new oil for fluorescence >> imaging at 37°C, called 37DF, that has very low auto fluorescence. It >> is not on their website, but I could get the product pdf sent to me by >> a Cargille person, so I can send it if someone is interested. I'm >> trying to get some here in France, but none of the french Cargille >> distributors has heard about it yet... It has been found to be quite >> good for fluorescence imaging of live cells at 37°C, according to this >> paper in J Microscopy : >> >> Immersion oil for high-resolution live-cell imaging at 37 degrees C: >> optical and physical characteristics. >> Oomen LC, Sacher R, Brocks HH, Zwier JM, Brakenhoff GJ, Jalink K. >> J Microsc. 2008 Nov;232(2):353-61. >> >> >> On Mon, Mar 23, 2009 at 11:18, Keith Morris >> <[hidden email]> wrote: >>> >>> Hi all, >>> >>> >>> >>> Using immersion oil at 37oC. >>> >>> >>> >>> Zeiss sell a range of low fluorescence immersion oils: bottles of >>> 20, 250 and 500 ml, unfortunately all filled with the same 518F >>> oil. According to Zeiss it’s apparently ‘compatible’ with our >>> Zeiss microscopes, but little extra information is offered on the >>> Zeiss website. >>> >>> >>> >>> The only info I can gleam from the web [other than the ubiquitous >>> 518F safety data Sheet] is the following >>> >>> Immersion oil "Immersol" 518 F fluorescence free, (ISO 8036-1/2, n >>> (e)=1.518 (23°C), halogen-free) >>> >>> >>> >>> So not much comment about 37oC use. PeCon’s site [who supply most >>> of Zeiss’s decent incubator stuff] have no opinion on the matter >>> either. >>> >>> >>> >>> The 518F oil safety sheet mentions “Use of the Substance / >>> Preparation: For application information, consult the processing >>> instructions. Processing instructions or technical >>> >>> information sheet available on request.”. I’ll ask our >>> knowledgeable local Zeiss Confocal rep for more details, as we are >>> just installing a Zeiss/PeCon 37oC live cell incubator here on our >>> Zeiss 510 MetaHead confocal. >>> >>> Cargilee’s site in comparison is passionate about all things >>> immersion oil: >>> http://www.cargille.com/immeroil.shtml >>> http://www.cargille.com/immeroilselection.shtml >>> and their excellent, if elderly, ‘primer’ >>> http://www.cargille.com/immersionoilmicroscope.shtml >>> >>> From postings to this server back in 2006, and Cargille’s site not >>> mentioning ‘non-fluorescence’ for type 37, it appears that >>> Cargille 37 probably autofluoresces, so it’s likely better for >>> live cell phase-contrast [halogen lamp] transmission microscopy >>> [but then often so are dry objectives]. I’ll stick with standard >>> 518F immersion oil at 37oC though, it’s always imaged well enough >>> on Zeiss microscopes at that temperature [and at least at 37oC it >>> never became cloudy due to crystal precipitation]. Similarly the >>> Cargille DF and FF seem fine to me at 37oC, and we stick >>> ruthlessly with the same brand on each microscope. A lot of our >>> live cell work is/was done with low power [20x Phase] air >>> objectives though. >>> >>> I do notice that immersion oil often ingresses into oil immersion >>> objective internal optics with time, although strangely it rarely >>> notices when imaging [like dirt on much of the internal optics, >>> you must mostly focus through it]. One microscopy core manager >>> even commented to me that he considers oil objectives >>> ‘consumables’. The cost of ‘repair’ [often the same price as a new >>> objective] is so high we frequently have to live with it [I advise >>> users that our say 40x objective image quality is suspect for this >>> reason]. In truth we don’t notice any drop in image quality when >>> it must have happened, although you can see it ‘smeared’ on the >>> optics when looking through the removed objective, using a >>> magnifying glass, bright room light and a ‘cleaned’ objective top >>> lens. >>> >>> Old listerver posting on Cargille 37 attached below. >>> >>> Regards >>> >>> Keith >>> >>> PS missed this link on the last post: >>> >>> Dissolving crystals in Zeiss 518F oil >>> http://www.zeiss.com/C1256F8500454979/0/ >>> E8CD09DFA520787FC1256F86004B5FFB/$file/immersol-crystals.pdf >>> >>> --------------------------------------------------------------------- >>> ------ >>> Dr Keith J. Morris, >>> Molecular Cytogenetics and Microscopy Core, >>> Laboratory 00/069 and 00/070, >>> The Wellcome Trust Centre for Human Genetics, >>> Roosevelt Drive, >>> Oxford OX3 7BN, >>> United Kingdom. >>> >>> Telephone: +44 (0)1865 287568 >>> Email: [hidden email] >>> Web-pages: http://www.well.ox.ac.uk/cytogenetics/ >>> >>> >>> >>> Subject: >>> >>> Re: Fluorescence from Type 37 Immersion Oil >>> >>> From: >>> >>> Vitaly Boyko <[hidden email]> >>> >>> Reply-To: >>> >>> Vitaly Boyko <[hidden email]> >>> >>> Date: >>> >>> Wed, 16 Aug 2006 10:23:20 -0400 >>> >>> Content-Type: >>> >>> text/plain >>> >>> >>> >>> Search the CONFOCAL archive at >>> >>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >>> >>> >>> >>> PS What would be more appropriate to compare is Zeiss 518F (not >>> 518C!) with >>> >>> the Nikon type A or Cargille type DF. >>> >>> >>> >>> >>> >>> >>> >>> Hi Jason, >>> >>> >>> >>> I wonder why you have tested the Cargille FF imm. oil, as the >>> refractive >>> >>> index of FF oil is too far off for the standard glass >>> (1.518) ?????!!!!! >>> >>> >>> >>> Actually, I haven't noticed the "very bad" autofluorescence of the >>> Cargille >>> >>> type DF versus Zeiss 518 in the CFP, YFP or FRET channels. The >>> difference is >>> >>> less than 10%. >>> >>> >>> >>> The problem with the Nikon oil is its dispenser and/or viscosity - >>> it is >>> >>> often annoying "fighting" with the air bubbles. >>> >>> >>> >>> What is about the Invitrogen/Mol.Probes mounting media >>> autofluorescence? >>> >>> >>> >>> I am fed up with the irreproducibility of the background >>> autofluorescence >>> >>> using the ProlongGold mounting media!!! That is a serious problem >>> compared >>> >>> to the issue of the immersion oils. >>> >>> >>> >>> Cheers, >>> >>> >>> >>> Vitaly >>> >>> >>> >>> NCI-Frederick >>> >>> >>> >>> >>> >>> ----- Original Message ----- >>> >>> From: "Kilgore, Jason" <[hidden email]> >>> >>> To: <[hidden email]> >>> >>> Sent: Tuesday, August 15, 2006 7:26 PM >>> >>> Subject: Re: Fluorescence from Type 37 Immersion Oil >>> >>> >>> >>> >>> >>>> Search the CONFOCAL archive at >>> >>>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >>> >>>> >>> >>>> Back in 2000 I performed a comparison of four different immersion >>>> oils >>> >>>> (Zeiss 518C, Nikon Type A, Cargille Type DF, and Cargille Type >>>> FF) for >>> >>>> autofluorescence (using DAPI, FITC, acridine orange, and TRITC >>>> filter >>> >>>> sets) and general resolution at 60x on a Nikon E400. I didn't test >>> >>>> Cargille 37. >>> >>>> >>> >>>> Green autofluorescence tended to be worst for all four, but was >>>> least >>> >>>> for Cargille FF, followed by Nikon Type A, then Zeiss, then (very >>>> bad) >>> >>>> Cargille DF. However, the resolution was unacceptable for >>>> Cargille FF. >>> >>>> Autofluorescence in the other channels was moderate (DAPI, TRITC) to >>> >>>> very low (acridine orange), with the same comparative trend >>>> applying. >>> >>>> >>> >>>> Thus, ever since we have been using Nikon Type A with no >>>> complaints for >>> >>>> most of our imaging. >>> >>>> >>> >>>> Feel free to contact me personally and I can send you the data. >>> >>>> >>> >>>> Jason >>> >>>> >>> >>>> Jason A. Kilgore >>> >>>> Cell Biology / Histology >>> >>>> Molecular Probes/Invitrogen >>> >>>> [hidden email] >>> >>>> >>> >>>> >>> >>>> -----Original Message----- >>> >>>> From: Confocal Microscopy List >>>> [mailto:[hidden email]] On >>> >>>> Behalf Of S. Brunet >>> >>>> Sent: Tuesday, August 15, 2006 11:19 AM >>> >>>> To: [hidden email] >>> >>>> Subject: Fluorescence from Type 37 Immersion Oil >>> >>>> >>> >>>> Search the CONFOCAL archive at >>> >>>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >>> >>>> >>> >>>> Hello to all, >>> >>>> >>> >>>> We recently began using the Cargille 37 oil with the temperature >>>> chamber >>> >>>> on the >>> >>>> microscope. I found significant fluorescence from the oil when >>>> exposed >>> >>>> to the >>> >>>> HBO lamp (I could not see the eYFP because of the blue background). >>> >>>> >>> >>>> So I used the 20x air objective and I was able to obtain an image >>>> from >>> >>>> the >>> >>>> fluorescence from a drop of 37 oil placed on a coverslip as a sample >>> >>>> (excitation at 488 or 458nm and pinhole at the usual confocal >>>> setting). >>> >>>> >>> >>>> Is this the norm? >>> >>>> >>> >>>> Regards, >>> >>>> Sophie >>> >>>> _______________________________________ >>> >>>> Sophie M.K. Brunet, Ph.D. >>> >>>> Research Officer >>> >>>> Optical Spectroscopist, Laser Systems and Applications >>> >>>> [hidden email] >>> >>>> 306-966-1719 (office) 306-966-1702 (fax) >>> >>>> _______________________________________ >>> >>>> Saskatchewan Structural Sciences Centre >>> >>>> University of Saskatchewan >>> >>>> Thorvaldson Bldg. >>> >>>> 110 Science Place >>> >>>> Saskatoon, SK S7N 5C9 >>> >>>> >>> >>> Dear list, This is a commercial response. Nikon's 'Perfect Focus System' corrects for focus drift by moving an internal lens located inside the PFS unit. Because the nosepiece does not move, viscosity of the immersion medium does not present a problem. Regarding Nikon immersion oil, my experience is that bubbles usually disappear after a few seconds and stubborn bubbles can be burst by touching them with the tip the applicator. Bubbles are rarely seen when the 8cc applicator is used. Jeffrey Larson Product Manager Nikon Confocal Systems Nikon Instruments Inc. (631) 547-8540 -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Chris Wood Sent: Tuesday, March 24, 2009 11:39 AM To: [hidden email] Subject: Re: Immersion Oil - 37oC Won't this hysteresis play havoc with software-based autofocus routines? Any ideas on compatibility with such techniques? Saludos Chris IBt/UNAM On Mon, 23 Mar 2009 08:56:00 -0400, John Oreopoulos <[hidden email]> wrote: >We purchased a bottle of this product a few months back and I've used >it a few times when doing live-cell imaging. One thing I noticed that >was different when using this compared to regular immersion oil for >fluorescence was that there was a short lag time in terms of >adjusting the focus of the microscope and the image coming into focus >on my preview computer screen (and in the eyepieces). I guess this >has to do with the viscosity of the oil. It took a while to get >adjusted to this because I would keep overshooting the fine focus, >but other than that it works great (no commercial interest). > >John Oreopoulos > > >On 23-Mar-09, at 6:42 AM, Christophe Leterrier wrote: > >> I would like to point out that Cargille has a new oil for fluorescence >> imaging at 37°C, called 37DF, that has very low auto fluorescence. It >> is not on their website, but I could get the product pdf sent to me by >> a Cargille person, so I can send it if someone is interested. I'm >> trying to get some here in France, but none of the french Cargille >> distributors has heard about it yet... It has been found to be quite >> good for fluorescence imaging of live cells at 37°C, according to this >> paper in J Microscopy : >> >> Immersion oil for high-resolution live-cell imaging at 37 degrees C: >> optical and physical characteristics. >> Oomen LC, Sacher R, Brocks HH, Zwier JM, Brakenhoff GJ, Jalink K. >> J Microsc. 2008 Nov;232(2):353-61. >> >> >> On Mon, Mar 23, 2009 at 11:18, Keith Morris >> <[hidden email]> wrote: >>> >>> Hi all, >>> >>> >>> >>> Using immersion oil at 37oC. >>> >>> >>> >>> Zeiss sell a range of low fluorescence immersion oils: bottles of >>> 20, 250 and 500 ml, unfortunately all filled with the same 518F >>> oil. According to Zeiss it's apparently 'compatible' with our >>> Zeiss microscopes, but little extra information is offered on the >>> Zeiss website. >>> >>> >>> >>> The only info I can gleam from the web [other than the ubiquitous >>> 518F safety data Sheet] is the following >>> >>> Immersion oil "Immersol" 518 F fluorescence free, (ISO 8036-1/2, n >>> (e)=1.518 (23°C), halogen-free) >>> >>> >>> >>> So not much comment about 37oC use. PeCon's site [who supply most >>> of Zeiss's decent incubator stuff] have no opinion on the matter >>> either. >>> >>> >>> >>> The 518F oil safety sheet mentions "Use of the Substance / >>> Preparation: For application information, consult the processing >>> instructions. Processing instructions or technical >>> >>> information sheet available on request.". I'll ask our >>> knowledgeable local Zeiss Confocal rep for more details, as we are >>> just installing a Zeiss/PeCon 37oC live cell incubator here on our >>> Zeiss 510 MetaHead confocal. >>> >>> Cargilee's site in comparison is passionate about all things >>> immersion oil: >>> http://www.cargille.com/immeroil.shtml >>> http://www.cargille.com/immeroilselection.shtml >>> and their excellent, if elderly, 'primer' >>> http://www.cargille.com/immersionoilmicroscope.shtml >>> >>> From postings to this server back in 2006, and Cargille's site not >>> mentioning 'non-fluorescence' for type 37, it appears that >>> Cargille 37 probably autofluoresces, so it's likely better for >>> live cell phase-contrast [halogen lamp] transmission microscopy >>> [but then often so are dry objectives]. I'll stick with standard >>> 518F immersion oil at 37oC though, it's always imaged well enough >>> on Zeiss microscopes at that temperature [and at least at 37oC it >>> never became cloudy due to crystal precipitation]. Similarly the >>> Cargille DF and FF seem fine to me at 37oC, and we stick >>> ruthlessly with the same brand on each microscope. A lot of our >>> live cell work is/was done with low power [20x Phase] air >>> objectives though. >>> >>> I do notice that immersion oil often ingresses into oil immersion >>> objective internal optics with time, although strangely it rarely >>> notices when imaging [like dirt on much of the internal optics, >>> you must mostly focus through it]. One microscopy core manager >>> even commented to me that he considers oil objectives >>> 'consumables'. The cost of 'repair' [often the same price as a new >>> objective] is so high we frequently have to live with it [I advise >>> users that our say 40x objective image quality is suspect for this >>> reason]. In truth we don't notice any drop in image quality when >>> it must have happened, although you can see it 'smeared' on the >>> optics when looking through the removed objective, using a >>> magnifying glass, bright room light and a 'cleaned' objective top >>> lens. >>> >>> Old listerver posting on Cargille 37 attached below. >>> >>> Regards >>> >>> Keith >>> >>> PS missed this link on the last post: >>> >>> Dissolving crystals in Zeiss 518F oil >>> http://www.zeiss.com/C1256F8500454979/0/ >>> E8CD09DFA520787FC1256F86004B5FFB/$file/immersol-crystals.pdf >>> >>> --------------------------------------------------------------------- >>> ------ >>> Dr Keith J. Morris, >>> Molecular Cytogenetics and Microscopy Core, >>> Laboratory 00/069 and 00/070, >>> The Wellcome Trust Centre for Human Genetics, >>> Roosevelt Drive, >>> Oxford OX3 7BN, >>> United Kingdom. >>> >>> Telephone: +44 (0)1865 287568 >>> Email: [hidden email] >>> Web-pages: http://www.well.ox.ac.uk/cytogenetics/ >>> >>> >>> >>> Subject: >>> >>> Re: Fluorescence from Type 37 Immersion Oil >>> >>> From: >>> >>> Vitaly Boyko <[hidden email]> >>> >>> Reply-To: >>> >>> Vitaly Boyko <[hidden email]> >>> >>> Date: >>> >>> Wed, 16 Aug 2006 10:23:20 -0400 >>> >>> Content-Type: >>> >>> text/plain >>> >>> >>> >>> Search the CONFOCAL archive at >>> >>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >>> >>> >>> >>> PS What would be more appropriate to compare is Zeiss 518F (not >>> 518C!) with >>> >>> the Nikon type A or Cargille type DF. >>> >>> >>> >>> >>> >>> >>> >>> Hi Jason, >>> >>> >>> >>> I wonder why you have tested the Cargille FF imm. oil, as the >>> refractive >>> >>> index of FF oil is too far off for the standard glass >>> (1.518) ?????!!!!! >>> >>> >>> >>> Actually, I haven't noticed the "very bad" autofluorescence of the >>> Cargille >>> >>> type DF versus Zeiss 518 in the CFP, YFP or FRET channels. The >>> difference is >>> >>> less than 10%. >>> >>> >>> >>> The problem with the Nikon oil is its dispenser and/or viscosity - >>> it is >>> >>> often annoying "fighting" with the air bubbles. >>> >>> >>> >>> What is about the Invitrogen/Mol.Probes mounting media >>> autofluorescence? >>> >>> >>> >>> I am fed up with the irreproducibility of the background >>> autofluorescence >>> >>> using the ProlongGold mounting media!!! That is a serious problem >>> compared >>> >>> to the issue of the immersion oils. >>> >>> >>> >>> Cheers, >>> >>> >>> >>> Vitaly >>> >>> >>> >>> NCI-Frederick >>> >>> >>> >>> >>> >>> ----- Original Message ----- >>> >>> From: "Kilgore, Jason" <[hidden email]> >>> >>> To: <[hidden email]> >>> >>> Sent: Tuesday, August 15, 2006 7:26 PM >>> >>> Subject: Re: