Dictyostelium confocal and heat
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Oshel, Philip Eugene |
Dictyostelium confocal and heat
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Confocalers,
Anyone here working on Dictyostelium? If yes, do you have problems with heat from the illumination source killing the critters? The cells are really sensitive to heat and light, so I'd like to hear some actual experiences with different systems looking at live Dictys, and how happy they were after you were done looking at them. A regular fluorescence 'scope can easily kill them. "Illumination source", as I'm wondering about both spinning-disc systems that use Hg or Xe lamps and scanned-laser systems, point scanners or line scanners. We're looking to get a new confocal, and what kind of system (disc, point scanner, line scanner, millions of nanognomes with LED flashlights, whatever) is an issue for the group here. I'm already dealing with vendors, so no vendor contacts off-list, please. Technical information appropriate for the list is appreciated. Thanks. Phil -- Philip Oshel Microscopy Facility Supervisor Biology Department 024C Brooks Hall Central Michigan University Mt. Pleasant, MI 48859 (989) 774-3576 |
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Knecht, David |
Re: Dictyostelium confocal and heat
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Hi Philip- We have been wrestling with this problem for years. We have heat reflection and heat absorption filters in the transmitted path of all our microscopes. We always try to minimize light levels for both fluorescence and transmitted as that has a bigger effect than exposure time. We have used various light sources and all are problematic. Dicty is a great test of the efficiency of a confocal system, as their rounding up response is so easy to see. We used that as a diagnostic in evaluating various systems. In the end, they can all be made to work, but it comes down to how often you take images and how low you can turn down the light and still see your probe. I think that in general, deconvolution or a laser scanning system in which you can open the pinhole and sacrifice some confocality are the best. Spinning disk is good, but we just got one so I don't have as much experience yet. Others have indicated it is an improvement and my initial tests indicate we can image faster. Our Leica confocal is probably the worst in our stable because you cannot open the pinhole and decrease confocality, however we use it regularly for xyt imaging by just not collecting images as often. Bottom line- no magic anywhere, only tradeoffs. Dave
On Dec 12, 2008, at 8:37 AM, Philip Oshel wrote:
Dr. David Knecht Department of Molecular and Cell Biology Co-head Flow Cytometry and Confocal Microscopy Facility U-3125 91 N. Eagleville Rd. University of Connecticut Storrs, CT 06269 860-486-2200 860-486-4331 (fax) |
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Phillips, Thomas E. |
Re: Dictyostelium confocal and heat
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David – based on your experience with Dicty, what temperature do
you think your confocals are getting the slides to? Ideally your answer
include the laser strength/scan speed/etc. thanks, Tom Thomas E. Phillips, Ph.D Professor of Biological Sciences Chair, MU Faculty Council Director, Molecular Cytology Core 2 Tucker Hall University of Missouri Columbia, MO 65211-7400 573-882-4712 (office) 573-882-0123 (fax) http://www.biology.missouri.edu/faculty/phillips.html From: Confocal Microscopy
List [mailto:[hidden email]] On Behalf Of David Knecht Hi Philip- We have been wrestling with this problem for
years. We have heat reflection and heat absorption filters in the
transmitted path of all our microscopes. We always try to minimize light
levels for both fluorescence and transmitted as that has a bigger effect than
exposure time. We have used various light sources and all are
problematic. Dicty is a great test of the efficiency of a confocal
system, as their rounding up response is so easy to see. We used that as
a diagnostic in evaluating various systems. In the end, they can all be
made to work, but it comes down to how often you take images and how low you
can turn down the light and still see your probe. I think that in
general, deconvolution or a laser scanning system in which you can open the
pinhole and sacrifice some confocality are the best. Spinning disk is
good, but we just got one so I don't have as much experience yet. Others
have indicated it is an improvement and my initial tests indicate we can image
faster. Our Leica confocal is probably the worst in our stable because
you cannot open the pinhole and decrease confocality, however we use it regularly
for xyt imaging by just not collecting images as often. Bottom line- no
magic anywhere, only tradeoffs. Dave On Dec 12, 2008, at 8:37 AM, Philip Oshel wrote:
Confocalers, Dr.
