fixed cells plasma membrane labeling
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lechristophe |
fixed cells plasma membrane labeling
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Dear all,
I would like to stain the plasma membrane of fixed adherent cells (COS, 293, hippocampal neurons in culture). The best would be a good primary antibody that labels the plasma membrane after fixation, either with paraformaldehyde or methanol, as we could just add the primary/corresponding secondary antibodies to the immunocytochemistry procedure, without adding any steps. I don't like lipid dyes such as liquid DiL because I never got good results with them for this kind of work, even with fixable versions. Thanks for your suggestions, Christophe Leterrier |
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xavier Sanjuan |
Re: fixed cells plasma membrane labeling
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Hi
Christophe,
years
ago one colleague used TRITC-concanavalin to stain the plasma membrane.
Concanavalin is a lectin that specifically binds alpha-mannose residues on
membrane gycoproteins. The only thing is that it probably will not work using
methanol as a fixative since to stain plasma membrane with concanavalin you
must avoid permeabilization.
Hope
this helps.
Regards,
Xavi.
___________________________________ Xavier
Sanjuan
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Jerry Sedgewick-2 |
Re: LSM multiphoton confocal not picking up reflection
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal I know this had been discussed in the past, but I didn't follow the thread. I seem to be getting more engineer types wanting to use our confocal. Because they often have ancillary equipment they need to set up to replace the stage, I am cornered into providing a spot for them on a custom-built multiphoton. They are forever wanting to see surfaces or reflection from particles: if my memory serves me well, reflection cannot be had unless the illumination wavelength matches the reflected wavelength. But I'm starting to wonder whether another phenomenon I am not aware of creeps in, for, on this custom multiphoton, I have replaced a cube with a 50/50 beamsplitter so that the reflected wavelength is the same as the illumination wavelength, and removed filters from in front of the PMT detector (sensitive out to 800nm or so), set the pulsed laser at 750nm, and even placed a 1/2 waveplate and glan polarizer in the laser path with another polarizer in front of the PMT to play with these (I believe the polarizer in front of the PMT, however, is a circular polarizer as the reflected light never quite extinguished with cross-polarization). Inexplicably, I got no reflection off surfaces. The custom multiphoton has two paths, one for the external detectors (where the 50/50 beamsplitter sent the reflected light) and internal PMTs in a Fluoview 300 confocal unit. Strangely, if I sent the reflected light to the internal PMTs, I was able to get a reflection, if I was willing to live with a bright spot in the middle of the field. This wasn't supposed to happen because the reflected light was (supposedly) blocked by bandpass filters, but, after placing another 650sp filter in the reflected light path, the reflection went away. (Clearly, a portion of the laser light bled through). In any event, it is confounding to get the reflection in one instance, and to not get it in the other even when optimized. Any ideas? Jerry Sedgewick University of Minnesota --- http://USFamily.Net/dialup.html - $8.25/mo! -- http://www.usfamily.net/dsl.html - $19.99/mo! --- |
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hello: The best troubleshooting I can recommend is to put a mirror at the focus of the objective. Track the reflected beam using an IR card or IR viewer. Our system has a dichroic which is intended to block the IR beam when I use the Mira and the internal PMTs. Extra filters are used in the non-descanned path. Check your default settings and your external detector path. Maybe something is not transmitting or not reflecting 750nm. Are you sure that you external PMT can detect 750nm? By the way, many bandpass filters will transmit IR light (usually 800 - 100nm). Good luck! Sophie ____________________________________________________ Sophie M. K. Brunet, Ph. D. Research Officer Optical Spectroscopy, Laser Systems and Applications Chemistry 112 sessional lecturer [hidden email] 306-966-1719 (office) 306-966-1702 (fax) ____________________________________________________ Saskatchewan Structural Sciences Centre University of Saskatchewan Thorvaldson Bldg. 110 Science Place Saskatoon, Sk S7N 5C9 ____________________________________________________ Quoting Jerry Sedgewick <[hidden email]>: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > I know this had been discussed in the past, but I didn't follow the thread. > I > seem to be getting more engineer types wanting to use our confocal. Because > they often have ancillary equipment they need to set up to replace the > stage, I > am cornered into providing a spot for them on a