5th colour?
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hello, Has anyone successfully imaged 5 colours in the same sample with their confocal? We have a user who is keen to do this. They are already using DAPI, AF488, AF555 & AF647 so if there's something that maybe excites in the blue and emits in the red/far red? Or if that's not possible a completely different set of 5. We have the usual range of excitation wavelengths from 405 up to 635 nm. Thanks, Simon |
 
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Kilgore, Jason-2 |
Re: 5th colour? ** Vendor response **
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** ** Vendor Response ** I've imaged up to 6 Qdots at a time on the same sample using confocal. Qdot nanocrystals will all excite at one low excitation (UV or 405nm), but have reasonably tight emission wavelengths, which allows better multiplexability. Cheers, Jason Jason A. Kilgore Technical Application Scientist Molecular Probes Labeling and Detection Technologies Cells Systems Division T 1 800 955 6288 then option 5 or 541 335 0353 . F 541 335 0238 29851 Willow Creek Rd . Eugene . OR . 97402-9132 . United States www.invitrogen.com/technicalsupport -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Simon Walker Sent: Friday, November 04, 2011 9:37 AM To: [hidden email] Subject: 5th colour? ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hello, Has anyone successfully imaged 5 colours in the same sample with their confocal? We have a user who is keen to do this. They are already using DAPI, AF488, AF555 & AF647 so if there's something that maybe excites in the blue and emits in the red/far red? Or if that's not possible a completely different set of 5. We have the usual range of excitation wavelengths from 405 up to 635 nm. Thanks, Simon |
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In reply to this post by simon walker (BI)-2
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Dyomics has some large stokes shift dyes that absorb in the green and emit in the red/far red. We looked into them a while ago for this purpose but never had a user who was sufficiently motivated to test them, so I don't know how well they work. See http://www.dyomics.com/dy-480xl.html?&L=1%2F%2Findex.php%3Foption%3Dcom_fabrik I think there may be another company with large stokes shift dyes, but I can't remember who. Kurt On 11/4/2011 9:36 AM, Simon Walker wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Hello, > Has anyone successfully imaged 5 colours in the same sample with their > confocal? We have a user who is keen to do this. They are already using > DAPI, AF488, AF555& AF647 so if there's something that maybe excites in the > blue and emits in the red/far red? Or if that's not possible a completely > different set of 5. We have the usual range of excitation wavelengths from > 405 up to 635 nm. > Thanks, > Simon > |
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In reply to this post by simon walker (BI)-2
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** We have, but using spectral confocal to separate out the different dyes. Craig On Fri, Nov 4, 2011 at 10:36 AM, Simon Walker <[hidden email]>wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Hello, > Has anyone successfully imaged 5 colours in the same sample with their > confocal? We have a user who is keen to do this. They are already using > DAPI, AF488, AF555 & AF647 so if there's something that maybe excites in > the > blue and emits in the red/far red? Or if that's not possible a completely > different set of 5. We have the usual range of excitation wavelengths from > 405 up to 635 nm. > Thanks, > Simon > |
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John Oreopoulos |
Re: 5th colour? - vendor response
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Commercial response: Hi Simon, Quite recently in the R+D lab over here we've been routinely 3D imaging Invitrorgen Alexa750, Anaspec HiLyte 750, or Li-Cor Dye800CW (no commercial interest for any of these probes) with a Yokogawa CSU spinning disk upgraded with our Borealis illumination system. This is possible since Borealis extends the range of possible wavelengths (UV and NIR) that can be passed through the CSU (730 nm or 785 nm laser excitation, >790 nm emission for the dyes just mentioned). While I haven't tried imaging a penta-labeled sample, it should be possible with the right set of emission filters and dichroic filter. We have yet to build a 5-band dichroic but what you could do in the meantime is use two different dichroic mirrors and switch them when needed for the appropriate channels. One of the other parts of the Borealis upgrade for the CSU is a motorized filter wheel and dichroic slider. All of the laser and filter combinations can be automated in software. If you were willing, the user in question could send us a penta-labled sample with the 5th label emitting somewhere above 750 nm and we could try imaging it with the system we have in house. Contact me offline if you're interested. Cheers, John Oreopoulos Research Assistant Spectral Applied Research Richmond Hill, Ontario Canada www.spectral.ca On 2011-11-04, at 4:18 PM, Craig Brideau wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > We have, but using spectral confocal to separate out the different dyes. > > Craig > > > > On Fri, Nov 4, 2011 at 10:36 AM, Simon Walker > <[hidden email]>wrote: > >> ***** >> To join, leave or search the confocal microscopy listserv, go to: >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >> ***** >> >> Hello, >> Has anyone successfully imaged 5 colours in the same sample with their >> confocal? We have a user who is keen to do this. They are already using >> DAPI, AF488, AF555 & AF647 so if there's something that maybe excites in >> the >> blue and emits in the red/far red? Or if that's not possible a completely >> different set of 5. We have the usual range of excitation wavelengths from >> 405 up to 635 nm. >> Thanks, >> Simon >> |