David Knecht Department
of Molecular and Cell Biology Co-head
Flow Cytometry and Confocal Microscopy Facility U-3125 91
N. Eagleville Rd. University
of Connecticut Storrs,
CT 06269 860-486-2200 860-486-4331
(fax)
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Michael Herron |
Re: Dictyostelium confocal and heat
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In reply to this post by Knecht, David
David,
Could you please keep us up-to-date with regards to your upcoming luck with the spinning disk system? We are trying to make the same decision now. Mike On Dec 12, 2008, at 9:48 AM, David Knecht wrote: > Hi Philip- We have been wrestling with this problem for years. We > have heat reflection and heat absorption filters in the transmitted > path of all our microscopes. We always try to minimize light > levels for both fluorescence and transmitted as that has a bigger > effect than exposure time. We have used various light sources and > all are problematic. Dicty is a great test of the efficiency of a > confocal system, as their rounding up response is so easy to see. > We used that as a diagnostic in evaluating various systems. In the > end, they can all be made to work, but it comes down to how often > you take images and how low you can turn down the light and still > see your probe. I think that in general, deconvolution or a laser > scanning system in which you can open the pinhole and sacrifice > some confocality are the best. Spinning disk is good, but we just > got one so I don't have as much experience yet. Others have > indicated it is an improvement and my initial tests indicate we can > image faster. Our Leica confocal is probably the worst in our > stable because you cannot open the pinhole and decrease > confocality, however we use it regularly for xyt imaging by just > not collecting images as often. Bottom line- no magic anywhere, > only tradeoffs. Dave > > On Dec 12, 2008, at 8:37 AM, Philip Oshel wrote: > >> Confocalers, >> >> Anyone here working on Dictyostelium? If yes, do you have problems >> with heat from the illumination source killing the critters? >> The cells are really sensitive to heat and light, so I'd like to >> hear some actual experiences with different systems looking at >> live Dictys, and how happy they were after you were done looking >> at them. A regular fluorescence 'scope can easily kill them. >> >> "Illumination source", as I'm wondering about both spinning-disc >> systems that use Hg or Xe lamps and scanned-laser systems, point >> scanners or line scanners. We're looking to get a new confocal, >> and what kind of system (disc, point scanner, line scanner, >> millions of nanognomes with LED flashlights, whatever) is an issue >> for the group here. >> >> I'm already dealing with vendors, so no vendor contacts off-list, >> please. Technical information appropriate for the list is >> appreciated. >> >> Thanks. >> >> Phil >> -- >> Philip Oshel >> Microscopy Facility Supervisor >> Biology Department >> 024C Brooks Hall >> Central Michigan University >> Mt. Pleasant, MI 48859 >> (989) 774-3576 > > Dr. David Knecht > Department of Molecular and Cell Biology > Co-head Flow Cytometry and Confocal Microscopy Facility > U-3125 > 91 N. Eagleville Rd. > University of Connecticut > Storrs, CT 06269 > 860-486-2200 > 860-486-4331 (fax) > > Michael J. Herron, U of MN, Dept. of Entomology [hidden email] 612-624-3688 (office) 612-625-5299 (FAX) |
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Phillips, Thomas E. |
Dictyostelium confocal and heat
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In reply to this post by Oshel, Philip Eugene
David - based on your experience with Dicty, what temperature do you think your confocals are getting the slides to? Ideally your answer include the laser strength/scan speed/etc. thanks, Tom
Thomas E. Phillips, Ph.D Professor of Biological Sciences Chair, MU Faculty Council Director, Molecular Cytology Core 2 Tucker Hall University of Missouri Columbia, MO 65211-7400 573-882-4712 (office) 573-882-0123 (fax) [hidden email] From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of David Knecht Sent: Friday, December 12, 2008 9:48 AM To: [hidden email] Subject: Re: Dictyostelium confocal and heat Hi Philip- We have been wrestling with this problem for years. We have heat reflection and heat absorption filters in the transmitted path of all our microscopes. We always try to minimize light levels for both fluorescence and transmitted as that has a bigger effect than exposure time. We have used various light sources and all are problematic. Dicty is a great test of the efficiency of a confocal system, as their rounding up response is so easy to see. We used that as a diagnostic in evaluating various systems. In the end, they can all be made to work, but it comes down to how often you take images and how low you can turn down the light and still see your probe. I think that in general, deconvolution or a laser scanning system in which you can open the pinhole and sacrifice some confocality are the best. Spinning disk is good, but we just got one so I don't have as much experience yet. Others have indicated it is an improvement and my initial tests indicate we can image faster. Our Leica confocal is probably the worst in our stable because you cannot open the pinhole and decrease confocality, however we use it regularly for xyt imaging by just not collecting images as often. Bottom line- no magic anywhere, only tradeoffs. Dave On Dec 12, 2008, at 8:37 AM, Philip Oshel wrote: Confocalers, Anyone