custom-built > multiphoton. They > are forever wanting to see surfaces or reflection from particles: if my > memory > serves me well, reflection cannot be had unless the illumination wavelength > matches the reflected wavelength. But I'm starting to wonder whether another > phenomenon I am not aware of creeps in, for, on this custom > multiphoton, I have > replaced a cube with a 50/50 beamsplitter so that the reflected wavelength is > the same as the illumination wavelength, and removed filters from in front of > the PMT detector (sensitive out to 800nm or so), set the pulsed laser > at 750nm, > and even placed a 1/2 waveplate and glan polarizer in the laser path with > another polarizer in front of the PMT to play with these (I believe the > polarizer in front of the PMT, however, is a circular polarizer as the > reflected light never quite extinguished with cross-polarization). > Inexplicably, I got no reflection off surfaces. > > The custom multiphoton has two paths, one for the external detectors > (where the > 50/50 beamsplitter sent the reflected light) and internal PMTs in a Fluoview > 300 confocal unit. Strangely, if I sent the reflected light to the internal > PMTs, I was able to get a reflection, if I was willing to live with a bright > spot in the middle of the field. This wasn't supposed to happen because the > reflected light was (supposedly) blocked by bandpass filters, but, after > placing another 650sp filter in the reflected light path, the reflection went > away. (Clearly, a portion of the laser light bled through). > > In any event, it is confounding to get the reflection in one instance, and to > not get it in the other even when optimized. Any ideas? > > Jerry Sedgewick > University of Minnesota > > > > > > --- http://USFamily.Net/dialup.html - $8.25/mo! -- > http://www.usfamily.net/dsl.html - $19.99/mo! --- > |
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Mayandi Sivaguru |
Re: LSM multiphoton confocal not picking up reflection
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Search the CONFOCAL archive at
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Jerry, it is good to check whether you have any IR blocker on the path of
external detectors as Sophie suggested. In addition, you can check what
kind of beam splitter you are using i.e to deflect the light to the
external pmts, usually in zeiss, you will be using a dichroic in the
filter turret assembled in a cube, which sends all the visible wavelength
from the emission but if I remember right, since you use the KP650 (may
be identical to what you mention 650sp), all the IR light is blocked and
the NDD filter turrets will have band pass filters, but also usually
coated with IR blocking coatings. To my knowledge the internal band pass
filters of an NLO system should have also coated with this, but since you
have custom made this system, your internal band pass filters might not
be IR coated and hence you get the reflection but the external filters
might be so it might block the signal.
Let us know what you have found. Thanks Shiv At 12:06 PM 2/13/2008, you wrote: Search the CONFOCAL archive at Microscopy Facility Manager 8, Institute for Genomic Biology University of Illinois at Urbana-Champaign 1206 West Gregory Dr. Urbana, IL 61801 USA Office: 217.333.1214 Fax: 217.244.2496 [hidden email] http://core.igb.uiuc.edu |
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Tim O'Brien Sr. |
Re: fixed cells plasma membrane labeling
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In reply to this post by lechristophe
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal If you don't want to stain the lipids, then you have to know a bit more about the specific cells you are trying to stain. If they make tight junctions, a ZO1 antibody might work, or a pancadherin antibody. Just a phalloidin stain for F-actin is fast and easy, and often stains the perimeter of confluent cells. You could also try antibodies to the neuronal forms of spectrin, which might light up the edges of the plasma mambrane. Hope this helps, and please post what ends up working best! Tim O'Brien UNC Chapel Hill On Feb 13, 2008, at 5:49 AM, Christophe Leterrier wrote: > Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi- > bin/wa?S1=confocal Dear all, > > I would like to stain the plasma membrane of fixed adherent cells > (COS, 293, hippocampal neurons in culture). The best would be a > good primary antibody that labels the plasma membrane after > fixation, either with paraformaldehyde or methanol, as we could > just add the primary/corresponding secondary antibodies to the > immunocytochemistry procedure, without adding any steps. I don't > like lipid dyes such as liquid DiL because I never got good results > with them for this kind of work, even with fixable versions. > > Thanks for your suggestions, > > Christophe Leterrier > > |
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In reply to this post by S. Brunet