Fluorescence from Type 37 Immersion Oil >>> >>> >>> >>> >>> >>>> Search the CONFOCAL archive at >>> >>>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >>> >>>> >>> >>>> Back in 2000 I performed a comparison of four different immersion >>>> oils >>> >>>> (Zeiss 518C, Nikon Type A, Cargille Type DF, and Cargille Type >>>> FF) for >>> >>>> autofluorescence (using DAPI, FITC, acridine orange, and TRITC >>>> filter >>> >>>> sets) and general resolution at 60x on a Nikon E400. I didn't test >>> >>>> Cargille 37. >>> >>>> >>> >>>> Green autofluorescence tended to be worst for all four, but was >>>> least >>> >>>> for Cargille FF, followed by Nikon Type A, then Zeiss, then (very >>>> bad) >>> >>>> Cargille DF. However, the resolution was unacceptable for >>>> Cargille FF. >>> >>>> Autofluorescence in the other channels was moderate (DAPI, TRITC) to >>> >>>> very low (acridine orange), with the same comparative trend >>>> applying. >>> >>>> >>> >>>> Thus, ever since we have been using Nikon Type A with no >>>> complaints for >>> >>>> most of our imaging. >>> >>>> >>> >>>> Feel free to contact me personally and I can send you the data. >>> >>>> >>> >>>> Jason >>> >>>> >>> >>>> Jason A. Kilgore >>> >>>> Cell Biology / Histology >>> >>>> Molecular Probes/Invitrogen >>> >>>> [hidden email] >>> >>>> >>> >>>> >>> >>>> -----Original Message----- >>> >>>> From: Confocal Microscopy List >>>> [mailto:[hidden email]] On >>> >>>> Behalf Of S. Brunet >>> >>>> Sent: Tuesday, August 15, 2006 11:19 AM >>> >>>> To: [hidden email] >>> >>>> Subject: Fluorescence from Type 37 Immersion Oil >>> >>>> >>> >>>> Search the CONFOCAL archive at >>> >>>> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal >>> >>>> >>> >>>> Hello to all, >>> >>>> >>> >>>> We recently began using the Cargille 37 oil with the temperature >>>> chamber >>> >>>> on the >>> >>>> microscope. I found significant fluorescence from the oil when >>>> exposed >>> >>>> to the >>> >>>> HBO lamp (I could not see the eYFP because of the blue background). >>> >>>> >>> >>>> So I used the 20x air objective and I was able to obtain an image >>>> from >>> >>>> the >>> >>>> fluorescence from a drop of 37 oil placed on a coverslip as a sample >>> >>>> (excitation at 488 or 458nm and pinhole at the usual confocal >>>> setting). >>> >>>> >>> >>>> Is this the norm? >>> >>>> >>> >>>> Regards, >>> >>>> Sophie >>> >>>> _______________________________________ >>> >>>> Sophie M.K. Brunet, Ph.D. >>> >>>> Research Officer >>> >>>> Optical Spectroscopist, Laser Systems and Applications >>> >>>> [hidden email] >>> >>>> 306-966-1719 (office) 306-966-1702 (fax) >>> >>>> _______________________________________ >>> >>>> Saskatchewan Structural Sciences Centre >>> >>>> University of Saskatchewan >>> >>>> Thorvaldson Bldg. >>> >>>> 110 Science Place >>> >>>> Saskatoon, SK S7N 5C9 >>> >>>> >>> >>> Hi Matiar It may be that the drive where your temp directory is located is running short of space thus preventing acquistion. LS2K writes files to c:\temp first, in your case, as they are acquired as untitledN ... and them copy (or move?) to the directory you specified. In general, acquistion does not depend on the amount of RAM installed. As Michael suggested, go to Tools | System configuration, look under the Scan System tab for Collection Limits to make sure that it is set properly. Depending on the verion of LS2K, it could be either 1 GB (pre 5.2, if I remember correctly) or 2 GB. This limit is hardcoded and cannot be increased. If your stack will go over that limit LS2K will not let you. You can also change where the Temporary Directory is located, may be point it to a larger disk. You do need to have system access level in LS2K to change things in System config, I think. You can check that in Tools | User Setup. Good luck. On Tue, 24 Mar 2009, Michael Bastiani wrote: > Try increasing the memory allocated to image acquisition. In Tools select > System Config and set memory to size smaller than Computer RAM. Also make > sure your computer hard drive has plenty of space. > You cannot collect images/stacks greater than the number set in System Config. > good luck, > michael > > > On Mar 24, 2009, at 2:57 AM, Matiar Jafari wrote: > > > I've been getting this error when i shoot larger z stacks and when i > > shoot longer time series > > > > Serious Error: NO file could be found matching the specification > > 'C:\Temp\untitled\untitled1_raw*.pic' > > > > then i either click ok or close the dialog box and lasersharp 2000 > > closes/crashes > > > > but when i shoot smaller stacks this does NOT happen any help would be > > greatly appreciated > > > > ive tried deleting the C:\Temp folder and this does nothing > > > > The reason i need this to finish is so that i can then export it to my > > desired location on a different server. > > > > Thank You > > --- > > Matiar Jafari Pang (Wai Pang Chan, [hidden email], PAB A087, 206-685-1519) The Biology Imaging Facility (http://staff.washington.edu/wpchan/if/) P ----- Original Message ----- From: Watkins, Simon C <[hidden email]> To: [hidden email] <[hidden email]> Sent: Tue Mar 24 09:55:14 2009 Subject: QFM 2009 second announcement Folks, As many of you know, Quantitative Fluorescence Microscopy is a course run at the Mount Desert Island Bio Labs annually. This is an intensive lab/lecture course, focusing on fluorescence microscopy in all its forms, including widefield, Confocal, Multiphoton, Decon, TIRF, FRET etc, though with an emphasis on live cell methods. We also encourage students to provide their own specimens such that the hands on components of the course are as enriching as possible. While we have had a healthy applicant pool, we have also had a surprising number of dropouts/cancellations,(we are guessing that the economy isnt exactly helping).... Right now we need to fill 3 of the 32 slots, so if you or anyone you work with/for might be interested please encourage them to apply. The dates for course are from Saturday May 30th 2009 to the following Friday (June 5th) . The url for the course is http://www.cbi.pitt.edu/qfm/index.html Thanks Simon Simon C. Watkins Ph.D, FRCPath Professor and Vice Chair, Cell Biology and Physiology Professor, Immunology Director, Center for Biologic Imaging BSTS 225, University of Pittsburgh 3500 Terrace St. Pittsburgh PA 15261 Tel: 412-352-2277 Fax:412-648-2797 URL: http://www.cbi.pitt.edu ************************************************************************************************** IMPORTANT: The contents of this email and any attachments are confidential. They are intended for the named recipient(s) only. If you have received this email in error, please notify the system manager or the sender immediately and do not disclose the contents to anyone or make copies thereof. *** eSafe scanned this email for viruses, vandals, and malicious content. *** ********************************************************************************************** I'm looking for a far red reagent for this that has an excitation range 633 - 647 to be used on a PE RS3 system. To image dictystelium. Or any label substance that these cells will take up. Joe Goodhouse
Visit us at http://www.molbio1.princeton.edu/facility/confocal/ Hi Matiar, Chang and others have beaten me too it but anyway: I've had this error before on our Radiance 2000 and it was fixed pretty easily and permanently after a chat to the Bio-Rad engineer, but it was sorted out years ago so I am rather fuzzy on the reason for it and the cure. I'm sure though it is a memory error of some sort [I assume you are still running LaserSharp 2000 under NT - our error wasn't on the latest version 6 but earlier, probably v4]. We did add more physical memory - to 768Mb or possibly 1Gb [try eBay for cheap s/h ECC memory, and check if you need matched pairs], plus we played around with the hard drive space, system page file, LaserSharp settings logged in as system, and we put the crucial :\temp on our very much larger D:\ drive [set via Lasersharp system] as the C:\ drive is always compromised to ~4Gb under NT, unless you increased it after SP6 is applied. We had two hard drives fitted, the first split into two partitions C:,E: and the much larger second one D: for user data and the Temp folder So I'm sure all the other postings are on the right track, it's system or hard drive memory and the 1Gb/2Gb limit - see Pangs post [no idea what error message we got though other than the Z-stacks/time-lapses never finished and we lost everything]. I think we still had to split very long time-lapses