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In reply to this post by simon walker (BI)-2
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi Simon, Spectral karyotyping is is now well past 700 publications since 1996 with 5 fluorophores plus DAPI. The 5 are (or are similar to): Spectrum Green (Rhodamine 110, Alexa Fluor 488, DyLight 488), Spectrum Orange (Cy3, Cy3B, Alexa Fluor 555, Alexa Fluor 568), Texas Red (Alexa Fluor 594), Cy5, Cy5.5. A spectral confocal ought to be able to match the performance of the SKY system. Tsurui et al used the SKY system for 7-color immunofluorescence (no DNA counterstain here): J Histochem Cytochem. <#> 2000 May;48(5):653-62. Seven-color fluorescence imaging of tissue samples based on Fourier spectroscopy and singular value decomposition. Tsurui H </pubmed?term=%22Tsurui%20H%22%5BAuthor%5D>, Nishimura H </pubmed?term=%22Nishimura%20H%22%5BAuthor%5D>, Hattori S </pubmed?term=%22Hattori%20S%22%5BAuthor%5D>, Hirose S </pubmed?term=%22Hirose%20S%22%5BAuthor%5D>, Okumura K </pubmed?term=%22Okumura%20K%22%5BAuthor%5D>, Shirai T </pubmed?term=%22Shirai%20T%22%5BAuthor%5D>. Source Department of Pathology, Juntendo University School of Medicine, Tokyo, Japan. [hidden email] Abstract Seven-color analyses of immunofluorescence-stained tissue samples were accomplished using Fourier spectroscopy-based hyperspectral imaging and singular value decomposition. This system consists of a combination of seven fluorescent dyes, three filtersets, an epifluorescence microscope, a spectral imaging system, a computer for data acquisition, and data analysis software. The spectra of all pixels in a multicolor image were taken simultaneously using a Sagnac type interferometer. The spectra were deconvolved to estimate the contribution of each component dye, and individual dye images were constructed based on the intensities of assigned signals. To obtain mixed spectra, three filter sets, i.e., Bl, Gr, and Rd for *Alexa488 and Alexa532*, for* Alexa546, Alexa568, and Alexa594*, and for *Cy5 and Cy5.5*, respectively, were used for simultaneous excitation of two or three dyes. These fluorophores have considerable spectral overlap which precludes their separation by conventional analysis. We resolved their relative contributions to the fluorescent signal by a method involving linear unmixing based on singular value decomposition of the matrices consisting of dye spectra. Analyses of mouse thymic tissues stained with seven different fluorescent dyes provided clear independent images, and any combination of two or three individual dye images could be used for constructing multicolor images. PMID: 10769049. Some simple suggestions for adding one fluorophore to your list - consider any one of: * Abberior STAR 440SX ... http://www.abberior.com/index.php?option=com_content&view=article&id=90&Itemid=88 * Lucifer Yellow * Pacific Orange * DyLight 405 * Alexa Fluor 594 ... if in a different location than 555 and 647 ... different as shown by single staining and FMO (fluorescence minus one) controls. Brilliant Violet 421 ... http://www.biolegend.com/brilliantviolet see also www.sirigen.com and www.i-cyt.com see http://www.i-cyt.com/icyt_whats_new.php and http://www.i-cyt.com/pdf/BV_Brochure_080811.pdf (keep going to page 5 to check out BV570 ... currently the acceptor is Cy3 [poor QY] but hopefully they will switch to Cy3B [7x better QY than Cy3, at least in solution] or an Alexa or DyLight etc dye). from http://www.biolegend.com/brilliantviolet Brilliant Violet 421™ has an extinction coefficient of 2,500,000 M^-1 cm^-1 at 405 nm, an aqueous solution quantum yield of 65 ± 5% and solubility in excess of 50 mg/mL in PBS. The extinction coefficient contributes to its superior brightness compared to Pacific Blue™, which has an extinction coefficient of 30,000 M^-1 cm^-1 . High Sensitivity Fluorescence™ polymers can also be modified with functional groups to produce high stokes shift emissions. Brilliant Violet 570™ is such a variant of the Brilliant Violet 421™ polymer, emitting maximally at 570 nm. The figures below provide the absorbance and emission spectra of BV421™ and BV570™, respectively. BioLegend anti-CD4-BV421 worked great on my Zeiss Axiovert 200M microscope (FluoArc Hg lamp, ORCA-II ERG and by eye) on PBMC's (specimen sent to me by BioLegend). Photobleached farily quickly on my Leica SP5 confocal microscope (right after a 50 mW 405 nm laser was installed under service contract), but I did get a nice optical section before bleaching. I have encouraged folks from all three companies to work on finding an optimized mounting medium for fluorescence microscopy. Their focus right now is on the flow cytometry market, so feel free to take the initiative. Sincerely, George p.s. full disclosure: I am a co-inventor on three spectral unmixing patents from the SKY manufacturer - I do not expect to see any royalties from these. Donations welcome. p.p.s. Jeremy - I was not teasing about colocalization being dead. If you have any more manuscripts on colocalization, I suggest you get them published before all the reviewers and editors catch up to me. On 11/4/2011 12:36 PM, Simon Walker wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Hello, > Has anyone successfully imaged 5 colours in the same sample with their > confocal? We have a user who is keen to do this. They are already using > DAPI, AF488, AF555& AF647 so if there's something that maybe excites in the > blue and emits in the red/far red? Or if that's not possible a completely > different set of 5. We have the usual range of excitation wavelengths from > 405 up to 635 nm. > Thanks, > Simon > > -- George McNamara, PhD Analytical Imaging Core Facility University of Miami |