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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hello again: I forgot to ask: can you check your external path with a visible light laser as well? This might tell you if it is an optical filter issue only. Good luck! Sophie ____________________________________________________ Sophie M. K. Brunet, Ph. D. Research Officer Optical Spectroscopy, Laser Systems and Applications Chemistry 112 sessional lecturer [hidden email] 306-966-1719 (office) 306-966-1702 (fax) ____________________________________________________ Saskatchewan Structural Sciences Centre University of Saskatchewan Thorvaldson Bldg. 110 Science Place Saskatoon, Sk S7N 5C9 ____________________________________________________ Quoting "S. Brunet" <[hidden email]>: > Search the CONFOCAL archive at > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > Hello: > > The best troubleshooting I can recommend is to put a mirror at the focus of > the > objective. Track the reflected beam using an IR card or IR viewer. Our > system > has a dichroic which is intended to block the IR beam when I use the Mira and > the internal PMTs. Extra filters are used in the non-descanned path. Check > your default settings and your external detector path. Maybe something is > not > transmitting or not reflecting 750nm. Are you sure that you external PMT can > detect 750nm? > > By the way, many bandpass filters will transmit IR light (usually 800 - > 100nm). > > Good luck! > Sophie > ____________________________________________________ > Sophie M. K. Brunet, Ph. D. > Research Officer > Optical Spectroscopy, Laser Systems and Applications > Chemistry 112 sessional lecturer > [hidden email] > 306-966-1719 (office) 306-966-1702 (fax) > ____________________________________________________ > Saskatchewan Structural Sciences Centre > University of Saskatchewan > Thorvaldson Bldg. > 110 Science Place > Saskatoon, Sk S7N 5C9 > ____________________________________________________ > > > Quoting Jerry Sedgewick <[hidden email]>: > > > Search the CONFOCAL archive at > > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal > > > > I know this had been discussed in the past, but I didn't follow the thread. > > I > > seem to be getting more engineer types wanting to use our confocal. > Because > > they often have ancillary equipment they need to set up to replace the > > stage, I > > am cornered into providing a spot for them on a custom-built > > multiphoton. They > > are forever wanting to see surfaces or reflection from particles: if my > > memory > > serves me well, reflection cannot be had unless the illumination wavelength > > matches the reflected wavelength. But I'm starting to wonder whether > another > > phenomenon I am not aware of creeps in, for, on this custom > > multiphoton, I have > > replaced a cube with a 50/50 beamsplitter so that the reflected wavelength > is > > the same as the illumination wavelength, and removed filters from in front > of > > the PMT detector (sensitive out to 800nm or so), set the pulsed laser > > at 750nm, > > and even placed a 1/2 waveplate and glan polarizer in the laser path with > > another polarizer in front of the PMT to play with these (I believe the > > polarizer in front of the PMT, however, is a circular polarizer as the > > reflected light never quite extinguished with cross-polarization). > > Inexplicably, I got no reflection off surfaces. > > > > The custom multiphoton has two paths, one for the external detectors > > (where the > > 50/50 beamsplitter sent the reflected light) and internal PMTs in a > Fluoview > > 300 confocal unit. Strangely, if I sent the reflected light to the > internal > > PMTs, I was able to get a reflection, if I was willing to live with a > bright > > spot in the middle of the field. This wasn't supposed to happen because > the > > reflected light was (supposedly) blocked by bandpass filters, but, after > > placing another 650sp filter in the reflected light path, the reflection > went > > away. (Clearly, a portion of the laser light bled through). > > > > In any event, it is confounding to get the reflection in one instance, and > to > > not get it in the other even when optimized. Any ideas? > > > > Jerry Sedgewick > > University of Minnesota > > > > > > > > > > > > --- http://USFamily.Net/dialup.html - $8.25/mo! -- > > http://www.usfamily.net/dsl.html - $19.99/mo! --- > > > |
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In reply to this post by Jerry Sedgewick-2