with z stacks though into two runs [2Gb limit]. Also allocated more virtual memory [page file] from the hard drive to help out the physical RAM [forgotten how to do that under NT, but it's probably under 'system']. I think there used to be a 2Gb limit on collections though even with LaserSharp 6 [1Gb default] even with the tweeks. Zeiss [Microscience] should still support the Radiance 2000 [not the 1024 though] so an email to their service support should clarify the solution. I don't know if upgrading to XP ever helped [you needed a new Firewire card and deep pockets for Bio-Rad to do this for you]. Again look at the old postings below that seem very relevant. Keith --------------------------------------------------------------------------- Dr Keith J. Morris, Molecular Cytogenetics and Microscopy Core, Laboratory 00/069 and 00/070, The Wellcome Trust Centre for Human Genetics, Roosevelt Drive, Oxford OX3 7BN, United Kingdom. Telephone: +44 (0)1865 287568 Email: [hidden email] Web-pages: http://www.well.ox.ac.uk/cytogenetics/ ------------------------------------------------------------------- Subject: Re: Radiance 2000 (was Re: [CONFOCAL] Bio-Rad 1024) From: W. Chan Reply-To: Confocal Microscopy List <[hidden email]> Date: Tue, 21 Mar 2006 16:59:39 -0800 Content-Type: TEXT/PLAIN Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal The Control Panel error message is quite misleading. For v5.2, it states that "Collection Requires Too Much Memory" when the predicted dataset size exceeds the collection limit, but in fact it refers to disk space. I agree. Even a 2 GB limit is really not much when you need multi-channels with a merge pane at 1024x1024 with multiple time points. May be a work around is not to have a merge pane which can be generated quite easily after you have the data. A few of our users do not use the merge pane option in their methods. They do not use LaserSharp to analyze their images so they can't read the merge pane .pmf file even if they want to. -- Pang (Wai Pang Chan, [hidden email], PAB A087, 206-685-1519) The Biology Imaging Facility (http://staff.washington.edu/wpchan/if/) On Sat, 18 Mar 2006, Michael Cammer wrote: > I'll check the upgrade software for writing to disk. But with multiple > probe files our problem was the merge file. For two probes, it needed > 150% more memory than the raw images themselves. Even changing the > configuration didn't provide enough memory. > > >> Are you referring to RAM or harddisk space? As far as I know, LaserSharps >> writes to temporary files rather than holding the experiment in RAM. We >> have a Radiance 2000 on NT (1 GB RAM, 1.5 GB page file) with LaserSharp >> 5.2 build 824. The collection limit is 2 GB. You may want to check >> "System Configuration | Scan system" for the setting of the collection >> limit. Previous versions may have a 1 GB limit. Also check the location >> of the temporary files which needs to have plenty of space (as much as you >> want to acquire each time or more). Good luck. >> >> -- >> Pang (Wai Pang Chan, [hidden email], PAB A087, 206-685-1519) >> The Biology Imaging Facility (http://staff.washington.edu/wpchan/if/) >> >> On Fri, 17 Mar 2006, Michael Cammer wrote: >> >>> We have the Radiance 2000. This is a major limitation of the system. >>> We've had to compromise by restarting long time lapse experiments in the >>> middle, by taking fewer Z planes per timepoint or longer intervals. >>> But when our BioRad confocals die, LaserSharp software should be >>> obsolete. >>> -Michael >>> >>> At 05:51 PM 03/14/06 -0800, you wrote: >>>> >>>> I have a question regarding a BioRad 1024 confocal system. This system >>>> is >>>> working fine. But one trouble is that people cannot run any scanning >>>> program (time lapse experiments) that contains more than 230 mb, >>>> although >>>> the system has plenty of free memory (we just added another 1gb to it). >>>> I >>>> will appreciate if anyone can share with us if you had same experience >>>> and >>>> managed to resolve it. RECENT POSTINGS -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Michael Bastiani Sent: 24 March 2009 13:57 To: [hidden email] Subject: Re: LaserSharp 2000 Error Try increasing the memory allocated to image acquisition. In Tools select System Config and set memory to size smaller than Computer RAM. Also make sure your computer hard drive has plenty of space. You cannot collect images/stacks greater than the number set in System Config. good luck, michael On Mar 24, 2009, at 2:57 AM, Matiar Jafari wrote: > I've been getting this error when i shoot larger z stacks and when i > shoot longer time series > > Serious Error: NO file could be found matching the specification > 'C:\Temp\untitled\untitled1_raw*.pic' > > then i either click ok or close the dialog box and lasersharp 2000 > closes/crashes > > but when i shoot smaller stacks this does NOT happen any help would be > greatly appreciated > > ive tried deleting the C:\Temp folder and this does nothing > > The reason i need this to finish is so that i can then export it to my > desired location on a different server. > > Thank You > --- > Matiar Jafari [hidden email] Hi Joe, What do you want to label exactly? Do the dicty need to be alive? Do you just need a dye that can show you individual cells? Cheers Cam Cameron J. Nowell Microscpy Manager Central Resource for Advanced Microscopy Ludwig Insttue for Cancer Research PO Box 2008 Royal Melbourne Hospital Victoria, 3050 AUSTRALIA Office: +61 3 9341 3155 Mobile: +61422882700 Fax: +61 3 9341 3104 http://www.ludwig.edu.au/branch/research/platform/microscopy.htm ________________________________ From: Confocal Microscopy List on behalf of Goodhouse, Joseph G. Sent: Wed 25/03/2009 4:54 AM To: [hidden email] Subject: Far Red Cell Tracker or Tracer. I'm looking for a far red reagent for this that has an excitation range 633 - 647 to be used on a PE RS3 system. To image dictystelium. Or any label substance that these cells will take up. Joe Goodhouse Confocal Core Lab Manager Dept. of Molecular Biology Princeton University 609-258-5432 Visit us at http://www.molbio1.princeton.edu/facility/confocal/ <http://www.molbio1.princeton.edu/facility/confocal/> You are invited to attend our live webinar on Friday,
3-April at 1 PM (New York time): "Recent Advancements in CCD and EMCCD Cameras for
Ultra-Low Light Microscopy: Technology Overview and Issues to Consider Before
Buying." See details below. Admission is free, but connection lines are limited so
reserve yours now. -------------------------------------------------------------- TO RESERVE YOUR CONNECTION LINE -------------------------------------------------------------- Complete the information at this link and you will be sent
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PALM, confocal and deconvolution techniques. Photometrics is presenting a
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advancements in science-grade CCD cameras for ultra-low light imaging and how
these technologies work, what their fundamental limitations are, and what
should be considered before acquiring such a camera for their lab should attend. Details
are below. There is no cost, but connection lines are limited so
reserve yours now. ---------------------------------------- MEETING SUMMARY ---------------------------------------- Name: "Recent Advancements in CCD Cameras for
Ultra-Low Light Microscopy: Technology Overview and Issues to Consider Before
Buying." Date: Friday, April 3, 2009 Time: 1:00 PM, New York Time Agenda: -Review of Key Concepts: -How Interline CCD Sensors Work -Limitations Imposed
by Sensitivity and Noise -Overview of Intensified CCDs -Overview of Electron Multiplied CCDs (EM-CCDs) -Recent Advancements in EM-CCDs -Comparison of Intensified and EM-CCDs -Practical Considerations -Guidance on Selecting a Camera Presented by Photometrics, makers of precision cameras for
microscopy, the information imparted will be broadly useful to anyone striving
to make the best camera choice for their imaging goals. This session requires attendees to use a Java-enabled browser
with a high bandwidth connection. Audio is via toll-free telephone
(allows two-way conversation) and computer streaming (only one-way). There is
