Re: recommendation for a "blue" secondary fluorochrome COMMERCIAL RESPONSE
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Jeffrey Carmichael |
Re: recommendation for a "blue" secondary fluorochrome COMMERCIAL 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 Post images on http://www.imgur.com and include the link in your posting. ***** This has been addressed more than once in the listserv, but below are some resources to look into the BrilliantViolet fluorophores which are much more than an order of magnitude brighter than AF405. You could potentially add two additional channels in this spectral "dead zone". Chroma Technology has no commercial interest in these fluorophores, only in the filter sets we've recommended for detecting them. https://www.jacksonimmuno.com/technical/products/conjugate-selection/brilliant-violet http://bit.ly/ChromaTechnology2MtEdC9 http://bit.ly/bdbiosciences2MEed69 Good luck and please share your results! Jeff *Jeff Carmichael* *Product & Technical Marketing Manager* *[hidden email] <[hidden email]> | 802-428-2528* * <[hidden email]>* On Mon, Aug 20, 2018 at 7:06 PM, Cromey, Douglas W - (dcromey) < [hidden email]> wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Is there a better or preferred "blue" fluorochrome? Mostly this is a > widefield fluorescence question (using a DAPI cube), but there is the > possibility for using a confocal with a 405nm laser line. > > I'm working with a group that is trying to work around a frustrating > autofluorescence issue in liver tissue. What we ended up doing on an > earlier project was not using any secondaries that fluoresced in the green > channel (no fitc/AF488/GFP) and instead used the green channel on our > widefield microscope as a control, ratioing this image so we could subtract > it out of the other channels to remove some of the broad autofluorescent > background. This worked nicely. In the published images we showed nuclei > (blue) and two specific stains using AF568 and AF647, with reduced > background autofluorescence. > > The PI now wants to have labelling with three specific fluorochromes, and > I am advocating using the green channel as the generic "everything lights > up" channel for context, so we can hopefully skip the Hoechst 3342 or DAPI. > Since we won't be using DNA dyes, is there a good blue fluorochrome that > can be used as a secondary for specific labelling of a marker found on an > intracellular organelle (other than the nucleus)? > > Thanks, > Doug > > ------------------------------------------------------------ > ------------------------------ > Douglas W. Cromey, M.S. - Associate Scientific Investigator > Dept. of Cellular & Molecular Medicine, University of Arizona > 1501 N. Campbell Ave, Tucson, AZ 85724-5044 USA > > office: LSN 463 email: [hidden email]<mailto: > [hidden email]> > voice: 520-626-2824 fax: 520-626-2097 > > http://microscopy.arizona.edu/learn/microscopy-imaging-resources-www > Home of: "Microscopy and Imaging Resources on the WWW" > > UA Microscopy Alliance - http://microscopy.arizona.edu< > http://microscopy.arizona.edu/> > -- <https://www.chroma.com/>CHROMA TECHNOLOGY CORP® *an employee owned company* 10 Imtec Lane, Bellows Falls, Vermont 05101 USA 800-824-7662 | FAX: 802-428-2525 www.chroma.com <https://www.chroma.com/> | [hidden email] <mailto:[hidden email]> |
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Moulding, Dale |
Re: recommendation for a "blue" secondary fluorochrome COMMERCIAL 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 Post images on http://www.imgur.com and include the link in your posting. ***** I've tried the BV dyes. I may have got the wrong impression about them, but thought they were relatively large? Certainly for whole mount staining I thought they didn’t penetrate tissue very well. I also found that while BV421 was useful, the longer wavelength BV dyes when used on monolayers gave quite considerable fluorescence when excited at 561, 594 or 633 nm, making them less useful than we had hoped for multichannel imaging without unmixing. I'd be very interested to hear if I'm mistaken, and they are in fact small molecules that can penetrate well into whole mounts. If so we would certainly switch to BV421 instead of Alexa405. Empirically we do find alexa405 is not particularly bright, but generally find it to be as about as photostable as Alexa568. Cheers Dale -----Original Message----- From: Confocal Microscopy List <[hidden email]> On Behalf Of Jeff Carmichael Sent: 21 August 2018 12:31 To: [hidden email] Subject: Re: recommendation for a "blue" secondary fluorochrome COMMERCIAL RESPONSE ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** This has been addressed more than once in the listserv, but below are some resources to look into the BrilliantViolet fluorophores which are much more than an order of magnitude brighter than AF405. You could potentially add two additional channels in this spectral "dead zone". Chroma Technology has no commercial interest in these fluorophores, only in the filter sets we've recommended for detecting them. https://www.jacksonimmuno.com/technical/products/conjugate-selection/brilliant-violet http://bit.ly/ChromaTechnology2MtEdC9 http://bit.ly/bdbiosciences2MEed69 Good luck and please share your results! Jeff *Jeff Carmichael* *Product & Technical Marketing Manager* *[hidden email] <[hidden email]> | 802-428-2528* * <[hidden email]>* On Mon, Aug 20, 2018 at 7:06 PM, Cromey, Douglas W - (dcromey) < [hidden email]> wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Is there a better or preferred "blue" fluorochrome? Mostly this is a > widefield fluorescence question (using a DAPI cube), but there is the > possibility for using a confocal with a 405nm laser line. > > I'm working with a group that is trying to work around a frustrating > autofluorescence issue in liver tissue. What we ended up doing on an > earlier project was not using any secondaries that fluoresced in the > green channel (no fitc/AF488/GFP) and instead used the green channel > on our widefield microscope as a control, ratioing this image so we > could subtract it out of the other channels to remove some of the > broad autofluorescent background. This worked nicely. In the published > images we showed nuclei > (blue) and two specific stains using AF568 and AF647, with reduced > background autofluorescence. > > The PI now wants to have labelling with three specific fluorochromes, > and I am advocating using the green channel as the generic "everything > lights up" channel for context, so we can hopefully skip the Hoechst 3342 or DAPI. > Since we won't be using DNA dyes, is there a good blue fluorochrome > that can be used as a secondary for specific labelling of a marker > found on an intracellular organelle (other than the nucleus)? > > Thanks, > Doug > > ------------------------------------------------------------ > ------------------------------ > Douglas W. Cromey, M.S. - Associate Scientific Investigator Dept. of > Cellular & Molecular Medicine, University of Arizona > 1501 N. Campbell Ave, Tucson, AZ 85724-5044 USA > > office: LSN 463 email: [hidden email]<mailto: > [hidden email]> > voice: 520-626-2824 fax: 520-626-2097 > > http://microscopy.arizona.edu/learn/microscopy-imaging-resources-www > Home of: "Microscopy and Imaging Resources on the WWW" > > UA Microscopy Alliance - http://microscopy.arizona.edu< > http://microscopy.arizona.edu/> > -- <https://www.chroma.com/>CHROMA TECHNOLOGY CORP® *an employee owned company* 10 Imtec Lane, Bellows Falls, Vermont 05101 USA 800-824-7662 | FAX: 802-428-2525 www.chroma.com <https://www.chroma.com/> | [hidden email] <mailto:[hidden email]> |
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Jeffrey Carmichael |
Re: recommendation for a "blue" secondary fluorochrome COMMERCIAL 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 Post images on http://www.imgur.com and include the link in your posting. ***** Dale, You're right, these are polymers and they are fairly large, but there should only one per antibody. Unfortunately I'm removed from hands-on sample processing these days at Chroma, but you might want to contact BD Biosciences directly or Jackson ImmunoResearch for their recommendations. I have scanned several slides of thin tissues labeled with both BV421 & BV480 (and AF488) in the same sample, and all were clearly present and spectrally separable. Jeff On Tue, Aug 21, 2018 at 8:15 AM, Moulding, Dale <[hidden email]> wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > I've tried the BV dyes. I may have got the wrong impression about them, > but thought they were relatively large? Certainly for whole mount staining > I thought they didn’t penetrate tissue very well. > I also found that while BV421 was useful, the longer wavelength BV dyes > when used on monolayers gave quite considerable fluorescence when excited > at 561, 594 or 633 nm, making them less useful than we had hoped for > multichannel imaging without unmixing. > I'd be very interested to hear if I'm mistaken, and they are in fact small > molecules that can penetrate well into whole mounts. If so we would > certainly switch to BV421 instead of Alexa405. Empirically we do find > alexa405 is not particularly bright, but