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal My guess is that your 50/50 beamsplitter is not splitting at the IR wavelength - as Sophie said, you should be able to check it with your IR viewer. But I must add that what you're trying to do sounds like an awfully bad idea unless you first un-modelock the beam so that you have CW illumination. Otherwise you risk doing very strange things to the specimens (put some carbon black on a slide by holding it above a match and then see what happens if you look at it in 2p mode). 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 -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Jerry Sedgewick Sent: Thursday, 14 February 2008 3:28 AM To: [hidden email] Subject: Re: LSM multiphoton confocal not picking up reflection Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal I know this had been discussed in the past, but I didn't follow the thread. I seem to be getting more engineer types wanting to use our confocal. Because they often have ancillary equipment they need to set up to replace the stage, I am cornered into providing a spot for them on a custom-built multiphoton. They are forever wanting to see surfaces or reflection from particles: if my memory serves me well, reflection cannot be had unless the illumination wavelength matches the reflected wavelength. But I'm starting to wonder whether another phenomenon I am not aware of creeps in, for, on this custom multiphoton, I have replaced a cube with a 50/50 beamsplitter so that the reflected wavelength is the same as the illumination wavelength, and removed filters from in front of the PMT detector (sensitive out to 800nm or so), set the pulsed laser at 750nm, and even placed a 1/2 waveplate and glan polarizer in the laser path with another polarizer in front of the PMT to play with these (I believe the polarizer in front of the PMT, however, is a circular polarizer as the reflected light never quite extinguished with cross-polarization). Inexplicably, I got no reflection off surfaces. The custom multiphoton has two paths, one for the external detectors (where the 50/50 beamsplitter sent the reflected light) and internal PMTs in a Fluoview 300 confocal unit. Strangely, if I sent the reflected light to the internal PMTs, I was able to get a reflection, if I was willing to live with a bright spot in the middle of the field. This wasn't supposed to happen because the reflected light was (supposedly) blocked by bandpass filters, but, after placing another 650sp filter in the reflected light path, the reflection went away. (Clearly, a portion of the laser light bled through). In any event, it is confounding to get the reflection in one instance, and to not get it in the other even when optimized. Any ideas? Jerry Sedgewick University of Minnesota --- http://USFamily.Net/dialup.html - $8.25/mo! -- http://www.usfamily.net/dsl.html - $19.99/mo! --- No virus found in this incoming message. Checked by AVG Free Edition. Version: 7.5.516 / Virus Database: 269.20.0/1268 - Release Date: 9/02/2008 11:54 AM No virus found in this outgoing message. Checked by AVG Free Edition. Version: 7.5.516 / Virus Database: 269.20.0/1268 - Release Date: 9/02/2008 11:54 AM |
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Julio Vazquez |
Re: LSM multiphoton confocal not picking up reflection
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In reply to this post by Jerry Sedgewick-2
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Hi Jerry, I don't know what your problem might be, but here are some numbers, using a tissue section (with a histochemical stain, but probably fluorescent too) as a sample, and a dry 20x/0.75 PlanApo: with Chameleon at 780 nm, 0.5% power at AOTF, using an 80/20 mirror and a LP 505 for detection, I get a reflected light image at a PMT gain of 275 with pinhole set at max, and PMT gain of 475 with pinhole set at 1 Airy unit. If I use a 500-550 for detection, under same conditions, I get no signal, so I am picking up reflected light, and not possible fluorescence of the sample (which I probably wouldn't pick at 0.5% laser power, but maybe at 5% and gain at 900 if I were imaging a fluorescent sample in two-photon mode). Since I get an image with pinhole set to 1 Airy unit, it further confirms I am not picking up two-photon fluorescence. The image with pinhole wide open is more fuzzy than the one with pinhole at 1 Airy, because the former is not confocal. On a non-descanned detector, you should get something similar to what you would get with pinhole wide open. Just as a side note, I must say the reflected light image with the 2P laser appears quite normal to me, and I wouldn't necessarily worry about damaging the sample at 0.5% laser intensity. I bet I could get an image at 0.1 %, but I didn't try. That would be about 0.1 mW of power at the sample (0.1 % of 1 Watt, plus about 10% overall efficiency of the light path for the IR) Your problem is strange, because on our microscope, with the 2P laser, unless we use the proper dichroics and bandpass filters for detection, we will generally get reflections, since the laser intensity is many orders of magnitude greater than the fluorescence we are trying to detect... The same is true in confocal mode, where one should avoid including the laser wavelength in the detection channel (even with the proper dichoic), since the small fraction of reflected light that would pass through is still strong enough to contaminate the image. This is with lasers that deliver about 1-10% the power of the 2P. I can't quite reproduce your set-up with NDD detector, because I don't have the option of removing the bandpass filter in front of the NDD. Since the NDDs are supposed to collect all light scattered by the sample, and since the power levels used in 2P are so high, I wouldn't be surprised they would have a strong IR blocking filter in front of them, but I really don't know. -- Julio Vazquez Fred Hutchinson Cancer Research Center Seattle, WA 98109-1024 == On Feb 13, 2008, at 8:27 AM, Jerry Sedgewick wrote:
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Julio Vazquez |
Re: LSM multiphoton confocal not picking up reflection
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In reply to this post by Jerry Sedgewick-2
Search the CONFOCAL archive at
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Hi Jerry, To follow up on this, I tried to get a reflected light image on the NDDs of our LSM 510. We actually have a 510 longpass filter in one of the positions. The settings were: 2P at 780 nm, 80/20 (or Short Pass 650) on the dichroic position, Long Pass 680 in the objective turret (to reflect <680 nm light to the NDDs), and 510 LP in front of the NDD. I could not get any reflected light image at reasonable laser power levels. I suppose that either the 680 LP in the turret is very very efficient, or the 510 LP includes an NIR filter, or possibly both. In any event, the 780 nm light seems to be blocked very efficiently (which is what you would need to perform NDD 2P fluorescence imaging. As I mentioned, I had no difficulty getting a reflected light image at 780 nm in normal confocal mode. -- Julio Vazquez, Fred Hutchinson Cancer Research Center Seattle, WA 98109-1024 On Feb 13, 2008, at 8:27 AM, Jerry Sedgewick wrote:
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Kilgore, Jason |
Re: fixed cells plasma membrane labeling *VENDOR RESPONSE*
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In reply to this post by lechristophe
Search the CONFOCAL archive at
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** VENDOR RESPONSE **
Dear Christophe,
A few years ago I optimized labeling conditions for
plasma membrane labeling of both live and formaldehyde-fixed cells using wheat
germ agglutinin products. We have many "colors" available from Molecular
Probes (probes.invitrogen.com). A protocol is available if you go to the
webpage for product I34406, the Image-iT LIVE Plasma Membrane and Nuclear
Labeling Kit:
You
can label already-fixed cells as long as you do not permeabilize first (or the
conjugate will also label intracellular targets), but then you can perm after
labeling and continue with other labels.
This
option will be considerably more targeted than general lipophilic dyes like
DiI.
Cheers,
Jason
Molecular Probes / Invitrogen
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Christophe Leterrier Sent: Wednesday, February 13, 2008 2:50 AM To: [hidden email] Subject: fixed cells plasma membrane labeling I would like to stain the plasma membrane of fixed adherent cells (COS, 293, hippocampal neurons in culture). The best would be a good primary antibody that labels the plasma membrane after fixation, either with paraformaldehyde or methanol, as we could just add the primary/corresponding secondary antibodies to the immunocytochemistry procedure, without adding any steps. I don't like lipid dyes such as liquid DiL because I never got good results with them for this kind of work, even with fixable versions. Thanks for your suggestions, Christophe Leterrier |
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Herman Favoreel |
CellCubator climate chamber question
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In reply to this post by lechristophe
Search the CONFOCAL archive at
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Dear all,
My apologies for this non-confocal related question. For live cell imaging experiments, we use a CellCubator climate chamber. When we put the CO2 level to 5%, and then close the CO2 faucet but keep the airstream going, which is supposed to be a closed loop, the CO2 level drops to below 1% within 10 to 15 minutes. That seems very fast to me and appears to point to quite a high CO2 consumption, and (with Al Gore in mind :) ) it makes me reluctant of using 5% CO2 conditions for long term experiments (e.g. several days). I wonder whether this is a normal speed of CO2 loss, could anyone share his or her experience with the CellCubator or other incubators and their CO2 consumption ? Many thanks in advance, Herman Herman Favoreel Department of Virology, Parasitology, and Immunology Faculty of Veterinary Medicine Ghent University Salisburylaan 133 9820 Merelbeke BELGIUM Phone: ++ 32 9 264 73 74 Fax: ++ 32 9 264 74 95 E-mail: [hidden email] website: http://www.vpi.ugent.be |
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Rietdorf, Jens |
Re: [SPAM] CellCubator climate chamber question
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Search the CONFOCAL archive at
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Dear Herman,
when we were constructing an incubator at embl, we
calculated the CO2 loss at 5% to be less than the CO2 produced by the operator
in front of the device. I admit, we didnt have Al in mind, but the fact that the
student inside the tiny little microscope room could fall asleep and never wake
up again, but we measured and thats what we found.