no charge to participate in this on-line seminar. -------------------------------------------------------------- TO RESERVE YOUR CONNECTION LINE -------------------------------------------------------------- Click this URL: http://www.photomet.com/resources/webinars/webinar_details.php?webinar_id=51 David I have a customer in Victoria that is purchasing a big confocal system from Nikon with a FN1. He is looking at the Rolara uncooled and was enquiring if there is a demo camera available for sale. We are waiting on the release of the CFI money now so it can happen fairly soon Chris Cathcart ----- Original Message ----- From: David Hitrys <[hidden email]> To: [hidden email] <[hidden email]> Sent: Tue Mar 24 17:41:45 2009 Subject: CCD and EMCCD cameras for ultra-low light microscopy: Educational web session You are invited to attend our live webinar on Friday, 3-April at 1 PM (New York time): "Recent Advancements in CCD and EMCCD Cameras for Ultra-Low Light Microscopy: Technology Overview and Issues to Consider Before Buying." See details below. Admission is free, but connection lines are limited so reserve yours now. -------------------------------------------------------------- TO RESERVE YOUR CONNECTION LINE -------------------------------------------------------------- Complete the information at this link and you will be sent access information for the session: http://www.photomet.com/resources/webinars/webinar_details.php?webinar_id=51 Details: Cameras utilizing Intensification and Electron-Multiplication technologies are used by life and physical science microscopists to enable the imaging of dynamic events under the most challenging low-light conditions such as those encountered with TIRF, FRET, PALM, confocal and deconvolution techniques. Photometrics is presenting a free, live, interactive, web-based seminar on these two signal amplification technologies on Friday, 3-April at 1:00 PM (New York time). Who should attend: Anyone interested in learning about the recent advancements in science-grade CCD cameras for ultra-low light imaging and how these technologies work, what their fundamental limitations are, and what should be considered before acquiring such a camera for their lab should attend. Details are below. There is no cost, but connection lines are limited so reserve yours now. ---------------------------------------- MEETING SUMMARY ---------------------------------------- Name: "Recent Advancements in CCD Cameras for Ultra-Low Light Microscopy: Technology Overview and Issues to Consider Before Buying." Date: Friday, April 3, 2009 Time: 1:00 PM, New York Time Agenda: -Review of Key Concepts: -How Interline CCD Sensors Work -Limitations Imposed by Sensitivity and Noise -Overview of Intensified CCDs -Overview of Electron Multiplied CCDs (EM-CCDs) -Recent Advancements in EM-CCDs -Comparison of Intensified and EM-CCDs -Practical Considerations -Guidance on Selecting a Camera Presented by Photometrics, makers of precision cameras for microscopy, the information imparted will be broadly useful to anyone striving to make the best camera choice for their imaging goals. This session requires attendees to use a Java-enabled browser with a high bandwidth connection. Audio is via toll-free telephone (allows two-way conversation) and computer streaming (only one-way). There is no charge to participate in this on-line seminar. -------------------------------------------------------------- TO RESERVE YOUR CONNECTION LINE -------------------------------------------------------------- Click this URL: http://www.photomet.com/resources/webinars/webinar_details.php?webinar_id=51 ************************************************************************************************** IMPORTANT: The contents of this email and any attachments are confidential. They are intended for the named recipient(s) only. If you have received this email in error, please notify the system manager or the sender immediately and do not disclose the contents to anyone or make copies thereof. *** eSafe scanned this email for viruses, vandals, and malicious content. *** ********************************************************************************************** My mistake . Was meant for David only Chris Cathcart ----- Original Message ----- From: Cathcart, Chris To: '[hidden email]' <[hidden email]> Sent: Tue Mar 24 17:48:58 2009 Subject: Qimaging. Rolara uncooled David I have a customer in Victoria that is purchasing a big confocal system from Nikon with a FN1. He is looking at the Rolara uncooled and was enquiring if there is a demo camera available for sale. We are waiting on the release of the CFI money now so it can happen fairly soon Chris Cathcart ----- Original Message ----- From: David Hitrys <[hidden email]> To: [hidden email] <[hidden email]> Sent: Tue Mar 24 17:41:45 2009 Subject: CCD and EMCCD cameras for ultra-low light microscopy: Educational web session You are invited to attend our live webinar on Friday, 3-April at 1 PM (New York time): "Recent Advancements in CCD and EMCCD Cameras for Ultra-Low Light Microscopy: Technology Overview and Issues to Consider Before Buying." See details below. Admission is free, but connection lines are limited so reserve yours now. -------------------------------------------------------------- TO RESERVE YOUR CONNECTION LINE -------------------------------------------------------------- Complete the information at this link and you will be sent access information for the session: http://www.photomet.com/resources/webinars/webinar_details.php?webinar_id=51 Details: Cameras utilizing Intensification and Electron-Multiplication technologies are used by life and physical science microscopists to enable the imaging of dynamic events under the most challenging low-light conditions such as those encountered with TIRF, FRET, PALM, confocal and deconvolution techniques. Photometrics is presenting a free, live, interactive, web-based seminar on these two signal amplification technologies on Friday, 3-April at 1:00 PM (New York time). Who should attend: Anyone interested in learning about the recent advancements in science-grade CCD cameras for ultra-low light imaging and how these technologies work, what their fundamental limitations are, and what should be considered before acquiring such a camera for their lab should attend. Details are below. There is no cost, but connection lines are limited so reserve yours now. ---------------------------------------- MEETING SUMMARY ---------------------------------------- Name: "Recent Advancements in CCD Cameras for Ultra-Low Light Microscopy: Technology Overview and Issues to Consider Before Buying." Date: Friday, April 3, 2009 Time: 1:00 PM, New York Time Agenda: -Review of Key Concepts: -How Interline CCD Sensors Work -Limitations Imposed by Sensitivity and Noise -Overview of Intensified CCDs -Overview of Electron Multiplied CCDs (EM-CCDs) -Recent Advancements in EM-CCDs -Comparison of Intensified and EM-CCDs -Practical Considerations -Guidance on Selecting a Camera Presented by Photometrics, makers of precision cameras for microscopy, the information imparted will be broadly useful to anyone striving to make the best camera choice for their imaging goals. This session requires attendees to use a Java-enabled browser with a high bandwidth connection. Audio is via toll-free telephone (allows two-way conversation) and computer streaming (only one-way). There is no charge to participate in this on-line seminar. -------------------------------------------------------------- TO RESERVE YOUR CONNECTION LINE -------------------------------------------------------------- Click this URL: http://www.photomet.com/resources/webinars/webinar_details.php?webinar_id=51 ************************************************************************************************** IMPORTANT: The contents of this email and any attachments are confidential. They are intended for the named recipient(s) only. If you have received this email in error, please notify the system manager or the sender immediately and do not disclose the contents to anyone or make copies thereof. *** eSafe scanned this email for viruses, vandals, and malicious content. *** ********************************************************************************************** Hello, We are having some issues with imaging of cells using FITC filter for GFP expression. We are seeing that fraction of cells are showing fluorescence when viewed via the FITC filter with no transfection reagent present. We have tried playing with exposure times, different samples, glass, plastic, etc. Can anybody advise. Thanks, -Prabhakar Hi all,
In Chapter 11 of the Handbook, Rimas Juskaitis goes into some
detail about measurements that he made on the relatively large changes
in RI that can occur when immersion oils are use at other than room
temp.