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In reply to this post by simon walker (BI)-2
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Simon, on a Leica SP5 you can do it for example with Dapi (405), Alexa 488, Cy3 (543 or 561), Texas Red (594) and Cy5 (633). Quantum dots certainly are an option, but personaly I don't have a lot experince with them. Steffen On 04.11.2011 17:36, Simon Walker wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Hello, > Has anyone successfully imaged 5 colours in the same sample with their > confocal? We have a user who is keen to do this. They are already using > DAPI, AF488, AF555& AF647 so if there's something that maybe excites in the > blue and emits in the red/far red? Or if that's not possible a completely > different set of 5. We have the usual range of excitation wavelengths from > 405 up to 635 nm. > Thanks, > Simon > -- ------------------------------------------------------------ Steffen Dietzel, PD Dr. rer. nat Ludwig-Maximilians-Universität München Walter-Brendel-Zentrum für experimentelle Medizin (WBex) Head of light microscopy Mail room: Marchioninistr. 15, D-81377 München Building location: Marchioninistr. 27, München-Großhadern |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Thanks to everyone who's responded to this for all the useful suggestions. I think we'll try throwing AF594 into the mix as a relatively easy option and take it from there. Simon -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Steffen Dietzel Sent: 08 November 2011 17:26 To: [hidden email] Subject: Re: 5th colour? ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Simon, on a Leica SP5 you can do it for example with Dapi (405), Alexa 488, Cy3 (543 or 561), Texas Red (594) and Cy5 (633). Quantum dots certainly are an option, but personaly I don't have a lot experince with them. Steffen On 04.11.2011 17:36, Simon Walker wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Hello, > Has anyone successfully imaged 5 colours in the same sample with their > confocal? We have a user who is keen to do this. They are already using > DAPI, AF488, AF555& AF647 so if there's something that maybe excites in the > blue and emits in the red/far red? Or if that's not possible a completely > different set of 5. We have the usual range of excitation wavelengths from > 405 up to 635 nm. > Thanks, > Simon > -- ------------------------------------------------------------ Steffen Dietzel, PD Dr. rer. nat Ludwig-Maximilians-Universität München Walter-Brendel-Zentrum für experimentelle Medizin (WBex) Head of light microscopy Mail room: Marchioninistr. 15, D-81377 München Building location: Marchioninistr. 27, München-Großhadern The Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT Registered Charity No. 1053902. The information transmitted in this email is directed only to the addressee. If you received this in error, please contact the sender and delete this email from your system. The contents of this e-mail are the views of the sender and do not necessarily represent the views of the Babraham Institute. Full conditions at: www.babraham.ac.uk<http://www.babraham.ac.uk/email_disclaimer.html> |
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In reply to this post by simon walker (BI)-2
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi Simon, With 5 colors I personally think that in most cases this will require linear unmixing algorithms. Many scopes nowadays have implemented that already in the acquisition software. martin |
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In reply to this post by simon walker (BI)-2
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hello You can use Chromeo 494. It has a long stokes shift (Em 628nm) that allow you to excite two dyes (any 488 et chromeo494) with the same 488 excitation laser. Hope this help. |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** There is another possibility - use lifetime imaging to distinguish between dyes with similar spectra but different lifetimes. Carlsson, K. and Liljeborg, A., 2009. Confocal fluorescence microscopy using intensity-modulated multiple-wavelength scanning (IMS): evaluation of results from spectral and lifetime imaging. Proceedings of SPIE 3261, 30 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) Australian Centre for Microscopy & Microanalysis, 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 Olivier Bardot Sent: Thursday, 10 November 2011 8:52 PM To: [hidden email] Subject: Re: 5th colour? ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hello You can use Chromeo 494. It has a long stokes shift (Em 628nm) that allow you to excite two dyes (any 488 et chromeo494) with the same 488 excitation laser. Hope this help. ----- No virus found in this message. Checked by AVG - www.avg.com Version: 10.0.1411 / Virus Database: 2092/4007 - Release Date: 11/09/11 ----- No virus found in this message. Checked by AVG - www.avg.com Version: 2012.0.1869 / Virus Database: 2092/4607 - Release Date: 11/09/11 |
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