regards, jens
--- Dr.
Jens Rietdorf[hidden email] From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Herman Favoreel Sent: Donnerstag, 21. Februar 2008 16:06 To: [hidden email] Subject: [SPAM] CellCubator climate chamber question My apologies for this non-confocal related question. For live cell imaging
experiments, we use a CellCubator climate chamber. When we put the CO2 level to
5%, and then close the CO2 faucet but keep the airstream going, which is
supposed to be a closed loop, the CO2 level drops to below 1% within 10 to 15
minutes.
That seems very fast to me and appears to point to quite a high CO2
consumption, and (with Al Gore in mind :) ) it makes me reluctant of using 5%
CO2 conditions for long term experiments (e.g. several days). I wonder whether
this is a normal speed of CO2 loss, could anyone share his or her experience
with the CellCubator or other incubators and their CO2 consumption ?
Many thanks in advance,
Herman
Herman Favoreel
Department of Virology, Parasitology, and Immunology
Faculty of Veterinary Medicine
Ghent University
Salisburylaan 133
9820 Merelbeke
BELGIUM
Phone: ++ 32 9 264 73 74
Fax: ++ 32 9 264 74 95
E-mail: [hidden email]
website: http://www.vpi.ugent.be |
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Excess CO2 in the room does not make you fall asleep and never wake up - you start hyperventilating and get a splitting headache. It's oxygen depletion that's dangerous - that is symptomless and fatal. You should be more concerned about the dry nitrogen passing through your TiS laser than the CO2 in your incubator! (And, of course, even more careful about liquid nitrogen). 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 <http://www.guycox.net/> ________________________________ From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Rietdorf, Jens Sent: Monday, 25 February 2008 7:39 AM To: [hidden email] Subject: Re: [SPAM] CellCubator climate chamber question Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear Herman, when we were constructing an incubator at embl, we calculated the CO2 loss at 5% to be less than the CO2 produced by the operator in front of the device. I admit, we didnt have Al in mind, but the fact that the student inside the tiny little microscope room could fall asleep and never wake up again, but we measured and thats what we found. regards, jens --- Dr. Jens Rietdorf <mailto:[hidden email]> Head Microscopy Novartis Research Foundation Friedrich-Miescher-Institute <http://www.fmi.ch/> , wro1066.2.16 Maulbeerstr.66, CH-4058 Basel, Switzerland phone +41(61)69-75172 mobil +41 798284737 Email:rietdorf(at)fmi.ch ________________________________ From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Herman Favoreel Sent: Donnerstag, 21. Februar 2008 16:06 To: [hidden email] Subject: [SPAM] CellCubator climate chamber question Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear all, My apologies for this non-confocal related question. For live cell imaging experiments, we use a CellCubator climate chamber. When we put the CO2 level to 5%, and then close the CO2 faucet but keep the airstream going, which is supposed to be a closed loop, the CO2 level drops to below 1% within 10 to 15 minutes. That seems very fast to me and appears to point to quite a high CO2 consumption, and (with Al Gore in mind :) ) it makes me reluctant of using 5% CO2 conditions for long term experiments (e.g. several days). I wonder whether this is a normal speed of CO2 loss, could anyone share his or her experience with the CellCubator or other incubators and their CO2 consumption ? Many thanks in advance, Herman Herman Favoreel Department of Virology, Parasitology, and Immunology Faculty of Veterinary Medicine Ghent University Salisburylaan 133 9820 Merelbeke BELGIUM Phone: ++ 32 9 264 73 74 Fax: ++ 32 9 264 74 95 E-mail: [hidden email] website: http://www.vpi.ugent.be No virus found in this incoming message. Checked by AVG Free Edition. Version: 7.5.516 / Virus Database: 269.20.9/1290 - Release Date: 20/02/2008 8:45 PM No virus found in this outgoing message. Checked by AVG Free Edition. Version: 7.5.516 / Virus Database: 269.21.0/1296 - Release Date: 24/02/2008 12:19 PM |
