[Spm] Re: live cell imaging-related

[Rohit Khanna]

Hello everybody,

Thanks Dipak and Guillaume for your quick response. The polystyrene
substrate i am using, are coated with poly-L-lysine and moreover, osteoblast
cells have a very flat morphology on such substrate. So cell attachment is
not a issue.

I am actually trying to image cell using contact mode but it seems that
there is a strong tip-cell adhesion due to which tip literally shakes plasma
membrane (outer boundary of cell) quite a bit.

I am not familiar with tapping mode imaging in liquid. Although, i take good
images using tapping mode in air. It will be great if somebody can help me
with the tapping mode imaging in liquid. Do you have any protocol?

As an alternative, i am planning to buy a much flexible cantilever with k=
0.01 N/m, but again i am not sure if this is going to solve my problem.

Please give your suggestions.

Thank you very much.

best regards,

Rohit




>
> Message: 1
> Date: Fri, 19 Dec 2008 19:38:11 +0530 (IST)
> From: Dipak Paramanik <dipak1205 at yahoo.co.in>
> Subject: [Spm] Re: Imaging of live osteoblast cells
> To: spm at spmlist.di.com
> Message-ID: <971982.74118.qm at web8601.mail.in.yahoo.com>
> Content-Type: text/plain; charset="utf-8"
>
> Hi Rohit,
>
> Here are your answers,
>
> 1. You can use Tapping mode AFM to image the living cell. Actually tapping
> mode is designed for the purpose to image soft samples like living cells.
> This mode make very less damage to the surfaces as the tip does not stay in
> contact with surface. You can do tapping mode imaging in air as well as in
> liquid environment. First do the imaging with less amplitude set point
> (generally 1) and gradually increase the value to get a better image.
>
> 2. To measure the volume of the cell: you can measure the height and area
> of each cell
> in the image using imageJ software. Then accourding to the geometry (
> cylidrcal or spherical ..etc) of the cell you aply simple formula to obtain
> the volume of the cell.
>
>
>
> Hope this would solve your problem
>
> good luck
> ---------------------------------------------------
> Dr. Dipak Paramanik
> Postdoctoral Scholar
> Atomic Beam Group
> Quantum Beam Center
> National Institute for Material Science
> 1-2-1 Sengen, Tsukuba, Ibaraki 305-0047
> Japan
> Phone: 81-29-851-3354 ext 2978
>  Personal homepage:
> http://www.iopb.res.in/~dipakpk
>
>
>
> --- On Fri, 19/12/08, spm-request at spmlist.di.com <
> spm-request at spmlist.di.com> wrote:
> From: spm-request at spmlist.di.com <spm-request at spmlist.di.com>
> Subject: Spm Digest, Vol 53, Issue 7
> To: spm at spmlist.di.com
> Date: Friday, 19 December, 2008, 3:36 PM
>
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> ----------------------------------------------------------------------
>
> Message: 1
> Date: Thu, 18 Dec 2008 19:04:04 -0600
> From: "Rohit Khanna" <rkhanna.iitk at gmail.com>
> Subject: [Spm] Re: Spm Digest, Vol 53, Issue 6
> To: spm at spmlist.di.com
> Message-ID:
>        <d92a40270812181704x4f55a67cr5c990de853131252 at mail.gmail.com>
> Content-Type: text/plain; charset="iso-8859-1"
>
> Hello everybody,
>
> I have two queries.
>
> 1. Imaging of live osteoblast cells (adherent cells).
> - i tried to image these cells using standard contact mode tip (V shape)
> with nominal radius of 20 nm, spring constant = 0.06N/m. Imaging of fixed
> cells on polystyrene substrate was quite easy to do both in air and liquid
> (using fluid cell).
>
> I could not image live cells, as tip while moving on the cell, actually
> shaked the plasma membrane quite a bit and after a few minutes, the cell
> got
> detached from the substrate.
>
> Can anybody suggest, what should be done...
>
> 2. How to calculate the cell volume from AFM height image.?
>
> Please give your suggestions.
>
> thank you very much.
>
> best,
>
> Rohit
>
>
>
>
> Message: 2
> Date: Fri, 19 Dec 2008 17:38:10 +0100
> From: Guillaume Lamour <guillaume.lamour at univ-paris5.fr>
> Subject: [Spm] Re: imaging of live osteoblast cells
> To: spm at spmlist.di.com
> Message-ID: <494BCDF2.8060407 at univ-paris5.fr>
> Content-Type: text/plain; charset=ISO-8859-1; format=flowed
>
> Rohit,
>
> Have you tried tapping mode? and maybe you could try to enhance the
> adhesion of your cells, using a biopolymer like poly-L-lysine to coat
> the polystyrene substrate.
>
> Good luck,
> Guillaume
>
>
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