Posted by
Axel Kurt Preuss on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Basic-live-cell-imaging-question-tp5687698p5704043.html
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Joel, I totally agree.
All what Gert needs is to avoid cell jitter or media perturbations. That s best achieved by having an immersion objective or by placing a coverslid on the cells with some space left for them to "breathe". In other words, not get squished or dry up. Or, to grow them on a coverslip and flip that in one way or the other upside down
There are three ways to achieve that
1) and 2) are leaving cells on their substrate and cover them with a coverslip
1) by placing spacers on the slid and place that on the cell culture
2) by placing the lid on the objective and glue it (reversibly with some non damaging glue or wax if it has to be) on the coverslip and make sure the edges of the slid are cemented in a way that they don't let media in
3)
The third way is the upside down approach . In this way, cells are grown on a coverslip and flipped upside down and best placed on some mold with enough medium volume. If the molded carrier is glass he may not need Hoffman. I think that s what Gert meant.
You also can buy a cheap small perfusion chamber which you can place upside down, (cells grown on coverslip are mounted into chamber, chamber gets flipped upside down, cells face downwards and their coverslip upwards towards objective, the whole chamber gets perfused and you can put it upside down and with some luck you don't get air bubbles killing your cells).
Colin needed to tell us whether he wants to stimulate the cells or not, and whether the BX has a water objective.
Thanks, Cheers
Best Regards
Axel cell +65 9271.5622
------------------
"We focus on your objectives!" -Axel K Preuss PhD, Central Imaging @IMCB, 6-19B, Singapore 138673
-----Original Message-----
From: Confocal Microscopy List [mailto:
[hidden email]] On Behalf Of JOEL B. SHEFFIELD
Sent: Thursday, November 04, 2010 9:40 AM
To:
[hidden email]
Subject: Re: Basic live cell imaging question...
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I think Gert's response makes a lot sense. For a newbie, with limited goals
(initially), it is not necessary to have an elaborate setup. In the (very)
old days, there was something called a "hanging drop culture" in which
tissue fragments were placed in a drop of medium on a slide, the slide was
inverted and examined with a conventional microscope. Gert is suggesting a
modern version of the same, assuming that you would visualize the cells
through the cover slip to which they are attached, Ujnfortunately, the
original writer did not specify the kind of optics they intended to use. If
the cells are, indeed, attached to the cover slip, it is possible to use
properly adjusted phase or Hoffman optics in transmissionto increase the
contrast. If fluorescence is needed, then you have to be concerned with
light toxicity, etc., but the optics should work fine, as long as you don't
want to see nuclear speckles, or details of mitochondria.
Joel
On Wed, Nov 3, 2010 at 9:03 PM, Axel Kurt Preuss <
[hidden email]> wrote:
> You need a water immersion object or have to build one
>
>
> Cheers
>
> Axel
> -----
> Axel K Preuss, PhD,
> Central Imaging, IMCB, A*Star, 61 Biopolis Dr, 6-19B, Singapore 138673,
> sent from 9271.5622
>
>
> On Nov 4, 2010, at 4:06 AM, Gert van Cappellen <
>
[hidden email]> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> >
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> > *****
> >
> > Culture your cells on a round coverslip. Take an object glas glue a
> > square piece of non-toxic rubber with a round hole on it. Fill this with
> > CO2 satured medium somewaht more as the volume of the hole. Put your
> > coverslip on it, with the cells to the medium off course. Press it
> > gently down and the glass will seal itself to the rubber ring. Now your
> > cells will survive for a couple of hours, so you can do the first
> > imaging. For real experiments you have to find a way to heat the object
> > glass to 37C.
> >
> > Good luck, Gert
> >
> > Op 29-10-2010 21:00, Dolphin, Colin schreef:
> >> *****
> >> To join, leave or search the confocal microscopy listserv, go to:
> >>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> >> *****
> >>
> >> We would like to do live cell imaging - mammalian cell lines - but only
> have direct access to an upright Olympus BX61. We don't really need
> complicated perfusion chambers, etc just something simple. We're real
> neophytes so all suggestions gratefully received.
> >>
> >> Colin
> >>
>
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Joel B. Sheffield, Ph.D
Department of Biology
Temple University
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