z stack fuzziness
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Hello,
Beginner's question .... I am starting to look at individual bacteria in a biofilm. The bacteria are around 2 microns long, by 1 micron wide. I am using a x60 (oil immersion) lens with N.A. 1.49 and I'm finding the image is clear near the coverslip but gets much fuzzier a few microns deeper. We also have a x40 oil immersion lens with NA 1.3, and a x40 non oil with NA 0.6. Two suggestions I have been given to improve things are 1) adjust the correction collar so that the best focus is above the coverslip (as though I had a thicker coverslip) 2) use a x40 lens which has better depth of focus and working distance. I wasn't sure about the second, I thought that with confocal I am looking at a plane which is in focus anyway, so better depth of focus wouldn't help, and the working distance is well above the depth I want to go to so that should be OK as well. The WD on the lenses are 0.2 and 0.12. If I could get a reasonably clear image 20-30 microns in I'd be pleased. My questions are, am I misunderstanding things, and is there anything else I could try? And do lower magnification lenses have advantages apart from wider field of view? Many thanks, Sarah |
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Hi Sarah -
If your biofilms are in water (flowcell or microtiter plate) then you need to use a water immersion lens to get that deep without aberration. The big questions here are 1) what exactly is the preparation you wish to examine and 2) what exactly do you want to with the data you collect. Both will factor into a decision on the axial resolution. You can contact me off-list for an in-depth discussion. Rob Palmer >Hello, > >Beginner's question .... > >I am starting to look at individual bacteria in a biofilm. The >bacteria are around 2 microns long, by 1 micron wide. > >I am using a x60 (oil immersion) lens with N.A. 1.49 and I'm finding >the image is clear near the coverslip but gets much fuzzier a few >microns deeper. > >We also have a x40 oil immersion lens with NA 1.3, and a x40 non oil >with NA 0.6. > >Two suggestions I have been given to improve things are > >1) adjust the correction collar so that the best focus is above the >coverslip (as though I had a thicker coverslip) >2) use a x40 lens which has better depth of focus and working distance. > >I wasn't sure about the second, I thought that with confocal I am >looking at a plane which is in focus anyway, so better depth of >focus wouldn't help, and the working distance is well above the >depth I want to go to so that should be OK as well. The WD on the >lenses are 0.2 and 0.12. If I could get a reasonably clear image >20-30 microns in I'd be pleased. > >My questions are, am I misunderstanding things, and is there >anything else I could try? And do lower magnification lenses have >advantages apart from wider field of view? > >Many thanks, > >Sarah > -- Robert J. Palmer Jr., Ph.D. Natl Inst Dental Craniofacial Res - Natl Insts Health Oral Infection and Immunity Branch Bldg 30, Room 310 30 Convent Drive Bethesda MD 20892 ph 301-594-0025 fax 301-402-0396 |
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In reply to this post by Sarah Chacko
Hello everybody,
when investigating various mouse samples with 2-photon excitation, I sometimes see very bright "artifact"-signals from what is probably dirt or skin pigments. These signals can be actually quite annoying, since they are often much brighter than fluorochromes and thus can e.g. easily trigger the safety shut off of sensitive PMTs. I assume it is some effect caused by strong absorption of these structures. I don't think it is fluorescence. It can be caused with very different wavelengths, it is present in pretty much all channels from blue to far red, and it feels too bright for fluorescence. It is also different from hair-autofluorescence. I am curious about the nature of this effect. Anybody any ideas? Steffen -- --------------------------------------------------------------------------------------------------- Steffen Dietzel, PD Dr. rer. nat Ludwig-Maximilians-Universität München Walter-Brendel-Zentrum für experimentelle Medizin (WBex) Marchioninistr. 15, D-81377 München |
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OK, this was something I've always been meaning to investigate ....
Here's the experiment: take a coverslip, smoke it very lightly by holding it above a match flame briefly.
Look at it in brightfield, and you will see, as expected, tiny carbon particles covering the field.
Image it in 2-photon and you will see what I can only describe as a firework display. Go back to BF and
the scanned area will be completely clean, no carbon remaining.
Un-modelock the laser, keeping the power the same, and the effect disappears, so it is a two-photon
process.
Anyone have an explanation?
Steffen, this may well be what you are seeing.
