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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 <
[log in to unmask]> 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 <[log in to unmask]> 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 <[log in to unmask]> wrote:
> >
> > From: Cammer, Michael <[log in to unmask]>
> > Subject: Re: nanotubes imaging
> > To: [log in to unmask]
> > 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:[log in to unmask]]
> On Behalf Of Charu Tanwar
> > Sent: Friday, February 11, 2011 5:25 AM
> > To: [log in to unmask]
> > 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.
> >
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-- 


Joel B. Sheffield, Ph.D
Department of Biology
Temple University
Philadelphia, PA 19122
Voice: 215 204 8839
e-mail: [log in to unmask]
URL:  http://astro.temple.edu/~jbs