generally find it to be as about > as photostable as Alexa568. > > Cheers > > Dale > > -----Original Message----- > From: Confocal Microscopy List <[hidden email]> On > Behalf Of Jeff Carmichael > Sent: 21 August 2018 12:31 > To: [hidden email] > Subject: Re: recommendation for a "blue" secondary fluorochrome COMMERCIAL > RESPONSE > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > This has been addressed more than once in the listserv, but below are some > resources to look into the BrilliantViolet fluorophores which are much more > than an order of magnitude brighter than AF405. You could potentially add > two additional channels in this spectral "dead zone". > > Chroma Technology has no commercial interest in these fluorophores, only > in the filter sets we've recommended for detecting them. > > https://www.jacksonimmuno.com/technical/products/conjugate- > selection/brilliant-violet > http://bit.ly/ChromaTechnology2MtEdC9 > http://bit.ly/bdbiosciences2MEed69 > > Good luck and please share your results! > Jeff > > *Jeff Carmichael* > *Product & Technical Marketing Manager* > > *[hidden email] <[hidden email]> | 802-428-2528* > > * <[hidden email]>* > > On Mon, Aug 20, 2018 at 7:06 PM, Cromey, Douglas W - (dcromey) < > [hidden email]> wrote: > > > ***** > > To join, leave or search the confocal microscopy listserv, go to: > > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > > Post images on http://www.imgur.com and include the link in your > posting. > > ***** > > > > Is there a better or preferred "blue" fluorochrome? Mostly this is a > > widefield fluorescence question (using a DAPI cube), but there is the > > possibility for using a confocal with a 405nm laser line. > > > > I'm working with a group that is trying to work around a frustrating > > autofluorescence issue in liver tissue. What we ended up doing on an > > earlier project was not using any secondaries that fluoresced in the > > green channel (no fitc/AF488/GFP) and instead used the green channel > > on our widefield microscope as a control, ratioing this image so we > > could subtract it out of the other channels to remove some of the > > broad autofluorescent background. This worked nicely. In the published > > images we showed nuclei > > (blue) and two specific stains using AF568 and AF647, with reduced > > background autofluorescence. > > > > The PI now wants to have labelling with three specific fluorochromes, > > and I am advocating using the green channel as the generic "everything > > lights up" channel for context, so we can hopefully skip the Hoechst > 3342 or DAPI. > > Since we won't be using DNA dyes, is there a good blue fluorochrome > > that can be used as a secondary for specific labelling of a marker > > found on an intracellular organelle (other than the nucleus)? > > > > Thanks, > > Doug > > > > ------------------------------------------------------------ > > ------------------------------ > > Douglas W. Cromey, M.S. - Associate Scientific Investigator Dept. of > > Cellular & Molecular Medicine, University of Arizona > > 1501 N. Campbell Ave, Tucson, AZ 85724-5044 USA > > > > office: LSN 463 email: [hidden email]<mailto: > > [hidden email]> > > voice: 520-626-2824 fax: 520-626-2097 > > > > http://microscopy.arizona.edu/learn/microscopy-imaging-resources-www > > Home of: "Microscopy and Imaging Resources on the WWW" > > > > UA Microscopy Alliance - http://microscopy.arizona.edu< > > http://microscopy.arizona.edu/> > > > > -- > <https://www.chroma.com/>CHROMA TECHNOLOGY CORP® *an employee owned > company* > 10 Imtec Lane, Bellows Falls, Vermont 05101 USA > 800-824-7662 | > FAX: 802-428-2525 > www.chroma.com <https://www.chroma.com/> | [hidden email] <mailto: > [hidden email]> > -- <https://www.chroma.com/>CHROMA TECHNOLOGY CORP® *an employee owned company* 10 Imtec Lane, Bellows Falls, Vermont 05101 USA 800-824-7662 | FAX: 802-428-2525 www.chroma.com <https://www.chroma.com/> | [hidden email] <mailto:[hidden email]> |
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Adrian Smith-6 |
Re: recommendation for a "blue" secondary fluorochrome COMMERCIAL RESPONSE
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In reply to this post by Moulding, Dale