The bottom line is that it can be very finicky: even one deg C
can make a noticeable difference. In addition, once the oil has
the WRONG RI, then the amount of spherical aberration produced will
depend on the free working distance (i.e., it may look better or worse
depending on the depth that you are focused into the specimen).
The solution as the Agard/Sedat group found years ago is to
always include sub-resolution beads in your specimen and focus
up-and-down on these in widefield to make sure that the point image
becomes blurry in the same way, whether you focus up or down from best
focus (i.e., blurry rings in both direction, not rings in one
direction and blobs in the other).
Agard and Sedat were early users of Cargille oils mixed to the
specific RI to give best results under the actual imaging
conditions.
If trying to do the same with a water objective looking into a
specimen with a higher RI than that of water, you can adjust the RI of
the coupling liquid to be the same as that of the specimen with
mixtures of water and glycerol, but then you will have to use the
correction collar (and coverslip thickness) to compensate for the fact
that you are no longer just looking into water.
Cheers,
Jim P.
**********************************************
Prof. James B. Pawley, Room 223, Zoology Research Building, 1117 Johnson Ave., Madison, WI, 53706 3D Microscopy of Living Cells Course, June 13-25, 2009, UBC, Vancouver Canada Info:
http://www.3dcourse.ubc.ca/
Hi all, Using immersion oil at 37oC. Zeiss sell a range of low fluorescence immersion oils: bottles of 20, 250 and 500 ml, unfortunately all filled with the same 518F oil. According to Zeiss it's apparently 'compatible' with our Zeiss microscopes, but little extra information is offered on the Zeiss website. The only info I can gleam from the web [other than the ubiquitous 518F safety data Sheet] is the following Immersion oil "Immersol" 518 F fluorescence free, (ISO 8036-1/2, n(e)=1.518 (23°C), halogen-free) So not much comment about 37oC use. PeCon's site [who supply most of Zeiss's decent incubator stuff] have no opinion on the matter either. The 518F oil safety sheet mentions "Use of the Substance / Preparation: For application information, consult the processing instructions. Processing instructions or technical information sheet available on request.". I'll ask our knowledgeable local Zeiss Confocal rep for more details, as we are just installing a Zeiss/PeCon 37oC live cell incubator here on our Zeiss 510 MetaHead confocal. Cargilee's site in comparison is passionate about all things immersion oil: http://www.cargille.com/immeroil.shtml From postings to this server back in 2006, and Cargille's site not mentioning 'non-fluorescence' for type 37, it appears that Cargille 37 probably autofluoresces, so it's likely better for live cell phase-contrast [halogen lamp] transmission microscopy [but then often so are dry objectives]. I'll stick with standard 518F immersion oil at 37oC though, it's always imaged well enough on Zeiss microscopes at that temperature [and at least at 37oC it never became cloudy due to crystal precipitation]. Similarly the Cargille DF and FF seem fine to me at 37oC, and we stick ruthlessly with the same brand on each microscope. A lot of our live cell work is/was done with low power [20x Phase] air objectives though. I do notice that immersion oil often ingresses into oil immersion objective internal optics with time, although strangely it rarely notices when imaging [like dirt on much of the internal optics, you must mostly focus through it]. One microscopy core manager even commented to me that he considers oil objectives 'consumables'. The cost of 'repair' [often the same price as a new objective] is so high we frequently have to live with it [I advise users that our say 40x objective image quality is suspect for this reason]. In truth we don't notice any drop in image quality when it must have happened, although you can see it 'smeared' on the optics when looking through the removed objective, using a magnifying glass, bright room light and a 'cleaned' objective top lens. Keith PS missed this link on the last post: Dissolving crystals in Zeiss 518F oil --------------------------------------------------------------------------- Subject: Re: Fluorescence from Type 37 Immersion Oil From: Vitaly Boyko <[hidden email]> Reply-To: Vitaly Boyko <[hidden email]> Date: Wed, 16 Aug 2006 10:23:20 -0400 Content-Type: text/plain Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal PS What would be more appropriate to compare is Zeiss 518F (not 518C!) with the Nikon type A or Cargille type DF. Hi Jason, I wonder why you have tested the Cargille FF imm. oil, as the refractive index of FF oil is too far off for the standard glass (1.518) ?????!!!!! Actually, I haven't noticed the "very bad" autofluorescence of the Cargille type DF versus Zeiss 518 in the CFP, YFP or FRET channels. The difference is less than 10%. The problem with the Nikon oil is its dispenser and/or viscosity - it is often annoying "fighting" with the air bubbles. What is about the Invitrogen/Mol.Probes mounting media autofluorescence? I am fed up with the irreproducibility of the background autofluorescence using the ProlongGold mounting media!!! That is a serious problem compared to the issue of the immersion oils. Cheers, Vitaly NCI-Frederick ----- Original Message ----- From: "Kilgore, Jason" <[hidden email]> To: <[hidden email]> Sent: Tuesday, August 15, 2006 7:26 PM Subject: Re: Fluorescence from Type 37 Immersion Oil > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Back in 2000 I performed a comparison of four different immersion oils > (Zeiss 518C, Nikon Type A, Cargille Type DF, and Cargille Type FF) for > autofluorescence (using DAPI, FITC, acridine orange, and TRITC filter > sets) and general resolution at 60x on a Nikon E400. I didn't test > Cargille 37. > > Green autofluorescence tended to be worst for all four, but was least > for Cargille FF, followed by Nikon Type A, then Zeiss, then (very bad) > Cargille DF. However, the resolution was unacceptable for Cargille FF. > Autofluorescence in the other channels was moderate (DAPI, TRITC) to > very low (acridine orange), with the same comparative trend applying. > > Thus, ever since we have been using Nikon Type A with no complaints for > most of our imaging. > > Feel free to contact me personally and I can send you the data. > > Jason > > Jason A. Kilgore > Cell Biology / Histology > Molecular Probes/Invitrogen > [hidden email] > > > -----Original Message----- > From: Confocal Microscopy List [mailto:[hidden email]] On > Behalf Of S. Brunet > Sent: Tuesday, August 15, 2006 11:19 AM > To: [hidden email] > Subject: Fluorescence from Type 37 Immersion Oil > > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Hello to all, > > We recently began using the Cargille 37 oil with the temperature chamber > on the > microscope. I found significant fluorescence from the oil when exposed > to the > HBO lamp (I could not see the eYFP because of the blue background). > > So I used the 20x air objective and I was able to obtain an image from > the > fluorescence from a drop of 37 oil placed on a coverslip as a sample > (excitation at 488 or 458nm and pinhole at the usual confocal setting). > > Is this the norm? > > Regards, > Sophie > _______________________________________ > Sophie M.K. Brunet, Ph.D. > Research Officer > Optical Spectroscopist, Laser Systems and Applications > [hidden email] > 306-966-1719 (office) 306-966-1702 (fax) > _______________________________________ > Saskatchewan Structural Sciences Centre > University of Saskatchewan > Thorvaldson Bldg. > 110 Science Place > Saskatoon, SK S7N 5C9 > -- I am going to guess that they will be fluorescent in a TRITC channel as well? Acquire both channels and apply a spectral unmixing algorithm. Or if you just want quick identification of GFP cells display the channels overlay: GFP will be green, the autofluorescent cells will be more yellow, like you would see them though a long pass filter. Anda Cornea, Ph.D. Director Imaging and Morphology Support Core Oregon National Primate Research Center Oregon Heath & Science University 503-690-5293 -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of B. Prabhakar Pandian Sent: Tuesday, March 24, 2009 3:30 PM To: [hidden email] Subject: Autofluorescence on cells using FITC filter Hello, We are having some issues with imaging of cells using FITC filter for GFP expression. We are seeing that fraction of cells are showing fluorescence when viewed via the FITC filter with no transfection reagent present. We have tried playing with exposure times, different samples, glass, plastic, etc. Can anybody advise. Thanks, -Prabhakar Dear all, So according to Chan the limit is hardcoded and cannot be changed.... we have multiple accounts on the computer and im wondering if im not able to change he memory option from 700 mb to the 1gb because its hardcoded or because i need the "admin" login user for lasersharp 