here working on Dictyostelium? If yes, do you have problems with heat from the illumination source killing the critters? The cells are really sensitive to heat and light, so I'd like to hear some actual experiences with different systems looking at live Dictys, and how happy they were after you were done looking at them. A regular fluorescence 'scope can easily kill them. "Illumination source", as I'm wondering about both spinning-disc systems that use Hg or Xe lamps and scanned-laser systems, point scanners or line scanners. We're looking to get a new confocal, and what kind of system (disc, point scanner, line scanner, millions of nanognomes with LED flashlights, whatever) is an issue for the group here. I'm already dealing with vendors, so no vendor contacts off-list, please. Technical information appropriate for the list is appreciated. Thanks. Phil -- Philip Oshel Microscopy Facility Supervisor Biology Department 024C Brooks Hall Central Michigan University Mt. Pleasant, MI 48859 (989) 774-3576 Dr. David Knecht Department of Molecular and Cell Biology Co-head Flow Cytometry and Confocal Microscopy Facility U-3125 91 N. Eagleville Rd. University of Connecticut Storrs, CT 06269 860-486-2200 860-486-4331 (fax) |
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Knecht, David |
Re: Dictyostelium confocal and heat
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I don't think this is about raising the temperature of the cells at all. We would see a shift in focal plane as the coverslip shifts if the temp were increasing significantly. I believe this is more about photodamage and photooxidation. If the cells are in HL5 medium which is very fluorescent, the cells are very sensitive to fluorescent excitation. If you change to LoFLo or buffer and allow them to recycle out their endosomes, they are dramatically less sensitive. Developing cells that have been out of medium for 6-8 hours are even easier to image. We also found that oxyrase will increase their resistance to light, indicating photooxidative damage. I have a time lapse movie where the cells are imaged on the Leica, where they rapidly round up and then when you turn the laser down, they very rapidly begin to extend and move again. We did some work with antioxidants, but not enough to find one that solves the problem. Dave
On Dec 12, 2008, at 11:39 AM, Phillips, Thomas E. wrote:
Dr. David Knecht Department of Molecular and Cell Biology Co-head Flow Cytometry and Confocal Microscopy Facility U-3125 91 N. Eagleville Rd. University of Connecticut Storrs, CT 06269 860-486-2200 860-486-4331 (fax) |
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Oshel, Philip Eugene |
Re: Dictyostelium confocal and heat
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Dave,
By "Leica", which one do you mean? A scanned-laser, or your new spinning-disc? Phil I don't think this is about raising the temperature of the cells at all. We would see a shift in focal plane as the coverslip shifts if the temp were increasing significantly. I believe this is more about photodamage and photooxidation. If the cells are in HL5 medium which is very fluorescent, the cells are very sensitive to fluorescent excitation. If you change to LoFLo or buffer and allow them to recycle out their endosomes, they are dramatically less sensitive. Developing cells that have been out of medium for 6-8 hours are even easier to image. We also found that oxyrase will increase their resistance to light, indicating photooxidative damage. I have a time lapse movie where the cells are imaged on the Leica, where they rapidly round up and then when you turn the laser down, they very rapidly begin to extend and move again. We did some work with antioxidants, but not enough to find one that solves the problem. Dave On Dec 12, 2008, at 11:39 AM, Phillips, Thomas E. wrote: David - based on your experience with Dicty, what temperature do you think your confocals are getting the slides to? Ideally your answer include the laser strength/scan speed/etc. thanks, Tom Thomas E. Phillips, Ph.D Professor of Biological Sciences Chair, MU Faculty Council Director, Molecular Cytology Core 2 Tucker Hall University of Missouri Columbia, MO 65211-7400 573-882-4712 (office) 573-882-0123 (fax) <mailto:[hidden email]>[hidden email] From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of David Knecht Sent: Friday, December 12, 2008 9:48 AM To: [hidden email] Subject: Re: Dictyostelium confocal and heat Hi Philip- We have been wrestling with this problem for years. We have heat reflection and heat absorption filters in the transmitted path of all our microscopes. We always try to minimize light levels for both fluorescence and transmitted as that has a bigger effect than exposure time. We have used various light sources and all are problematic. Dicty is a great test of the efficiency of a confocal system, as their rounding up response is so easy to see. We used that as a diagnostic in evaluating various systems. In the end, they can all be made to work, but it comes down to how often you take images and how low you can turn down the light and still see your probe. I think that in general, deconvolution or a laser scanning system in which you can open the pinhole and sacrifice some confocality are the best. Spinning disk is good, but we just got one so I don't have as much experience yet. Others have indicated it is an improvement and my initial tests indicate we can image faster. Our Leica confocal is probably the worst in our stable because you cannot open the