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Most of the longer wavelength Brilliant Violet reagents are tandems, ie a BV-polymer backbone as a donor with a second molecule as acceptor - hence the excitation at 561/594/633. Since Sirigin was purchased by Becton Dickinson they have come out with additional base polymers - Brilliant UV, Brilliant Blue, Brilliant Yellow/Green (may not be commercially available yet). Generally in each series the first reagent is a straight polymer and subsequent ones are tandems. As I know only BD is producing these and the focus has been on reagents for flow cytometry. http://www.bdbiosciences.com/anz/go/brilliant/ (this is the ANZ site - US site is down for me at the moment) Regards, Adrian On Tue, 21 Aug 2018 at 22:15, Moulding, Dale <[hidden email]> wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > I've tried the BV dyes. I may have got the wrong impression about them, > but thought they were relatively large? Certainly for whole mount staining > I thought they didn’t penetrate tissue very well. > I also found that while BV421 was useful, the longer wavelength BV dyes > when used on monolayers gave quite considerable fluorescence when excited > at 561, 594 or 633 nm, making them less useful than we had hoped for > multichannel imaging without unmixing. > I'd be very interested to hear if I'm mistaken, and they are in fact small > molecules that can penetrate well into whole mounts. If so we would > certainly switch to BV421 instead of Alexa405. Empirically we do find > alexa405 is not particularly bright, but generally find it to be as about > as photostable as Alexa568. > > Cheers > > Dale > > -----Original Message----- > From: Confocal Microscopy List <[hidden email]> On > Behalf Of Jeff Carmichael > Sent: 21 August 2018 12:31 > To: [hidden email] > Subject: Re: recommendation for a "blue" secondary fluorochrome COMMERCIAL > RESPONSE > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > This has been addressed more than once in the listserv, but below are some > resources to look into the BrilliantViolet fluorophores which are much more > than an order of magnitude brighter than AF405. You could potentially add > two additional channels in this spectral "dead zone". > > Chroma Technology has no commercial interest in these fluorophores, only > in the filter sets we've recommended for detecting them. > > > https://www.jacksonimmuno.com/technical/products/conjugate-selection/brilliant-violet > http://bit.ly/ChromaTechnology2MtEdC9 > http://bit.ly/bdbiosciences2MEed69 > > Good luck and please share your results! > Jeff > > *Jeff Carmichael* > *Product & Technical Marketing Manager* > > *[hidden email] <[hidden email]> | 802-428-2528* > > * <[hidden email]>* > > On Mon, Aug 20, 2018 at 7:06 PM, Cromey, Douglas W - (dcromey) < > [hidden email]> wrote: > > > ***** > > To join, leave or search the confocal microscopy listserv, go to: > > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > > Post images on http://www.imgur.com and include the link in your > posting. > > ***** > > > > Is there a better or preferred "blue" fluorochrome? Mostly this is a > > widefield fluorescence question (using a DAPI cube), but there is the > > possibility for using a confocal with a 405nm laser line. > > > > I'm working with a group that is trying to work around a frustrating > > autofluorescence issue in liver tissue. What we ended up doing on an > > earlier project was not using any secondaries that fluoresced in the > > green channel (no fitc/AF488/GFP) and instead used the green channel > > on our widefield microscope as a control, ratioing this image so we > > could subtract it out of the other channels to remove some of the > > broad autofluorescent background. This worked nicely. In the published > > images we showed nuclei > > (blue) and two specific stains using AF568 and AF647, with reduced > > background autofluorescence. > > > > The PI now wants to have labelling with three specific fluorochromes, > > and I am advocating using the green channel as the generic "everything > > lights up" channel for context, so we can hopefully skip the Hoechst > 3342 or DAPI. > > Since we won't be using DNA dyes, is there a good blue fluorochrome > > that can be used as a secondary for specific labelling of a marker > > found on an intracellular organelle (other than the nucleus)? > > > > Thanks, > > Doug > > > > ------------------------------------------------------------ > > ------------------------------ > > Douglas W. Cromey, M.S. - Associate Scientific Investigator Dept. of > > Cellular & Molecular Medicine, University of Arizona > > 1501 N. Campbell Ave, Tucson, AZ 85724-5044 USA > > > > office: LSN 463 email: [hidden email]<mailto: > > [hidden email]> > > voice: 520-626-2824 fax: 520-626-2097 > > > > http://microscopy.arizona.edu/learn/microscopy-imaging-resources-www > > Home of: "Microscopy and Imaging Resources on the WWW" > > > > UA Microscopy Alliance - http://microscopy.arizona.edu< > > http://microscopy.arizona.edu/> > > > > -- > <https://www.chroma.com/>CHROMA TECHNOLOGY CORP® *an employee owned > company* > 10 Imtec Lane, Bellows Falls, Vermont 05101 USA > 800-824-7662 | > FAX: 802-428-2525 > www.chroma.com <https://www.chroma.com/> | [hidden email] <mailto: > [hidden email]> > -- <https://www.centenary.org.au> |