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 From: Confocal Microscopy List on behalf of Steffen Dietzel Sent: Fri 19/02/2010 3:43 AM To: [hidden email] Subject: bright signals from "dirt" Hello everybody, |
 
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Martin Wessendorf-2 |
Re: bright signals from "dirt"
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Hey, Guy--
Guy Cox wrote: > Here's the experiment: take a coverslip, smoke it very lightly by > holding it above a match flame briefly. And then mount it to a slide? Or (as I assume) leave it dry and exposed to air? > Look at it in brightfield, and you will see, as expected, tiny carbon > particles covering the field. > > Image it in 2-photon and you will see what I can only describe as a > firework display. Go back to BF and > the scanned area will be completely clean, no carbon remaining. ...So it sounds as if the carbon has oxidized. > Un-modelock the laser, keeping the power the same, and the effect > disappears, so it /is/ a two-photon process. > > Anyone have an explanation? Does anyone know the absorbance spectrum of carbon? One explanation would be that it absorbs much more strongly in the visible than in the near IR but I have no idea of whether that's true. Another explanation is pure speculation: that there's something more than pure carbon on the coverslip--some complex organic that absorbs strongly in the visible range. Interesting phenomenon--I've only CREATED carbon on my specimens, not removed it! Martin -- Martin Wessendorf, PhD (612) 626 0145 (office) Associate Professor (612) 624 2991 (lab) Dept Neuroscience (612) 624 8118 (FAX) Univ Minnesota e-mail: martinw(at)umn.edu |
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Steffen Dietzel |
Re: bright signals from "dirt"
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In reply to this post by Guy Cox-2
At 09:47 19.02.2010, you wrote:
OK, this was something I've always been meaning to investigate .... Yup, that sounds pretty much like it. I am not sure though that this is (only) a two-photon effect. The only other signal that bright I have experienced was from metall particles which are known to produce plasmon-induced luminescence. I don't claim I fully understood the theory behind that but apparently this is a "multi-photon" induced process. However, I didn't find anything about plasmon-like effects in non-metalls. Steffen
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In reply to this post by Guy Cox-2
I would guess that the smoke particles have a very high 2-P
crossection and are incinerated, a process that evidently emils a lot
of light.
Cheers,
JP
**********************************************
James B.
Pawley,
Room 223, Zoology Research
Building,
1117 Johnson Ave., Madison, WI, 53706 3D Microscopy of Living Cells Course, June 12-24, 2010, UBC, Vancouver Canada Info: http://www.3dcourse.ubc.ca/ OK, this was something I've always been meaning to investigate .... Here's the experiment: take a coverslip, smoke it very lightly by holding it above a match flame briefly. Look at it in brightfield, and you will see, as expected, tiny carbon particles covering the field. Image it in 2-photon and you will see what I can only describe as a firework display. Go back to BF and the scanned area will be completely clean, no carbon remaining. Un-modelock the laser, keeping the power the same, and the effect disappears, so it is a two-photon process. Anyone have an explanation? Steffen, this may well be what you are seeing. Guy Optical Imaging Techniques in Cell Biology http://www.guycox.com/optical.htm From: Confocal Microscopy List on behalf of Steffen Dietzel Hello everybody, --
**********************************************
Prof. James B. Pawley, Room 223, Zoology Research Building, 1117 Johnson Ave., Madison, WI, 53706 3D Microscopy of Living Cells Course, June 12-24, 2010, UBC, Vancouver Canada Info: http://www.3dcourse.ubc.ca/ |
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In reply to this post by Guy Cox-2
Hi Guy
I've seen the same thing with strong absorbers. Since the emission seems broadband, I'd guess that some sort of plasma forms? In a mountant bubbles can also form when this takes place. Cheers Mark Cox wrote: > OK, this was something I've always been meaning to investigate .... > > Here's the experiment: take a coverslip, smoke it very lightly by > holding it above a match flame briefly. > > Look at it in brightfield, and you will see, as expected, tiny carbon > particles covering the field. > > Image it in 2-photon and you will see what I can only describe as a > firework display. Go back to BF and > the scanned area will be completely clean, no carbon remaining. > > Un-modelock the laser, keeping the power the same, and the effect > disappears, so it /is/ a two-photon > process. > > Anyone have an explanation? > > Steffen, this may well be what you are seeing. > > > 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 > <https://www.mcws.usyd.edu.au/exchweb/bin/redir.asp?URL=https://www.mcws.usyd.edu.au/exchweb/bin/redir.asp?URL=http://www.guycox.net> > > ------------------------------------------------------------------------ > *From:* Confocal Microscopy List on behalf of Steffen Dietzel > *Sent:* Fri 19/02/2010 3:43 AM > *To:* [hidden email] > *Subject:* bright signals from "dirt" > > Hello everybody, > > when investigating various mouse samples with > 2-photon excitation, I sometimes see very bright > "artifact"-signals from what is probably dirt or > skin pigments. These signals can be actually > quite annoying, since they are often much > brighter than fluorochromes and thus can e.g. > easily trigger the safety shut off of sensitive PMTs. > > I assume it is some effect caused by strong > absorption of these structures. I don't think it > is fluorescence. It can be caused with very > different wavelengths, it is present in pretty > much all channels from blue to far red, and it > feels too bright for fluorescence. It is also > different from hair-autofluorescence. I am > curious about the nature of this effect. Anybody any ideas? > > Steffen > > -- > --------------------------------------------------------------------------------------------------- > Steffen Dietzel, PD Dr. rer. nat > Ludwig-Maximilians-Universität München > Walter-Brendel-Zentrum für experimentelle Medizin (WBex) > Marchioninistr. 15, D-81377 München > |
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Axel Kurt Preuss |
Re: bright signals from "dirt"
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In reply to this post by James Pawley
I suspect Jahn Teller distortion (plasmonic distortion) plays a role in the MP induced glare of ...what I should call nano carbon particles (generated by combustion and nicely spraying or depositing themselves onto a coverslip as Guy suggested, or the particles found by Steffen in mouse samples)
Hi Steffen viele Gruesse nach Muenchen! Dear all who responded to my LSM 710 question. We just finished the demo and I ll respond this coming week. These were all extremely helpful comments, and maybe I can add ________________________________________ From: Confocal Microscopy List [[hidden email]] On Behalf Of James Pawley [[hidden email]] Sent: 19 February 2010 23:57 To: [hidden email] Subject: Re: bright signals from "dirt" I would guess that the smoke particles have a very high 2-P crossection and are incinerated, a process that evidently emils a lot of light. Cheers, JP ********************************************** James B. Pawley, Ph. 608-263-3147 Room 223, Zoology Research Building, FAX 608-265-5315 1117 Johnson Ave., Madison, WI, 53706 [hidden email] 3D Microscopy of Living Cells Course, June 12-24, 2010, UBC, Vancouver Canada Info: http://www.3dcourse.ubc.ca/ Applications due by March 15, 2010 "If it ain't diffraction, it must be statistics." Anon. OK, this was something I've always been meaning to investigate .... Here's the experiment: take a coverslip, smoke it very lightly by holding it above a match flame briefly. Look at it in brightfield, and you will see, as expected, tiny carbon particles covering the field. Image it in 2-photon and you will see what I can only describe as a firework display. Go back to BF and the scanned area will be completely clean, no carbon remaining. Un-modelock the laser, keeping the power the same, and the effect disappears, so it is a two-photon process. Anyone have an explanation? Steffen, this may well be what you are seeing. 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<https://www.mcws.usyd.edu.au/exchweb/bin/redir.asp?URL=https://www.mcws.usyd.edu.au/exchweb/bin/redir.asp?URL=http://www.guycox.net> ________________________________ From: Confocal Microscopy List on behalf of Steffen Dietzel Sent: Fri 19/02/2010 3:43 AM To: [hidden email] Subject: bright signals from "dirt" Hello everybody, when investigating various mouse samples with 2-photon excitation, I sometimes see very bright "artifact"-signals from what is probably dirt or skin pigments. These signals can be actually quite annoying, since they are often much brighter than fluorochromes and thus can e.g. easily trigger the safety shut off of sensitive PMTs. I assume it is some effect caused by strong absorption of these structures. I don't think it is fluorescence. It can be caused with very different wavelengths, it is present in pretty much all channels from blue to far red, and it feels too bright for fluorescence. It is also different from hair-autofluorescence. I am curious about the nature of this effect. Anybody any ideas? Steffen -- --------------------------------------------------------------------------------------------------- Steffen Dietzel, PD Dr. rer. nat Ludwig-Maximilians-Universität München Walter-Brendel-Zentrum für experimentelle Medizin (WBex) Marchioninistr. 