nanotubes imaging
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Dear List This is not a direct confocal related query but one of the applications of live cell imaging. One of our facility user is working on uptake of carbon nanoparticles (CNP) which are tagged with alexa 488, from the media inside the cells and she wants to monitor the uptake with due course of time. Her problem is when she image the cells with media containing CNP she could not see anything except whole background as green leaving the cellular area totally black. However, when she washes the same cells with media containing no CNP immediately, she observes very good accumulation of CNP inside the cells (with the same microscope and lasers settings as before). Because she wants to do time lapse, so it is neccessary for her to monitor the cells in the media containing CNP (as she has to study the uptake of CNP from the media) but as soon as she puts in the media containing CNP she faces the above problem. She has tried this experiment with different concentration of CNP in the media but of no use. Her experimental media is RPMI with 10%FCS. Any advice or suggestion would be highly appreciated. Thanks to list in advance. Charu Tanwar Imaging Specialist Advanced Instrumentation Research Facility Jawaharlal Nehru University New Delhi India. |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Does she need to turn the gain way up and allow the media to be very saturated in order to see the particles inside the cells? -mc -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Charu Tanwar Sent: Friday, February 11, 2011 5:25 AM To: [hidden email] Subject: nanotubes imaging ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Dear List This is not a direct confocal related query but one of the applications of live cell imaging. One of our facility user is working on uptake of carbon nanoparticles (CNP) which are tagged with alexa 488, from the media inside the cells and she wants to monitor the uptake with due course of time. Her problem is when she image the cells with media containing CNP she could not see anything except whole background as green leaving the cellular area totally black. However, when she washes the same cells with media containing no CNP immediately, she observes very good accumulation of CNP inside the cells (with the same microscope and lasers settings as before). Because she wants to do time lapse, so it is neccessary for her to monitor the cells in the media containing CNP (as she has to study the uptake of CNP from the media) but as soon as she puts in the media containing CNP she faces the above problem. She has tried this experiment with different concentration of CNP in the media but of no use. Her experimental media is RPMI with 10%FCS. Any advice or suggestion would be highly appreciated. Thanks to list in advance. Charu Tanwar Imaging Specialist Advanced Instrumentation Research Facility Jawaharlal Nehru University New Delhi India. ------------------------------------------------------------ This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email. ================================= |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** No, whatever gain or laser power she uses, media gets saturated but she is not able to visualize anything inside the cells. The cells are always complete black as something is totally excluded from them.But with the same settings of gain and laser she could see CNP inside the cells after washing the cells with media NOT having CNP.Thanks CHARU TANWAR Imaging Specialist Advanced Instrumentation Research Facility Jawaharlal Nehru University New Delhi 110067 India. --- On Fri, 11/2/11, Cammer, Michael <[hidden email]> wrote: From: Cammer, Michael <[hidden email]> Subject: Re: nanotubes imaging To: [hidden email] Date: Friday, 11 February, 2011, 6:49 PM ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Does she need to turn the gain way up and allow the media to be very saturated in order to see the particles inside the cells? -mc -----Original Message----- From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Charu Tanwar Sent: Friday, February 11, 2011 5:25 AM To: [hidden email] Subject: nanotubes imaging ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Dear List This is not a direct confocal related query but one of the applications of live cell imaging. One of our facility user is working on uptake of carbon nanoparticles (CNP) which are tagged with alexa 488, from the media inside the cells and she wants to monitor the uptake with due course of time. Her problem is when she image the cells with media containing CNP she could not see anything except whole background as green leaving the cellular area totally black. However, when she washes the same cells with media containing no CNP immediately, she