405 or 440 laser for light sheet system
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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 am putting together a light sheet system to do live imaging of zebrafish development. I am planning to use a laser engine like the Vortran Versalase. This allows for 4 laser lines. I had initially planned to get 440, 488, 515, and 516 to match the most common fluorescent proteins used in zebrafish lines (basically CFP, GFP, YFP, and RFP derivatives). However, I have noticed that BFP proteins have become more popular, particularly tagBFPII. So now I am stuck deciding between 405 or 440 lasers. A 405 laser can still excite CFPs, but about half optimal, but probably good enough do both CFP and BFP day n most cases. Also, though I know 405 light tends to be pretty harsh on live cells, but believe the less harsh exposure of light sheet will overcome this. I am also a bit worried about how far 405nm light will penetrate tissue. I have only used it to image cells, but know once you get into UV tissue or trance drops off quickly. So if you had to choose 405 or 440 which way would you go? I know there is always the option to through money at the problem and get both, but trying to avoid that because systems that support more than 4 lasers have a big bump in price. Steve |
 
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Tim Feinstein |
Re: 405 or 440 laser for light sheet system
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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 Steve, Good luck with the light sheet! IMO the answer depends on whether you plan on using CFP for FRET or FRET-based biosensors. I would avoid 405nm for CFP excitation based on my own experience so I'd lean 440 if you have FRET in mind. Of course in a pinch 405 is better than nothing. In favor of the 405nm, this line would let you do three-color imaging of fluorescent proteins, or four channels if recently developed IR emitters work in zebrafish. The emission of CFP overlaps strongly with YFP and especially GFP, making it unsuitable for co-imaging with other FPs except for red proteins. 405nm also lets you use DAPI when a want to image traditional whole mount specimens, though penetration is poor and you're better off using an IR dye like TO-PRO when possible. For what it's worth we use a 4-line versalase (405/488/561/633) on a home-built light sheet and we are very satisfied. No commercial interest, just a customer. Best, T Timothy Feinstein, Ph.D. Research Scientist Department of Developmental Biology University of Pittsburgh On 4/16/19, 5:06 PM, "Confocal Microscopy List on behalf of Steve H" <[hidden email] on behalf of [hidden email]> wrote: ***** To join, leave or search the confocal microscopy listserv, go to: https://nam05.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=02%7C01%7Ctnf8%40PITT.EDU%7C278bb38947ad46a89a8c08d6c2af5b30%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C1%7C636910455767735533&sdata=PxBIhgAnYtVoiXXwWlB2jkERL0ZSUqssJ1UlvbyydEc%3D&reserved=0 Post images on https://nam05.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&data=02%7C01%7Ctnf8%40PITT.EDU%7C278bb38947ad46a89a8c08d6c2af5b30%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C1%7C636910455767735533&sdata=urie%2F65mhIcij3CGTSs9SAYriiArL5FnrJIQhVBcXD8%3D&reserved=0 and include the link in your posting. ***** I am putting together a light sheet system to do live imaging of zebrafish development. I am planning to use a laser engine like the Vortran Versalase. This allows for 4 laser lines. I had initially planned to get 440, 488, 515, and 516 to match the most common fluorescent proteins used in zebrafish lines (basically CFP, GFP, YFP, and RFP derivatives). However, I have noticed that BFP proteins have become more popular, particularly tagBFPII. So now I am stuck deciding between 405 or 440 lasers. A 405 laser can still excite CFPs, but about half optimal, but probably good enough do both CFP and BFP day n most cases. Also, though I know 405 light tends to be pretty harsh on live cells, but believe the less harsh exposure of light sheet will overcome this. I am also a bit worried about how far 405nm light will penetrate tissue. I have only used it to image cells, but know once you get into UV tissue or trance drops off quickly. So if you had to choose 405 or 440 which way would you go? I know there is always the option to through money at the problem and get both, but trying to avoid that because systems that support more than 4 lasers have a big bump in price. Steve |