2000....help? also when i change the directory for the temp files it doesn't allow me to click ok thus it doesnt change where its located....any help??? On Tue, Mar 24, 2009 at 11:12 AM, Keith Morris <[hidden email]> wrote: > Hi Matiar, > Chang and others have beaten me too it but anyway: > > I've had this error before on our Radiance 2000 and it was fixed pretty > easily and permanently after a chat to the Bio-Rad engineer, but it was > sorted out years ago so I am rather fuzzy on the reason for it and the cure. > I'm sure though it is a memory error of some sort [I assume you are still > running LaserSharp 2000 under NT - our error wasn't on the latest version 6 > but earlier, probably v4]. We did add more physical memory - to 768Mb or > possibly 1Gb [try eBay for cheap s/h ECC memory, and check if you need > matched pairs], plus we played around with the hard drive space, system page > file, LaserSharp settings logged in as system, and we put the crucial :\temp > on our very much larger D:\ drive [set via Lasersharp system] as the C:\ > drive is always compromised to ~4Gb under NT, unless you increased it after > SP6 is applied. We had two hard drives fitted, the first split into two > partitions C:,E: and the much larger second one D: for user data and the > Temp folder > > So I'm sure all the other postings are on the right track, it's system or > hard drive memory and the 1Gb/2Gb limit - see Pangs post [no idea what error > message we got though other than the Z-stacks/time-lapses never finished and > we lost everything]. I think we still had to split very long time-lapses > with z stacks though into two runs [2Gb limit]. Also allocated more virtual > memory [page file] from the hard drive to help out the physical RAM > [forgotten how to do that under NT, but it's probably under 'system']. I > think there used to be a 2Gb limit on collections though even with > LaserSharp 6 [1Gb default] even with the tweeks. > > Zeiss [Microscience] should still support the Radiance 2000 [not the 1024 > though] so an email to their service support should clarify the solution. I > don't know if upgrading to XP ever helped [you needed a new Firewire card > and deep pockets for Bio-Rad to do this for you]. > > Again look at the old postings below that seem very relevant. > > Keith > > --------------------------------------------------------------------------- > Dr Keith J. Morris, > Molecular Cytogenetics and Microscopy Core, > Laboratory 00/069 and 00/070, > The Wellcome Trust Centre for Human Genetics, > Roosevelt Drive, > Oxford OX3 7BN, > United Kingdom. > > Telephone: +44 (0)1865 287568 > Email: [hidden email] > Web-pages: http://www.well.ox.ac.uk/cytogenetics/ > > > > > > > > ------------------------------------------------------------------- > Subject: Re: Radiance 2000 (was Re: [CONFOCAL] Bio-Rad 1024) > From: W. Chan > Reply-To: Confocal Microscopy List <[hidden email]> > Date: Tue, 21 Mar 2006 16:59:39 -0800 > Content-Type: TEXT/PLAIN > > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > The Control Panel error message is quite misleading. For v5.2, it states > that "Collection Requires Too Much Memory" when the predicted dataset size > exceeds the collection limit, but in fact it refers to disk space. > > I agree. Even a 2 GB limit is really not much when you need > multi-channels with a merge pane at 1024x1024 with multiple time points. > May be a work around is not to have a merge pane which can be generated > quite easily after you have the data. A few of our users do not use > the merge pane option in their methods. They do not use LaserSharp to > analyze their images so they can't read the merge pane .pmf file even if > they want to. > > -- > Pang (Wai Pang Chan, [hidden email], PAB A087, 206-685-1519) > The Biology Imaging Facility (http://staff.washington.edu/wpchan/if/) > > On Sat, 18 Mar 2006, Michael Cammer wrote: > >> I'll check the upgrade software for writing to disk. But with multiple >> probe files our problem was the merge file. For two probes, it needed >> 150% more memory than the raw images themselves. Even changing the >> configuration didn't provide enough memory. >> >> >>> Are you referring to RAM or harddisk space? As far as I know, > LaserSharps >>> writes to temporary files rather than holding the experiment in RAM. We >>> have a Radiance 2000 on NT (1 GB RAM, 1.5 GB page file) with LaserSharp >>> 5.2 build 824. The collection limit is 2 GB. You may want to check >>> "System Configuration | Scan system" for the setting of the collection >>> limit. Previous versions may have a 1 GB limit. Also check the location >>> of the temporary files which needs to have plenty of space (as much as > you >>> want to acquire each time or more). Good luck. >>> >>> -- >>> Pang (Wai Pang Chan, [hidden email], PAB A087, 206-685-1519) >>> The Biology Imaging Facility (http://staff.washington.edu/wpchan/if/) >>> >>> On Fri, 17 Mar 2006, Michael Cammer wrote: >>> >>>> We have the Radiance 2000. This is a major limitation of the system. >>>> We've had to compromise by restarting long time lapse experiments in the >>>> middle, by taking fewer Z planes per timepoint or longer intervals. >>>> But when our BioRad confocals die, LaserSharp software should be >>>> obsolete. >>>> -Michael >>>> >>>> At 05:51 PM 03/14/06 -0800, you wrote: >>>>> >>>>> I have a question regarding a BioRad 1024 confocal system. This system >>>>> is >>>>> working fine. But one trouble is that people cannot run any scanning >>>>> program (time lapse experiments) that contains more than 230 mb, >>>>> although >>>>> the system has plenty of free memory (we just added another 1gb to it). >>>>> I >>>>> will appreciate if anyone can share with us if you had same experience >>>>> and >>>>> managed to resolve it. > > > > > > > > > RECENT POSTINGS > -----Original Message----- > From: Confocal Microscopy List [mailto:[hidden email]] On > Behalf Of Michael Bastiani > Sent: 24 March 2009 13:57 > To: [hidden email] > Subject: Re: LaserSharp 2000 Error > > Try increasing the memory allocated to image acquisition. In Tools > select System Config and set memory to size smaller than Computer > RAM. Also make sure your computer hard drive has plenty of space. > You cannot collect images/stacks greater than the number set in System > Config. > good luck, > michael > > > On Mar 24, 2009, at 2:57 AM, Matiar Jafari wrote: > >> I've been getting this error when i shoot larger z stacks and when i >> shoot longer time series >> >> Serious Error: NO file could be found matching the specification >> 'C:\Temp\untitled\untitled1_raw*.pic' >> >> then i either click ok or close the dialog box and lasersharp 2000 >> closes/crashes >> >> but when i shoot smaller stacks this does NOT happen any help would be >> greatly appreciated >> >> ive tried deleting the C:\Temp folder and this does nothing >> >> The reason i need this to finish is so that i can then export it to my >> desired location on a different server. >> >> Thank You >> --- >> Matiar Jafari > > Michael Bastiani > [hidden email] > -- Matiar Jafari Prabhakar .... How do you know any of your cells are positive for GFP and it isn't all just autofluorescence? Therefore it wouldn't be the scope but your transfection/expression. Could it also be a problem with your prep? Are the cells being imaged live or fixed? Did you also run a control with mock transfection (aka no DNA or better yet GFPless construct but with transfection reagent)? -----Original Message----- From: Anda Cornea <[hidden email]> Date: Tue, 24 Mar 2009 17:04:34 To: <[hidden email]> Subject: Re: Autofluorescence on cells using FITC filter I am going to guess that they will be fluorescent in a TRITC channel as well? Acquire both channels and apply a spectral unmixing algorithm. Or if you just want quick identification of GFP cells display the channels overlay: GFP will be green, the autofluorescent cells will be more yellow, like you would see them though a long pass filter. Anda Cornea, Ph.D. Director Imaging and Morphology Support Core Oregon National Primate Research Center Oregon Heath & Science University 503-690-5293 -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of B. Prabhakar Pandian Sent: Tuesday, March 24, 2009 3:30 PM To: [hidden email] Subject: Autofluorescence on cells using FITC filter Hello, We are having some issues with imaging of cells using FITC filter for GFP expression. We are seeing that fraction of cells are showing fluorescence when viewed via the FITC filter with no transfection reagent present. We have tried playing with exposure times, different samples, glass, plastic, etc. Can anybody advise. Thanks, -Prabhakar Hi Matiar What I meant was e.g., in pre-5.2, you can only give max 1 GB to the application. It is not RAM related. Since you did not mention what version and OS you're running, I just assumed you're still using NT. LaserSharp 6 and XP run really well together; but that's another story. I only have access to v6 now so the terms in the menu may be different than earlier