observes very good accumulation of CNP inside the cells (with the same microscope and lasers settings as before). Because she wants to do time lapse, so it is neccessary for her to monitor the cells in the media containing CNP (as she has to study the uptake of CNP from the media) but as soon as she puts in the media containing CNP she faces the above problem. She has tried this experiment with different concentration of CNP in the media but of no use. Her experimental media is RPMI with 10%FCS. Any advice or suggestion would be highly appreciated. Thanks to list in advance. Charu Tanwar Imaging Specialist Advanced Instrumentation Research Facility Jawaharlal Nehru University New Delhi India. ------------------------------------------------------------ This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email. ================================= |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** It sounds however like she is completely missing the uptake event since the desired signal appears immediately after the washing step. Even though it is a confocal measurement, could not the overwhelming signal from the solution above and below the cell be creating what looks like a uniform black background in the cell? Does the intensity level in the cell drop or increase or stay the same after the washing step (assuming PMT voltages and gains stay the same), and how does this intensity compare to the intensities outside the cell after washing? Again, based on what you described, it seems like the uptake of the particles has already happened before washing, it cannot be instantaneous even if the agitation caused by flushing the system somehow promotes uptake. Why not try repeating the experiment with successively lower concentrations of the solution nanoparticles, even to the point where the regions outside the cell do not saturate so easily. What you dump into the imaging chamber does not equal what gets inside the cells (concentration-wise). Do let us know if you figure this one out. I think other people have tried these types of imaging experiments before or would be interested in them. John Oreopoulos Research Assistant Spectral Applied Research On 2011-02-11, at 8:35 AM, charu tanwar <[hidden email]> wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > No, whatever gain or laser power she uses, media gets saturated but she is not able to visualize anything inside the cells. The cells are always complete black as something is totally excluded from them.But with the same settings of gain and laser she could see CNP inside the cells after washing the cells with media NOT having CNP.Thanks > > CHARU TANWAR > Imaging Specialist > Advanced Instrumentation Research Facility > Jawaharlal Nehru University > New Delhi 110067 > India. > > --- On Fri, 11/2/11, Cammer, Michael <[hidden email]> wrote: > > From: Cammer, Michael <[hidden email]> > Subject: Re: nanotubes imaging > To: [hidden email] > Date: Friday, 11 February, 2011, 6:49 PM > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Does she need to turn the gain way up and allow the media to be very saturated in order to see the particles inside the cells? > -mc > > -----Original Message----- > From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Charu Tanwar > Sent: Friday, February 11, 2011 5:25 AM > To: [hidden email] > Subject: nanotubes imaging > > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Dear List > > This is not a direct confocal related query but one of the applications of > live cell imaging. One of our facility user is working on uptake of carbon > nanoparticles (CNP) which are tagged with alexa 488, from the media > inside the cells and she wants to monitor the uptake with due course of > time. Her problem is when she image the cells with media containing CNP > she could not see anything except whole background as green leaving the > cellular area totally black. However, when she washes the same cells with > media containing no CNP immediately, she observes very good > accumulation of CNP inside the cells (with the same microscope and lasers > settings as before). > > Because she wants to do time lapse, so it is neccessary for her to > monitor the cells in the media containing CNP (as she has to study the > uptake of CNP from the media) but as soon as she puts in the media > containing CNP she faces the above problem. > > She has tried this experiment with different concentration of CNP in the > media but of no use. > Her experimental media is RPMI with 10%FCS. > > Any advice or suggestion would be highly appreciated. > > Thanks to list in advance. > > Charu Tanwar > Imaging Specialist > Advanced Instrumentation Research Facility > Jawaharlal Nehru University > New Delhi > India. > > ------------------------------------------------------------ > This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email. > ================================= > > > |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** You mention the use of different concentrations of the CNP, but you don't give the range. I think that John has the right idea. Try a series of 10-fold dilutions of the tubes. If the cells require such massive amounts of CNP, the whole thing may be an artifact. Another approach, if a large amount of particles is indeed necessary, is to dilute the fluorescent particles with unlabeled ones. That way, the extracellular label can be reduced to manageable levels and still keep the concentration of tubes up. A third trick might be to run the experiment at lower temperature, so as to reduce the speed of the reactions. Joel On Fri, Feb 11, 2011 at 9:12 AM, John Oreopoulos < [hidden email]> wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > It sounds however like she is completely missing the uptake event since the > desired signal appears immediately after the washing step. Even though it is > a confocal measurement, could not the overwhelming signal from the solution > above and below the cell be creating what looks like a uniform black > background in the cell? Does the intensity level in the cell drop or > increase or stay the same after the washing step (assuming PMT voltages and > gains stay the same), and how does this intensity compare to the intensities > outside the cell after washing? > > Again, based on what you described, it seems like the uptake of the > particles has already happened before washing, it cannot be instantaneous > even if the agitation caused by flushing the system somehow promotes uptake. > > Why not try repeating the experiment with successively lower concentrations > of the solution nanoparticles, even to the point where the regions outside > the cell do not saturate so easily. What you dump into the imaging chamber > does not equal what gets inside the cells (concentration-wise). > > Do let us know if you figure this one out. I think other people have tried > these types of imaging experiments before or would be interested in them. > > John Oreopoulos > Research Assistant > Spectral Applied Research > > On 2011-02-11, at 8:35 AM, charu tanwar <[hidden email]> wrote: > > > ***** > > To join, leave or search the confocal microscopy listserv, go to: > > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > > ***** > > > > No, whatever gain or laser power she uses, media gets saturated but she > is not able to visualize anything inside the cells. The cells are always > complete black as something is totally excluded from them.But with the same > settings of gain and laser she could see CNP inside the cells after washing > the cells with media NOT having CNP.Thanks > > > > CHARU TANWAR > > Imaging Specialist > > Advanced Instrumentation Research Facility > > Jawaharlal Nehru University > > New Delhi 110067 > > India. > > > > --- On Fri, 11/2/11, Cammer, Michael <[hidden email]> wrote: > > > > From: Cammer, Michael <[hidden email]> > > Subject: Re: nanotubes imaging > > To: [hidden email] > > Date: Friday, 11 February, 2011, 6:49 PM > > > > ***** > > To join, leave or search the confocal microscopy listserv, go to: > > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > > ***** > > > > Does she need to turn the gain way up and allow the media to be very > saturated in order to see the particles inside the cells? > > -mc > > > > -----Original Message----- > > From: Confocal Microscopy List [mailto:[hidden email]] > On Behalf Of Charu Tanwar > > Sent: Friday, February 11, 2011 5:25 AM > > To: [hidden email] > > Subject: nanotubes imaging > > > > ***** > > To join, leave or search the confocal microscopy listserv, go to: > > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > > ***** > > > > Dear List > > > > This is not a direct confocal related query but one of the applications > of > > live cell imaging. One of our facility user is working on uptake of > carbon > > nanoparticles (CNP) which are tagged with alexa 488, from the media > > inside the cells and she wants to monitor the uptake with due course of > > time. Her problem is when she image the cells with media containing CNP > > she could not see anything except whole background as green leaving the > > cellular area totally black. However, when she washes the same cells with > > media containing no CNP immediately, she observes very good > > accumulation of CNP inside the cells (with the same microscope and lasers > > settings as before). > > > > Because she wants to do time lapse, so it is neccessary for her to > > monitor the cells in the media containing CNP (as she has to study the > > uptake of CNP from the media) but as soon as she puts in the media > > containing CNP she faces the above problem. > > > > She has tried this