pinhole and decrease confocality, however we use it regularly for xyt imaging by just not collecting images as often. Bottom line- no magic anywhere, only tradeoffs. Dave On Dec 12, 2008, at 8:37 AM, Philip Oshel wrote: Confocalers, Anyone here working on Dictyostelium? If yes, do you have problems with heat from the illumination source killing the critters? The cells are really sensitive to heat and light, so I'd like to hear some actual experiences with different systems looking at live Dictys, and how happy they were after you were done looking at them. A regular fluorescence 'scope can easily kill them. "Illumination source", as I'm wondering about both spinning-disc systems that use Hg or Xe lamps and scanned-laser systems, point scanners or line scanners. We're looking to get a new confocal, and what kind of system (disc, point scanner, line scanner, millions of nanognomes with LED flashlights, whatever) is an issue for the group here. I'm already dealing with vendors, so no vendor contacts off-list, please. Technical information appropriate for the list is appreciated. Thanks. Phil -- Philip Oshel Microscopy Facility Supervisor Biology Department 024C Brooks Hall Central Michigan University Mt. Pleasant, MI 48859 (989) 774-3576 Dr. David Knecht Department of Molecular and Cell Biology Co-head Flow Cytometry and Confocal Microscopy Facility U-3125 91 N. Eagleville Rd. University of Connecticut Storrs, CT 06269 860-486-2200 860-486-4331 (fax) Dr. David Knecht Department of Molecular and Cell Biology Co-head Flow Cytometry and Confocal Microscopy Facility U-3125 91 N. Eagleville Rd. University of Connecticut Storrs, CT 06269 860-486-2200 860-486-4331 (fax) -- Philip Oshel Microscopy Facility Supervisor Biology Department 024C Brooks Hall Central Michigan University Mt. Pleasant, MI 48859 (989) 774-3576 |
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Knecht, David |
Re: Dictyostelium confocal and heat
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Leica SP2 laser scanning system. Our new spinning disk is an Andor system. Dave
On Dec 12, 2008, at 12:16 PM, Philip Oshel wrote:
Dr. David Knecht Department of Molecular and Cell Biology Co-head Flow Cytometry and Confocal Microscopy Facility U-3125 91 N. Eagleville Rd. University of Connecticut Storrs, CT 06269 860-486-2200 860-486-4331 (fax) |
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George Peeters-2 |
Re: Dictyostelium confocal and heat, from a Vendor
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In reply to this post by Oshel, Philip Eugene
Dr. John Heuser and Dr. Margret Clark successfully imaged Dicty 6 years ago by using a more sensitive ICCD camera with the Yokogawa CSU-10b. Cameras, spinning disks, and software have improved since then so I do not feel this as a difficult problem to solve. An oxygen membrane overlying the critters was a big help per Dr. Heuser. Perhaps he might comment
I think the old pictures are on our web site. George George A. Peeters MD, MS President, Solamere Technology Group Inc 1427 Perry Ave Salt Lake City, UT 84103 801 322-2645 office 801 322-2645 fax 801 232-6911 cell On Dec 12, 2008, at 6:37 AM, Philip Oshel wrote:
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Rietdorf, Jens |
Re: Dictyostelium confocal and heat
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In reply to this post by Oshel, Philip Eugene
Dear Phil,
Ralph Graeph from Univ of Potsdam has compared Yokogawa spinning disk and single beam confocal microscopy with respect to live imaging capabilities for Dictyostelium. In these experiments, the laser intensity was reduced to allow gfp labeled centrosomes to be imaged over extended periods of time and the contrast achieved with both systems was compared (Adv Biochem Eng Biotechnol. 2005;95:57-75.) the CSU22 based system was outcompeting the single beam confocal (a Zeiss LSM 510). Though it was clearly possible to image live Dicty over extended periods of time even with both, a single beam scanner and a multi beam scanner already with the technology available in 2005, recent single beam scanners have improved in sensitivity, and EMCCD cameras have further improved the capabilities of spinning disc based systems. I can ask Ralph for further details of his experiments if required. Best, jens -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Philip Oshel Sent: Friday, December 12, 2008 2:37 PM To: [hidden email] Subject: Dictyostelium confocal and heat Confocalers, Anyone here working on Dictyostelium? If yes, do you have problems with heat from the illumination source killing the critters? The cells are really sensitive to heat and light, so I'd like to hear some actual experiences with different systems looking at live Dictys, and how happy they were after you were done looking at them. A regular fluorescence 'scope can easily kill them. "Illumination source", as I'm wondering about both spinning-disc systems that use Hg or Xe lamps and scanned-laser systems, point scanners or line scanners. We're looking to get a new confocal, and what kind of system (disc, point scanner, line scanner, millions of nanognomes with LED flashlights, whatever) is an issue for the group here. I'm already dealing with vendors, so no vendor contacts off-list, please. Technical information appropriate for the list is appreciated. Thanks. Phil -- Philip Oshel Microscopy Facility Supervisor Biology Department 024C Brooks Hall Central Michigan University Mt. Pleasant, MI 48859 (989) 774-3576 |
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