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Vickie Frohlich |
Re: recommendation for a "blue" secondary fluorochrome COMMERCIAL RESPONSE
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Jeffrey Carmichael |
Re: recommendation for a "blue" secondary fluorochrome COMMERCIAL RESPONSE
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In reply to this post by Adrian Smith-6
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** This is correct. My understanding is that BV421, BV480 & BB515 are all "native", primary fluors without "acceptors" and the additional, attendant fluorescence wavelengths. I've been out of the loop lately, but additional "primary" Brillinat Horizon fluors are likely to be introduced by BD Biosciences. What is often overlooked with these fluors is that they are so bright and photostable that their real potential value is to use them for directly labelled, primary antibodies, without need for amplification and the issues that can arise from using secondary antibodies. I think it remains to be seen how useful they might ultimately be as primary probes. Perhaps the problems associated with direct primary antibody conjugation such as: - maintaining the antigenicity of the antibody - permeability into tissue due to size might be too difficult to overcome, but because of their brightness & photostability, it seems worth the effort to try for those folks who are interested in developing new/improved methodologies. Jeff *Jeff Carmichael* *Product & Technical Marketing Manager* *[hidden email] <[hidden email]> | 802-428-2528* * <[hidden email]>* On Tue, Aug 21, 2018 at 8:32 AM, Adrian Smith <[hidden email] > wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > Post images on http://www.imgur.com and include the link in your posting. > ***** > > Most of the longer wavelength Brilliant Violet reagents are tandems, ie a > BV-polymer backbone as a donor with a second molecule as acceptor - hence > the excitation at 561/594/633. > > Since Sirigin was purchased by Becton Dickinson they have come out with > additional base polymers - Brilliant UV, Brilliant Blue, Brilliant > Yellow/Green (may not be commercially available yet). Generally in each > series the first reagent is a straight polymer and subsequent ones are > tandems. As I know only BD is producing these and the focus has been on > reagents for flow cytometry. > > http://www.bdbiosciences.com/anz/go/brilliant/ > > (this is the ANZ site - US site is down for me at the moment) > > Regards, > > Adrian > > > > On Tue, 21 Aug 2018 at 22:15, Moulding, Dale <[hidden email]> wrote: > > > ***** > > To join, leave or search the confocal microscopy listserv, go to: > > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > > Post images on http://www.imgur.com and include the link in your > posting. > > ***** > > > > I've tried the BV dyes. I may have got the wrong impression about them, > > but thought they were relatively large? Certainly for whole mount > staining > > I thought they didn’t penetrate tissue very well. > > I also found that while BV421 was useful, the longer wavelength BV dyes > > when used on monolayers gave quite considerable fluorescence when excited > > at 561, 594 or 633 nm, making them less useful than we had hoped for > > multichannel imaging without unmixing. > > I'd be very interested to hear if I'm mistaken, and they are in fact > small > > molecules that can penetrate well into whole mounts. If so we would > > certainly switch to BV421 instead of Alexa405. Empirically we do find > > alexa405 is not particularly bright, but generally find it to be as about > > as photostable as Alexa568. > > > > Cheers > > > > Dale > > > > -----Original Message----- > > From: Confocal Microscopy List <[hidden email]> On > > Behalf Of Jeff Carmichael > > Sent: 21 August 2018 12:31 > > To: [hidden email] > > Subject: Re: recommendation for a "blue" secondary fluorochrome > COMMERCIAL > > RESPONSE > > > > ***** > > To join, leave or search the confocal microscopy listserv, go to: > > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > > Post images on http://www.imgur.com and include the link in your > posting. > > ***** > > > > This has been addressed more than once in the listserv, but below are > some > > resources to look into the BrilliantViolet fluorophores which are much > more > > than an order of magnitude brighter than AF405. You could potentially add > > two additional channels in this spectral "dead zone". > > > > Chroma Technology has no commercial interest in these fluorophores, only > > in the filter sets we've recommended for detecting them. > > > > > > https://www.jacksonimmuno.com/technical/products/conjugate- > selection/brilliant-violet > > http://bit.ly/ChromaTechnology2MtEdC9 > > http://bit.ly/bdbiosciences2MEed69 > > > > Good luck and please share your results! > > Jeff > > > > *Jeff Carmichael* > > *Product & Technical Marketing Manager* > > > > *[hidden email] <[hidden email]> | 802-428-2528* > > > > * <[hidden email]>* > > > > On Mon, Aug 20, 2018 at 7:06 PM, Cromey, Douglas W - (dcromey) < > > [hidden email]> wrote: > > > > > ***** > > > To join, leave or search the confocal microscopy listserv, go to: > > > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > > > Post images on http://www.imgur.com and include the link in your > > posting. > > > ***** > > > > > > Is there a better or preferred "blue" fluorochrome? Mostly this is a > > > widefield fluorescence question (using a DAPI cube), but there is the > > > possibility for using a confocal with a 405nm laser line. > > > > > > I'm working with a group that is trying to work around a frustrating > > > autofluorescence issue in liver tissue. What we ended up doing on an > > > earlier project was not using any secondaries that fluoresced in the > > > green channel (no fitc/AF488/GFP) and instead used the green channel > > > on our widefield microscope as a control, ratioing this image so we > > > could subtract it out of the other channels to remove some of the > > > broad autofluorescent background. This worked nicely. In the published > > > images we showed nuclei > > > (blue) and two specific stains using AF568 and AF647, with reduced > > > background autofluorescence. > > > > > > The PI now wants to have labelling with three specific fluorochromes, > > > and I am advocating using the green channel as the generic "everything > > > lights up" channel for context, so we can hopefully skip the Hoechst > > 3342 or DAPI. > > > Since we won't be using DNA dyes, is there a good blue fluorochrome > > > that can be used as a secondary for specific labelling of a marker > > > found on an intracellular organelle (other than the nucleus)? > > > > > > Thanks, > > > Doug > > > > > > ------------------------------------------------------------ > > > ------------------------------ > > > Douglas W. Cromey, M.S. - Associate Scientific Investigator Dept. of > > > Cellular & Molecular Medicine, University of Arizona > > > 1501 N. Campbell Ave, Tucson, AZ 85724-5044 USA > > > > > > office: LSN 463 email: [hidden email]<mailto: > > > [hidden email]> > > > voice: 520-626-2824 fax: 520-626-2097 > > > > > > http://microscopy.arizona.edu/learn/microscopy-imaging-resources-www > > > Home of: "Microscopy and Imaging Resources on the WWW" > > > > > > UA Microscopy Alliance - http://microscopy.arizona.edu< > > > http://microscopy.arizona.edu/> > > > > > > > -- > > <https://www.chroma.com/>CHROMA TECHNOLOGY CORP® *an employee owned > > company* > > 10 Imtec Lane, Bellows Falls, Vermont 05101 USA > > 800-824-7662 | > > FAX: 802-428-2525 > > www.chroma.com <https://www.chroma.com/> | [hidden email] <mailto: > > [hidden email]> > > > > -- > <https://www.centenary.org.au> > -- <https://www.chroma.com/>CHROMA TECHNOLOGY CORP® *an employee owned company* 10 Imtec Lane, Bellows Falls, Vermont 05101 USA 800-824-7662 | FAX: 802-428-2525 www.chroma.com <https://www.chroma.com/> | [hidden email] <mailto:[hidden email]> |
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Nicolai.Urban@mpfi.org |
Re: recommendation for a "blue" secondary fluorochrome COMMERCIAL RESPONSE
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In reply to this post by Vickie Frohlich
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. ***** Hi Doug, I have had decent success using Atto 390 (https://www.atto-tec.com/attotecshop/product_info.php?language=en&info=p1_ATTO-390.html), both in live brain tissue and fixed cells. One thing to really watch out for, though, when using these near-UV dyes for multicolor imaging, is that they frequently show much stronger chromatic effects than the other color channels. This depends strongly on the optics you are using (especially the tube and objective lenses), as the chromatic correction usually gets worse the closer you get to the UV. In the past I have had severe problems with chromatic shift along the optical axis, as well as much stronger aberrations (especially coma and