experiment with different concentration of CNP in the > > media but of no use. > > Her experimental media is RPMI with 10%FCS. > > > > Any advice or suggestion would be highly appreciated. > > > > Thanks to list in advance. > > > > Charu Tanwar > > Imaging Specialist > > Advanced Instrumentation Research Facility > > Jawaharlal Nehru University > > New Delhi > > India. > > > > ------------------------------------------------------------ > > This email message, including any attachments, is for the sole use of the > intended recipient(s) and may contain information that is proprietary, > confidential, and exempt from disclosure under applicable law. Any > unauthorized review, use, disclosure, or distribution is prohibited. If you > have received this email in error please notify the sender by return email > and delete the original message. Please note, the recipient should check > this email and any attachments for the presence of viruses. The organization > accepts no liability for any damage caused by any virus transmitted by this > email. > > ================================= > > > > > > > -- Joel B. Sheffield, Ph.D Department of Biology Temple University Philadelphia, PA 19122 Voice: 215 204 8839 e-mail: [hidden email] URL: http://astro.temple.edu/~jbs |
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Hi, Another "trick" is to do incubate with the labeled tubes at 4C, where they will bind but scarcely be taken up in to the cell. Then wash, and image at cell temp (37C or whatever). That way you can follow the course of uptake in a background of unlabeled medium. Tobias >***** >To join, leave or search the confocal microscopy listserv, go to: >http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >***** > >You mention the use of different concentrations of the CNP, but you don't >give the range. I think that John has the right idea. Try a series of >10-fold dilutions of the tubes. If the cells require such massive amounts >of CNP, the whole thing may be an artifact. > >Another approach, if a large amount of particles is indeed necessary, is to >dilute the fluorescent particles with unlabeled ones. That way, the >extracellular label can be reduced to manageable levels and still keep the >concentration of tubes up. > >A third trick might be to run the experiment at lower temperature, so as to >reduce the speed of the reactions. > >Joel > > >On Fri, Feb 11, 2011 at 9:12 AM, John Oreopoulos < >[hidden email]> wrote: > >> ***** >> To join, leave or search the confocal microscopy listserv, go to: >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >> ***** >> >> It sounds however like she is completely missing the uptake event since the >> desired signal appears immediately after the washing step. Even though it is >> a confocal measurement, could not the overwhelming signal from the solution >> above and below the cell be creating what looks like a uniform black >> background in the cell? Does the intensity level in the cell drop or >> increase or stay the same after the washing step (assuming PMT voltages and >> gains stay the same), and how does this intensity compare to the intensities >> outside the cell after washing? >> >> Again, based on what you described, it seems like the uptake of the >> particles has already happened before washing, it cannot be instantaneous >> even if the agitation caused by flushing the system somehow promotes uptake. >> >> Why not try repeating the experiment with successively lower concentrations >> of the solution nanoparticles, even to the point where the regions outside >> the cell do not saturate so easily. What you dump into the imaging chamber >> does not equal what gets inside the cells (concentration-wise). >> >> Do let us know if you figure this one out. I think other people have tried >> these types of imaging experiments before or would be interested in them. >> >> John Oreopoulos >> Research Assistant >> Spectral Applied Research >> >> On 2011-02-11, at 8:35 AM, charu tanwar <[hidden email]> wrote: >> >> > ***** >> > To join, leave or search the confocal microscopy listserv, go to: >> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >> > ***** >> > >> > No, whatever gain or laser power she uses, media gets saturated but she >> is not able to visualize anything inside the cells. The cells are always >> complete black as something is totally excluded from them.But with the same >> settings of gain and laser she could see CNP inside the cells after washing >> the cells with media NOT having CNP.Thanks >> > >> > CHARU TANWAR >> > Imaging Specialist >> > Advanced Instrumentation Research Facility >> > Jawaharlal Nehru University >> > New Delhi 110067 >> > India. >> > >> > --- On Fri, 11/2/11, Cammer, Michael <[hidden email]> wrote: >> > >> > From: Cammer, Michael <[hidden email]> >> > Subject: Re: nanotubes