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Heddleston, John |
Re: 405 or 440 laser for light sheet system
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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 Steve, We try to stay away from wavelengths shorter than 488 (unless you want a 440 for FRET, as previously mentioned). The short wavelengths don’t penetrate well. I would recommend having a 488, 560, and 642. Then fourth line could be for whatever you think you would use more (440 or 513 or 589). There are more far red fluors coming in use (iRFP670, comes to mind, in addition to Halo dyes like Janelia Fluor 646) that are better for animal imaging. One of our light sheet systems uses an Omicron box with 488, 560, 594, 642, and 689, which is a good combo for us. Regards, John Application Scientist Advanced Imaging Center at HHMI Janelia E: [hidden email]<mailto:[hidden email]> P: (571) 209-4000, ext 3356 W: www.janelia.org/aic<http://www.janelia.org/aic> From: Confocal Microscopy List <[hidden email]<mailto:[hidden email]>> on behalf of "Feinstein, Timothy N" <[hidden email]<mailto:[hidden email]>> Reply-To: Confocal Microscopy List <[hidden email]<mailto:[hidden email]>> Date: Thursday, April 18, 2019 at 8:17 AM To: "[hidden email]<mailto:[hidden email]>" <[hidden email]<mailto:[hidden email]>> Subject: Re: 405 or 440 laser for light sheet system ***** 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 Steve, Good luck with the light sheet! IMO the answer depends on whether you plan on using CFP for FRET or FRET-based biosensors. I would avoid 405nm for CFP excitation based on my own experience so I'd lean 440 if you have FRET in mind. Of course in a pinch 405 is better than nothing. In favor of the 405nm, this line would let you do three-color imaging of fluorescent proteins, or four channels if recently developed IR emitters work in zebrafish. The emission of CFP overlaps strongly with YFP and especially GFP, making it unsuitable for co-imaging with other FPs except for red proteins. 405nm also lets you use DAPI when a want to image traditional whole mount specimens, though penetration is poor and you're better off using an IR dye like TO-PRO when possible. For what it's worth we use a 4-line versalase (405/488/561/633) on a home-built light sheet and we are very satisfied. No commercial interest, just a customer. Best, T Timothy Feinstein, Ph.D. Research Scientist Department of Developmental Biology University of Pittsburgh On 4/16/19, 5:06 PM, "Confocal Microscopy List on behalf of Steve H" <[hidden email]<mailto:[hidden email]> on behalf of [hidden email]<mailto:[hidden email]>> wrote: ***** To join, leave or search the confocal microscopy listserv, go to: https://nam05.safelinks.protection.outlook.com/?url=http%3A%2F%2Flists.umn.edu%2Fcgi-bin%2Fwa%3FA0%3Dconfocalmicroscopy&data=02%7C01%7Ctnf8%40PITT.EDU%7C278bb38947ad46a89a8c08d6c2af5b30%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C1%7C636910455767735533&sdata=PxBIhgAnYtVoiXXwWlB2jkERL0ZSUqssJ1UlvbyydEc%3D&reserved=0 Post images on https://nam05.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.imgur.com&data=02%7C01%7Ctnf8%40PITT.EDU%7C278bb38947ad46a89a8c08d6c2af5b30%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C1%7C636910455767735533&sdata=urie%2F65mhIcij3CGTSs9SAYriiArL5FnrJIQhVBcXD8%3D&reserved=0 and include the link in your posting. ***** I am putting together a light sheet system to do live imaging of zebrafish development. I am planning to use a laser engine like the Vortran Versalase. This allows for 4 laser lines. I had initially planned to get 440, 488, 515, and 516 to match the most common fluorescent proteins used in zebrafish lines (basically CFP, GFP, YFP, and RFP derivatives). However, I have noticed that BFP proteins have become more popular, particularly tagBFPII. So now I am stuck deciding between 405 or 440 lasers. A 405 laser can still excite CFPs, but about half optimal, but probably good enough do both CFP and BFP day n most cases. Also, though I know 405 light tends to be pretty harsh on live cells, but believe the less harsh exposure of light sheet will overcome this. I am also a bit worried about how far 405nm light will penetrate tissue. I have only used it to image cells, but know once you get into UV tissue or trance drops off quickly. So if you had to choose 405 or 440 which way would you go? I know there is always the option to through money at the problem and get both, but trying to avoid that because systems that support more than 4 lasers have a big bump in price. Steve |
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