astigmatism) in the deep blue channel. If you go (deep) into tissue, this can become prohibitive very quickly. Not to discourage you, just be sure to keep an eye out for potential issues and test your samples, dyes and system thoroughly with this in mind. Good luck! Nicolai Urban >>>>>>>>>><<<<<<<<>>>>>>>>>><<<<<<<<<<>>>>>>>>>><<<<<<<<<< Dr. Nicolai T. Urban Max Planck Florida Institute Jupiter, 33458 FL, USA -----Original Message----- From: Confocal Microscopy List <[hidden email]> On Behalf Of Frohlich, Victoria Sent: Dienstag, 21. August 2018 08:33 To: [hidden email] Subject: Re: recommendation for a "blue" secondary fluorochrome COMMERCIAL RESPONSE ***** To join, leave or search the confocal microscopy listserv, go to: https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7Cbca72c348b58419452c708d60766a3b3%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636704534756076388&sdata=nmGnYdg71423j2J6FzqAz%2FtBoDncfqNxpsLyU1SUZlY%3D&reserved=0 Post images on https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7Cbca72c348b58419452c708d60766a3b3%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636704534756076388&sdata=M%2Bo69yJjhaqpGhI46moEzhlfaOP0yPRXPvSyJXsU7%2Fk%3D&reserved=0 and include the link in your posting. ***** Try CF405 from Biotium. Bright and robust Vickie Sent from my iPhone > On Aug 21, 2018, at 7:32 AM, Jeff Carmichael <[hidden email]> wrote: > > ***** > To join, leave or search the confocal microscopy listserv, go to: > https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists. > umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=02%7C01%7CNi > colai.Urban%40MPFI.ORG%7Cbca72c348b58419452c708d60766a3b3%7C947b45517d > b44636a5fd1bdcad603ed0%7C0%7C0%7C636704534756076388&sdata=nmGnYdg7 > 1423j2J6FzqAz%2FtBoDncfqNxpsLyU1SUZlY%3D&reserved=0 > Post images on https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7Cbca72c348b58419452c708d60766a3b3%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636704534756076388&sdata=M%2Bo69yJjhaqpGhI46moEzhlfaOP0yPRXPvSyJXsU7%2Fk%3D&reserved=0 and include the link in your posting. > ***** > > This has been addressed more than once in the listserv, but below are > some resources to look into the BrilliantViolet fluorophores which are > much more than an order of magnitude brighter than AF405. You could > potentially add two additional channels in this spectral "dead zone". > > Chroma Technology has no commercial interest in these fluorophores, > only in the filter sets we've recommended for detecting them. > > https://na01.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.j > acksonimmuno.com%2Ftechnical%2Fproducts%2Fconjugate-selection%2Fbrilli > ant-violet&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7Cbca72c348b584 > 19452c708d60766a3b3%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C63670 > 4534756076388&sdata=7sB%2BwkffqcA3znOzQxc1fJ6jowr7AdbY78x9S62VBs0% > 3D&reserved=0 > https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fbit.ly > %2FChromaTechnology2MtEdC9&data=02%7C01%7CNicolai.Urban%40MPFI.ORG > %7Cbca72c348b58419452c708d60766a3b3%7C947b45517db44636a5fd1bdcad603ed0 > %7C0%7C0%7C636704534756076388&sdata=5A6mmulPlN5N9PVCohPfs7YQL5gHcZ > kIqg%2B5nlNc2ys%3D&reserved=0 > https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fbit.ly > %2Fbdbiosciences2MEed69&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C > bca72c348b58419452c708d60766a3b3%7C947b45517db44636a5fd1bdcad603ed0%7C > 0%7C0%7C636704534756076388&sdata=rjKyHFXEsTV%2FOwaMHHGv3y0EeykYZ8g > gZdEFnWenvhc%3D&reserved=0 > > Good luck and please share your results! > Jeff > > *Jeff Carmichael* > *Product & Technical Marketing Manager* > > *[hidden email] <[hidden email]> | 802-428-2528* > > * <[hidden email]>* > > On Mon, Aug 20, 2018 at 7:06 PM, Cromey, Douglas W - (dcromey) < > [hidden email]> wrote: > >> ***** >> To join, leave or search the confocal microscopy listserv, go to: >> https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists >> .umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=02%7C01%7C >> Nicolai.Urban%40MPFI.ORG%7Cbca72c348b58419452c708d60766a3b3%7C947b455 >> 17db44636a5fd1bdcad603ed0%7C0%7C0%7C636704534756076388&sdata=nmGn >> Ydg71423j2J6FzqAz%2FtBoDncfqNxpsLyU1SUZlY%3D&reserved=0 >> Post images on https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7Cbca72c348b58419452c708d60766a3b3%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636704534756076388&sdata=M%2Bo69yJjhaqpGhI46moEzhlfaOP0yPRXPvSyJXsU7%2Fk%3D&reserved=0 and include the link in your posting. >> ***** >> >> Is there a better or preferred "blue" fluorochrome? Mostly this is a >> widefield