version. System access in lasersharp is completely independent of NT's (or whatever window's) user privilege. Usually, the software is set up with an user called Administrator that have system access, other users for the software may only have read & write access. After you log into lasersharp, go to Tools | User Setup, the second column will tell you what Access level you have. Whoever install the software should have the password to log in as Administrator. There is a way to find that out. Everything for this software are stored in a database e.g., c:\LaserSharpNT\Database\lsMessages.mdb. If you have microsoft access, you can open a COPY of this file and look at the table called LaserSharpUsers and you can see all the user passwords in plain text. Access level 0 is for system. It is probably overly cautious to open a copy because the database will likely be opened as read-only; but why take a chance. Once you log in as an user with system access level, you should be able to change all the configuration parameters. But be very careful, you can mess up things really bad. I've learned that the hard way. Hope this help. On Tue, 24 Mar 2009, Matiar Jafari wrote: > Dear all, > > So according to Chan the limit is hardcoded and cannot be changed.... > > we have multiple accounts on the computer and im wondering if im not > able to change he memory option from 700 mb to the 1gb because its > hardcoded or because i need the "admin" login user for lasersharp > 2000....help? also when i change the directory for the temp files it > doesn't allow me to click ok thus it doesnt change where its > located....any help??? > > On Tue, Mar 24, 2009 at 11:12 AM, Keith Morris <[hidden email]> wrote: > > Hi Matiar, > > Chang and others have beaten me too it but anyway: > > > > I've had this error before on our Radiance 2000 and it was fixed pretty > > easily and permanently after a chat to the Bio-Rad engineer, but it was > > sorted out years ago so I am rather fuzzy on the reason for it and the cure. > > I'm sure though it is a memory error of some sort [I assume you are still > > running LaserSharp 2000 under NT - our error wasn't on the latest version 6 > > but earlier, probably v4]. We did add more physical memory - to 768Mb or > > possibly 1Gb [try eBay for cheap s/h ECC memory, and check if you need > > matched pairs], plus we played around with the hard drive space, system page > > file, LaserSharp settings logged in as system, and we put the crucial :\temp > > on our very much larger D:\ drive [set via Lasersharp system] as the C:\ > > drive is always compromised to ~4Gb under NT, unless you increased it after > > SP6 is applied. We had two hard drives fitted, the first split into two > > partitions C:,E: and the much larger second one D: for user data and the > > Temp folder > > > > So I'm sure all the other postings are on the right track, it's system or > > hard drive memory and the 1Gb/2Gb limit - see Pangs post [no idea what error > > message we got though other than the Z-stacks/time-lapses never finished and > > we lost everything]. I think we still had to split very long time-lapses > > with z stacks though into two runs [2Gb limit]. Also allocated more virtual > > memory [page file] from the hard drive to help out the physical RAM > > [forgotten how to do that under NT, but it's probably under 'system']. I > > think there used to be a 2Gb limit on collections though even with > > LaserSharp 6 [1Gb default] even with the tweeks. > > > > Zeiss [Microscience] should still support the Radiance 2000 [not the 1024 > > though] so an email to their service support should clarify the solution. I > > don't know if upgrading to XP ever helped [you needed a new Firewire card > > and deep pockets for Bio-Rad to do this for you]. > > > > Again look at the old postings below that seem very relevant. > > > > Keith > > > > --------------------------------------------------------------------------- > > Dr Keith J. Morris, > > Molecular Cytogenetics and Microscopy Core, > > Laboratory 00/069 and 00/070, > > The Wellcome Trust Centre for Human Genetics, > > Roosevelt Drive, > > Oxford OX3 7BN, > > United Kingdom. > > > > Telephone: +44 (0)1865 287568 > > Email: [hidden email] > > Web-pages: http://www.well.ox.ac.uk/cytogenetics/ > > > > > > > > > > > > > > > > ------------------------------------------------------------------- > > Subject: Re: Radiance 2000 (was Re: [CONFOCAL] Bio-Rad 1024) > > From: W. Chan > > Reply-To: Confocal Microscopy List <[hidden email]> > > Date: Tue, 21 Mar 2006 16:59:39 -0800 > > Content-Type: TEXT/PLAIN > > > > Search the CONFOCAL archive at > > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > > > The Control Panel error message is quite misleading. For v5.2, it states > > that "Collection Requires Too Much Memory" when the predicted dataset size > > exceeds the collection limit, but in fact it refers to disk space. > > > > I agree. Even a 2 GB limit is really not much when you need > > multi-channels with a merge pane at 1024x1024 with multiple time points. > > May be a work around is not to have a merge pane which can be generated > > quite easily after you have the data. A few of our users do not use > > the merge pane option in their methods. They do not use LaserSharp to > > analyze their images so they can't read the merge pane .pmf file even if > > they want to. > > > > -- > > Pang (Wai Pang Chan, [hidden email], PAB A087, 206-685-1519) > > The Biology Imaging Facility (http://staff.washington.edu/wpchan/if/) > > > > On Sat, 18 Mar 2006, Michael Cammer wrote: > > > >> I'll check the upgrade software for writing to disk. But with multiple > >> probe files our problem was the merge file. For two probes, it needed > >> 150% more memory than the raw images themselves. Even changing the > >> configuration didn't provide enough memory. > >> > >> > >>> Are you referring to RAM or harddisk space? As far as I know, > > LaserSharps > >>> writes to temporary files rather than holding the experiment in RAM. We > >>> have a Radiance 2000 on NT (1 GB RAM, 1.5 GB page file) with LaserSharp > >>> 5.2 build 824. The collection limit is 2 GB. You may want to check > >>> "System Configuration | Scan system" for the setting of the collection > >>> limit. Previous versions may have a 1 GB limit. Also check the location > >>> of the temporary files which needs to have plenty of space (as much as > > you > >>> want to acquire each time or more). Good luck. > >>> > >>> -- > >>> Pang (Wai Pang Chan, [hidden email], PAB A087, 206-685-1519) > >>> The Biology Imaging Facility (http://staff.washington.edu/wpchan/if/) > >>> > >>> On Fri, 17 Mar 2006, Michael Cammer wrote: > >>> > >>>> We have the Radiance 2000. This is a major limitation of the system. > >>>> We've had to compromise by restarting long time lapse experiments in the > >>>> middle, by taking fewer Z planes per timepoint or longer intervals. > >>>> But when our BioRad confocals die, LaserSharp software should be > >>>> obsolete. > >>>> -Michael > >>>> > >>>> At 05:51 PM 03/14/06 -0800, you wrote: > >>>>> > >>>>> I have a question regarding a BioRad 1024 confocal system. This system > >>>>> is > >>>>> working fine. But one trouble is that people cannot run any scanning > >>>>> program (time lapse experiments) that contains more than 230 mb, > >>>>> although > >>>>> the system has plenty of free memory (we just added another 1gb to it). > >>>>> I > >>>>> will appreciate if anyone can share with us if you had same experience > >>>>> and > >>>>> managed to resolve it. > > > > RECENT POSTINGS > > -----Original Message----- > > From: Confocal Microscopy List [mailto:[hidden email]] On > > Behalf Of Michael Bastiani > > Sent: 24 March 2009 13:57 > > To: [hidden email] > > Subject: Re: LaserSharp 2000 Error > > > > Try increasing the memory allocated to image acquisition. In Tools > > select System Config and set memory to size smaller than Computer > > RAM. Also make sure your computer hard drive has plenty of space. > > You cannot collect images/stacks greater than the number set in System > > Config. > > good luck, > > michael > > > > > > On Mar 24, 2009, at 2:57 AM, Matiar Jafari wrote: > > > >> I've been getting this error when i shoot larger z stacks and when i > >> shoot longer time series > >> > >> Serious Error: NO file could be found matching the specification > >> 'C:\Temp\untitled\untitled1_raw*.pic' > >> > >> then i either click ok or close the dialog box and lasersharp 2000 > >> closes/crashes > >> > >> but when i shoot smaller stacks this does NOT happen any help would be > >> greatly appreciated > >> > >> ive tried deleting the C:\Temp folder and this does nothing > >> > >> The reason i need this to finish is so that i can then export it to my > >> desired location on a different server. > >> > >> Thank You > >> --- > >> Matiar Jafari Pang (Wai Pang Chan, [hidden email], PAB A087, 206-685-1519) The Biology Imaging Facility (http://staff.washington.edu/wpchan/if/) |
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