imaging >> > To: [hidden email] >> > Date: Friday, 11 February, 2011, 6:49 PM >> > >> > ***** >> > To join, leave or search the confocal microscopy listserv, go to: >> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >> > ***** >> > >> > Does she need to turn the gain way up and allow the media to be very >> saturated in order to see the particles inside the cells? >> > -mc >> > >> > -----Original Message----- >> > From: Confocal Microscopy List [mailto:[hidden email]] > > On Behalf Of Charu Tanwar >> > Sent: Friday, February 11, 2011 5:25 AM >> > To: [hidden email] >> > Subject: nanotubes imaging >> > >> > ***** >> > To join, leave or search the confocal microscopy listserv, go to: >> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >> > ***** >> > >> > Dear List >> > >> > This is not a direct confocal related query but one of the applications >> of >> > live cell imaging. One of our facility user is working on uptake of >> carbon >> > nanoparticles (CNP) which are tagged with alexa 488, from the media >> > inside the cells and she wants to monitor the uptake with due course of >> > time. Her problem is when she image the cells with media containing CNP >> > she could not see anything except whole background as green leaving the >> > cellular area totally black. However, when she washes the same cells with >> > media containing no CNP immediately, she observes very good >> > accumulation of CNP inside the cells (with the same microscope and lasers >> > settings as before). >> > >> > Because she wants to do time lapse, so it is neccessary for her to >> > monitor the cells in the media containing CNP (as she has to study the >> > uptake of CNP from the media) but as soon as she puts in the media >> > containing CNP she faces the above problem. >> > >> > She has tried this experiment with different concentration of CNP in the >> > media but of no use. >> > Her experimental media is RPMI with 10%FCS. >> > >> > Any advice or suggestion would be highly appreciated. >> > >> > Thanks to list in advance. >> > >> > Charu Tanwar >> > Imaging Specialist >> > Advanced Instrumentation Research Facility >> > Jawaharlal Nehru University >> > New Delhi >> > India. >> > >> > ------------------------------------------------------------ >> > This email message, including any attachments, is for the sole use of the >> intended recipient(s) and may contain information that is proprietary, >> confidential, and exempt from disclosure under applicable law. Any >> unauthorized review, use, disclosure, or distribution is prohibited. If you >> have received this email in error please notify the sender by return email >> and delete the original message. Please note, the recipient should check >> this email and any attachments for the presence of viruses. The organization >> accepts no liability for any damage caused by any virus transmitted by this >> email. >> > ================================= >> > >> > >> > >> > > > >-- > > >Joel B. Sheffield, Ph.D >Department of Biology >Temple University >Philadelphia, PA 19122 >Voice: 215 204 8839 >e-mail: [hidden email] >URL: http://astro.temple.edu/~jbs -- _ ____ __ ____ / \ / / \ / \ \ Tobias I. Baskin / / / / \ \ \ Biology Department /_ / __ /__ \ \ \__ 611 N. Pleasant St. / / / \ \ \ University of Massachusetts / / / \ \ \ Amherst, MA, 01003 / / ___ / \ \__/ \ ____ www.bio.umass.edu/biology/baskin Voice: 413 - 545 - 1533 Fax: 413 - 545 - 3243 |
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In reply to this post by Charu Tanwar
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** I am not sure precisely what you are trying to see with this experiment. We have done this kind of imaging with fluorescent dextran and the fact that the media is fluorescent is not a problem. You see the black hole of the cell initially and over time, you see vesicles accumulate inside that black hole. It is just the inverse of normal imaging. There is no reason that the sea of external should inhibit visualization of the internalized material. Your external and internal fluorescence should be of similar intensity so washing should make no difference. Only if you are using a pH quenchable dye would the internalized fluorescence be different. The only reason I can see for you having a problem is if your CNP concentration is so high that you have an "inner filter effect" such that the CNP's are absorbing all the excitation light before it gets to the cells. If you are on an inverted and looking through the coverslip, then this is unlikely. However, if it is the problem, reducing the CNP concentration should eventually put you in a range where you can see the internalized fluorescence. Dave On Feb 11, 2011, at 5:25 AM, Charu Tanwar wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Dear List > > This is not a direct confocal related query