fluorescence question (using a DAPI cube), but there is the >> possibility for using a confocal with a 405nm laser line. >> >> I'm working with a group that is trying to work around a frustrating >> autofluorescence issue in liver tissue. What we ended up doing on an >> earlier project was not using any secondaries that fluoresced in the >> green channel (no fitc/AF488/GFP) and instead used the green channel >> on our widefield microscope as a control, ratioing this image so we >> could subtract it out of the other channels to remove some of the >> broad autofluorescent background. This worked nicely. In the >> published images we showed nuclei >> (blue) and two specific stains using AF568 and AF647, with reduced >> background autofluorescence. >> >> The PI now wants to have labelling with three specific fluorochromes, >> and I am advocating using the green channel as the generic >> "everything lights up" channel for context, so we can hopefully skip the Hoechst 3342 or DAPI. >> Since we won't be using DNA dyes, is there a good blue fluorochrome >> that can be used as a secondary for specific labelling of a marker >> found on an intracellular organelle (other than the nucleus)? >> >> Thanks, >> Doug >> >> ------------------------------------------------------------ >> ------------------------------ >> Douglas W. Cromey, M.S. - Associate Scientific Investigator Dept. of >> Cellular & Molecular Medicine, University of Arizona >> 1501 N. Campbell Ave, Tucson, AZ 85724-5044 USA >> >> office: LSN 463 email: [hidden email]<mailto: >> [hidden email]> >> voice: 520-626-2824 fax: 520-626-2097 >> >> https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fmicro >> scopy.arizona.edu%2Flearn%2Fmicroscopy-imaging-resources-www&data >> =02%7C01%7CNicolai.Urban%40MPFI.ORG%7Cbca72c348b58419452c708d60766a3b >> 3%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636704534756076388& >> ;sdata=Uers6TQVsdERd5TLgwFD4cRAgVS3m1ipSnARrNu5vIc%3D&reserved=0 >> Home of: "Microscopy and Imaging Resources on the WWW" >> >> UA Microscopy Alliance - >> https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fmicro >> scopy.arizona.edu&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7Cbca72 >> c348b58419452c708d60766a3b3%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C >> 0%7C636704534756076388&sdata=EPgfSrktJXjrmKGMx0wJXVYFGhIQbmCALyFk >> AWK2xd8%3D&reserved=0< >> https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fmicro >> scopy.arizona.edu%2F&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7Cbc >> a72c348b58419452c708d60766a3b3%7C947b45517db44636a5fd1bdcad603ed0%7C0 >> %7C0%7C636704534756076388&sdata=YMHFXCumxURKtEFsVi2kO6ew3QNAc%2Bb >> gJaqj97qHG30%3D&reserved=0> >> > > -- > <https://na01.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww. > chroma.com%2F&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7Cbca72c348b > 58419452c708d60766a3b3%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C63 > 6704534756076388&sdata=Ka%2BHihmwcUI7oNUbP9AwQc4OqU6zUvSOH76YS1zIT > DI%3D&reserved=0>CHROMA TECHNOLOGY CORP® *an employee owned > company* > 10 Imtec Lane, Bellows Falls, Vermont 05101 USA > 800-824-7662 | > FAX: 802-428-2525 > https://na01.safelinks.protection.outlook.com/?url=www.chroma.com& > data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7Cbca72c348b58419452c708d60766 > a3b3%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636704534756076388&a > mp;sdata=gtqpye2lYkRdMkwo9oBd42ruo%2Fjve0MLUXFB%2BAip%2BDE%3D&rese > rved=0 > <https://na01.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww. > chroma.com%2F&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7Cbca72c348b > 58419452c708d60766a3b3%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C63 > 6704534756076388&sdata=Ka%2BHihmwcUI7oNUbP9AwQc4OqU6zUvSOH76YS1zIT > DI%3D&reserved=0> | [hidden email] <mailto:[hidden email]> ________________________________ Email Disclaimer: https://na01.safelinks.protection.outlook.com/?url=www.stjude.org%2Femaildisclaimer&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7Cbca72c348b58419452c708d60766a3b3%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636704534756076388&sdata=ehRhSKaa6vodis%2BNBoM4oZvVpHY12%2BMvh4Md6yiUABY%3D&reserved=0 Consultation Disclaimer: https://na01.safelinks.protection.outlook.com/?url=www.stjude.org%2Fconsultationdisclaimer&data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7Cbca72c348b58419452c708d60766a3b3%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C636704534756076388&sdata=4k96m0HHCF9Tov0qSGH9Wq7W%2FooI374uBjelTgDeUFo%3D&reserved=0 |
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