15, D-81377 München -- ********************************************** Prof. James B. Pawley, Ph. 608-263-3147 Room 223, Zoology Research Building, FAX 608-265-5315 1117 Johnson Ave., Madison, WI, 53706 [hidden email] 3D Microscopy of Living Cells Course, June 12-24, 2010, UBC, Vancouver Canada Info: http://www.3dcourse.ubc.ca/ Applications due by March 15, 2010 "If it ain't diffraction, it must be statistics." Anon. Note: This message may contain confidential information. If this Email/Fax has been sent to you by mistake, please notify the sender and delete it immediately. Thank you. |
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Lingqing Zhang |
Re: bright signals from "dirt"
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In reply to this post by Steffen Dietzel
Hi Steffen,
I saw these effect often when doing two-photon imaging when encountering carbon-rich molecules or structures. It was sure quite annoying. Because it's so bright, that also gives us a way to separate it from real fluorescence signals. Those "artifact" seems always saturated. If we adjust the signal level just under saturation, then we can filter out those "artifact" by post processing. I think those "artifact" are from emissions of two-photon induced carbon plasmas. In the Guy's observation, the carbon deposition disappeared after 2-P excitation in a glass slide. It was because carbon was ionized by 2-P laser effect. Those laser produced plasma fly away from the slide surface. So the slide surface became clear. Lingqing ************************************* Lingqing Zhang, PhD Nonlinear Optical Imaging Lab LM/EM Imaging Core Facility West Virginia University 1 Medical Center Drive Morgantown, WV 26506 Phone: 304-293-5253 Email: [hidden email] On Thu, Feb 18, 2010 at 11:43 AM, Steffen Dietzel <[hidden email]> wrote: Hello everybody, |
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Steffen Dietzel |
Re: bright signals from "dirt"
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Thank you, Lingqing and everybody else for their input on this.
Maybe we have a mixture of effects here. Sometimes, I do induce large gas bubbles, easily seen in ordinary transmission light, so that's probably from plasma production. (Maybe incineration?). But sometimes, especially with rather low light intensity (5-10 mW, measured behind the objective) the signals seem rather stable - or at least not vanishing over time. So there may be something else at work. I did some literature search on the Jahn-Teller-effect that Axel suggested but my physics isn't good enough to conclude from what I've read wheather or not this might be responsible. It appears this 'firework display", as Guy aptly called it, is a well-known (by people working with multi-photon) but not well-explained phenomenon. Thanks again, any additional thoughts are appreciated. Steffen At 17:34 22.02.2010, you wrote: Hi Steffen, |
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Evelyn Ralston |
Re: bright signals from "dirt"
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In reply to this post by Steffen Dietzel
Sorry, I was off the listserv for a while. I saw your question but I
don't know if you got an answer. Mouse hair contains pigments which reflect light very brightly. Evelyn Ralston, Ph.D. Head, Light Imaging Section, Office of Science & Technology, NIAMS National Institutes of Health, Bldg 50, Rm 1535 Bethesda, MD 20892-8023 tel 301-496-6164; FAX 301-402-3417 On Feb 18, 2010, at 11:43 AM, Steffen Dietzel wrote: > Hello everybody, > > when investigating various mouse samples with > 2-photon excitation, I sometimes see very bright > "artifact"-signals from what is probably dirt or > skin pigments. These signals can be actually > quite annoying, since they are often much > brighter than fluorochromes and thus can e.g. > easily trigger the safety shut off of sensitive PMTs. > > I assume it is some effect caused by strong > absorption of these structures. I don't think it > is fluorescence. It can be caused with very > different wavelengths, it is present in pretty > much all channels from blue to far red, and it > feels too bright for fluorescence. It is also > different from hair-autofluorescence. I am > curious about the nature of this effect. Anybody any ideas? > > Steffen > > -- > --------------------------------------------------------------------------------------------------- > Steffen Dietzel, PD Dr. rer. nat > Ludwig-Maximilians-Universität München > Walter-Brendel-Zentrum für experimentelle Medizin (WBex) > Marchioninistr. 15, D-81377 München |
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