but one of the applications of > live cell imaging. One of our facility user is working on uptake of carbon > nanoparticles (CNP) which are tagged with alexa 488, from the media > inside the cells and she wants to monitor the uptake with due course of > time. Her problem is when she image the cells with media containing CNP > she could not see anything except whole background as green leaving the > cellular area totally black. However, when she washes the same cells with > media containing no CNP immediately, she observes very good > accumulation of CNP inside the cells (with the same microscope and lasers > settings as before). > > Because she wants to do time lapse, so it is neccessary for her to > monitor the cells in the media containing CNP (as she has to study the > uptake of CNP from the media) but as soon as she puts in the media > containing CNP she faces the above problem. > > She has tried this experiment with different concentration of CNP in the > media but of no use. > Her experimental media is RPMI with 10%FCS. > > Any advice or suggestion would be highly appreciated. > > Thanks to list in advance. > > Charu Tanwar > Imaging Specialist > Advanced Instrumentation Research Facility > Jawaharlal Nehru University > New Delhi > India. 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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In reply to this post by Tobias Baskin
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To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** The other comments below make me also want to ask this question: What is the size of the confocal pinhole during the experiment? John Oreopoulos Research Assistant Spectral Applied Research On 2011-02-11, at 9:45 AM, Tobias Baskin wrote: > ***** > To join, leave or search the confocal microscopy listserv, go to: > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy > ***** > > Hi, > Another "trick" is to do incubate with the labeled tubes at 4C, where they will bind but scarcely be taken up in to the cell. Then wash, and image at cell temp (37C or whatever). That way you can follow the course of uptake in a background of unlabeled medium. > > Tobias > >> ***** >> To join, leave or search the confocal microscopy listserv, go to: >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >> ***** >> >> You mention the use of different concentrations of the CNP, but you don't >> give the range. I think that John has the right idea. Try a series of >> 10-fold dilutions of the tubes. If the cells require such massive amounts >> of CNP, the whole thing may be an artifact. >> >> Another approach, if a large amount of particles is indeed necessary, is to >> dilute the fluorescent particles with unlabeled ones. That way, the >> extracellular label can be reduced to manageable levels and still keep the >> concentration of tubes up. >> >> A third trick might be to run the experiment at lower temperature, so as to >> reduce the speed of the reactions. >> >> Joel >> >> >> On Fri, Feb 11, 2011 at 9:12 AM, John Oreopoulos < >> [hidden email]> wrote: >> >>> ***** >>> To join, leave or search the confocal microscopy listserv, go to: >>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >>> ***** >>> >>> It sounds however like she is completely missing the uptake event since the >>> desired signal appears immediately after the washing step. Even though it is >>> a confocal measurement, could not the overwhelming signal from the solution >>> above and below the cell be creating what looks like a uniform black >>> background in the cell? Does the intensity level in the cell drop or >>> increase or stay the same after the washing step (assuming PMT voltages and >>> gains stay the same), and how does this intensity compare to the intensities >>> outside the cell after washing? >>> >>> Again, based on what you described, it seems like the uptake of the >>> particles has already happened before washing, it cannot be instantaneous >>> even if the agitation caused by flushing the system somehow promotes uptake. >>> >>> Why not try repeating the experiment with successively lower concentrations >>> of the solution nanoparticles, even to the point where the regions outside >>> the cell do not saturate so easily. What you dump into the imaging chamber >>> does not equal what gets inside the cells (concentration-wise). >>> >>> Do let us know if you figure this one out. I think other people have tried >>> these types of imaging experiments before or would be interested in them. >>> >>> John Oreopoulos >>> Research Assistant >>> Spectral Applied Research >>> >>> On 2011-02-11, at 8:35 AM, charu tanwar <[hidden email]> wrote: >>> >>> > ***** >>> > To join, leave or search the confocal microscopy listserv, go to: >>> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >>> > ***** >>> > >>> > No, whatever