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Deanne Veronica Catmull |
Re: [SPAM] CellCubator climate chamber question
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal I do not know much about this incubator system however does it use tubing at any end/s? You might want to check what the tubing material is composed of and their rate of gas exchange. The Cole Parmer catalogues contain a lot of information on this subject, it may be worth a look if you think this could be a problem. It may also be an idea to fit some sort of gas sensor in the room, just from an OH&S point of view to monitor the CO2 levels or monitor other gas levels in the room that may be used. It may save you or somebody else from a potential disaster! Deanne Catmull Research Assistant School of Dental Science University of Melbourne 720 Swanston St Carlton 3010 Fax: 9341-1597 Ph: 9341-1577 -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Guy Cox Sent: Monday, February 25, 2008 10:02 AM To: [hidden email] Subject: Re: [SPAM] CellCubator climate chamber question Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Excess CO2 in the room does not make you fall asleep and never wake up - you start hyperventilating and get a splitting headache. It's oxygen depletion that's dangerous - that is symptomless and fatal. You should be more concerned about the dry nitrogen passing through your TiS laser than the CO2 in your incubator! (And, of course, even more careful about liquid nitrogen). 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 <http://www.guycox.net/> ________________________________ From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Rietdorf, Jens Sent: Monday, 25 February 2008 7:39 AM To: [hidden email] Subject: Re: [SPAM] CellCubator climate chamber question Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear Herman, when we were constructing an incubator at embl, we calculated the CO2 loss at 5% to be less than the CO2 produced by the operator in front of the device. I admit, we didnt have Al in mind, but the fact that the student inside the tiny little microscope room could fall asleep and never wake up again, but we measured and thats what we found. regards, jens --- Dr. Jens Rietdorf <mailto:[hidden email]> Head Microscopy Novartis Research Foundation Friedrich-Miescher-Institute <http://www.fmi.ch/> , wro1066.2.16 Maulbeerstr.66, CH-4058 Basel, Switzerland phone +41(61)69-75172 mobil +41 798284737 Email:rietdorf(at)fmi.ch ________________________________ From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Herman Favoreel Sent: Donnerstag, 21. Februar 2008 16:06 To: [hidden email] Subject: [SPAM] CellCubator climate chamber question Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear all, My apologies for this non-confocal related question. For live cell imaging experiments, we use a CellCubator climate chamber. When we put the CO2 level to 5%, and then close the CO2 faucet but keep the airstream going, which is supposed to be a closed loop, the CO2 level drops to below 1% within 10 to 15 minutes. That seems very fast to me and appears to point to quite a high CO2 consumption, and (with Al Gore in mind :) ) it makes me reluctant of using 5% CO2 conditions for long term experiments (e.g. several days). I wonder whether this is a normal speed of CO2 loss, could anyone share his or her experience with the CellCubator or other incubators and their CO2 consumption ? Many thanks in advance, Herman Herman Favoreel Department of Virology, Parasitology, and Immunology Faculty of Veterinary Medicine Ghent University Salisburylaan 133 9820 Merelbeke BELGIUM Phone: ++ 32 9 264 73 74 Fax: ++ 32 9 264 74 95 E-mail: [hidden email] website: http://www.vpi.ugent.be No virus found in this incoming message. Checked by AVG Free Edition. Version: 7.5.516 / Virus Database: 269.20.9/1290 - Release Date: 20/02/2008 8:45 PM No virus found in this outgoing message. Checked by AVG Free Edition. Version: 7.5.516 / Virus Database: 269.21.0/1296 - Release Date: 24/02/2008 12:19 PM |
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