gain or laser power she uses, media gets saturated but she >>> is not able to visualize anything inside the cells. The cells are always >>> complete black as something is totally excluded from them.But with the same >>> settings of gain and laser she could see CNP inside the cells after washing >>> the cells with media NOT having CNP.Thanks >>> > >>> > CHARU TANWAR >>> > Imaging Specialist >>> > Advanced Instrumentation Research Facility >>> > Jawaharlal Nehru University >>> > New Delhi 110067 >>> > India. >>> > >>> > --- On Fri, 11/2/11, Cammer, Michael <[hidden email]> wrote: >>> > >>> > From: Cammer, Michael <[hidden email]> >>> > Subject: Re: nanotubes imaging >>> > To: [hidden email] >>> > Date: Friday, 11 February, 2011, 6:49 PM >>> > >>> > ***** >>> > To join, leave or search the confocal microscopy listserv, go to: >>> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >>> > ***** >>> > >>> > Does she need to turn the gain way up and allow the media to be very >>> saturated in order to see the particles inside the cells? >>> > -mc >>> > >>> > -----Original Message----- >>> > From: Confocal Microscopy List [mailto:[hidden email]] >> > On Behalf Of Charu Tanwar >>> > Sent: Friday, February 11, 2011 5:25 AM >>> > To: [hidden email] >>> > Subject: nanotubes imaging >>> > >>> > ***** >>> > To join, leave or search the confocal microscopy listserv, go to: >>> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy >>> > ***** >>> > >>> > Dear List >>> > >>> > This is not a direct confocal related query but one of the applications >>> of >>> > live cell imaging. One of our facility user is working on uptake of >>> carbon >>> > nanoparticles (CNP) which are tagged with alexa 488, from the media >>> > inside the cells and she wants to monitor the uptake with due course of >>> > time. Her problem is when she image the cells with media containing CNP >>> > she could not see anything except whole background as green leaving the >>> > cellular area totally black. However, when she washes the same cells with >>> > media containing no CNP immediately, she observes very good >>> > accumulation of CNP inside the cells (with the same microscope and lasers >>> > settings as before). >>> > >>> > Because she wants to do time lapse, so it is neccessary for her to >>> > monitor the cells in the media containing CNP (as she has to study the >>> > uptake of CNP from the media) but as soon as she puts in the media >>> > containing CNP she faces the above problem. >>> > >>> > She has tried this experiment with different concentration of CNP in the >>> > media but of no use. >>> > Her experimental media is RPMI with 10%FCS. >>> > >>> > Any advice or suggestion would be highly appreciated. >>> > >>> > Thanks to list in advance. >>> > >>> > Charu Tanwar >>> > Imaging Specialist >>> > Advanced Instrumentation Research Facility >>> > Jawaharlal Nehru University >>> > New Delhi >>> > India. >>> > >>> > ------------------------------------------------------------ >>> > This email message, including any attachments, is for the sole use of the >>> intended recipient(s) and may contain information that is proprietary, >>> confidential, and exempt from disclosure under applicable law. Any >>> unauthorized review, use, disclosure, or distribution is prohibited. If you >>> have received this email in error please notify the sender by return email >>> and delete the original message. Please note, the recipient should check >>> this email and any attachments for the presence of viruses. The organization >>> accepts no liability for any damage caused by any virus transmitted by this >>> email. >>> > ================================= >>> > >>> > >>> > >>> >> >> >> >> -- >> >> >> Joel B. Sheffield, Ph.D >> Department of Biology >> Temple University >> Philadelphia, PA 19122 >> Voice: 215 204 8839 >> e-mail: [hidden email] >> URL: http://astro.temple.edu/~jbs > > > -- > _ ____ __ ____ / \ / / \ / \ \ Tobias I. Baskin > / / / / \ \ \ Biology Department > /_ / __ /__ \ \ \__ 611 N. 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In reply to this post by Charu Tanwar
*****
To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy ***** Three ideas: She could tag the CNT with something that is not fluorescent until the cell takes it up. For example a caged fluorescent molecule that loses the caging group when cells take it up. Not sure if there is a way to do that in endosomes. or She could tag the particles with QDots and dilute way down to where she can see individual QDots (cf. Tom Jovin with EGFR a few years back). or She can try adding the CNT with the cells on salted ice at 4°C to block endocytosis, wait for them to bind to the cell surface, rinse well with ice cold medium and then return to 37 °C and watch uptake. |
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