Re: cytoo chamber assay

[Paloma Domínguez Giménez]

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Dear all,

I contact you because I am experiencing some problems in a migration assay and I wonder whether any of you have use these kind of plates before so you can give an idea of what is going on.

We  are trying to analyze a cell migration assay using Phase contrast illumination with a 10x objective.  The sample is a Cytoo chamber 1 well.
However, we have observed differences in the illumination depending on the position at the well, as if the illumination were more brilliant in some parts of the plate than others. Phase contrast is properly done at the centre of the well but light changes when moving slightly to the corners of the well. As if the phase rings were not properly aligned.

First of all I have checked the Phase rings and they are all correctly aligned. I wonder if we might be experiencing some kind of reflection....
The plate is black and I wonder if, since we are working on an inverted microscope, light might reflect at the wall of the well and we cannot get phase contrast illumination away from the center of the well.
If this is so, then the region available for image acquisition is really reduced...
Most probably the problem will be solved using any kind of fluorescence labeling in the cells, but my researcher is interested on PH contrast illumination.

The plate is this:
30-010

CYTOOchamber 1 well

Ext. diameter 35 mm; with lid; compatible with CYTOOchip for Live Cell Imaging; 1 well; Max. imaging area 16 x 16 mm²



All suggestions are very welcome!
Thank you in advanced.
Sincerely,

Paloma Dominguez Gimenez, PhD
Microscopy Facility
Andalusian Center for Molecular Biology and Regenerative Medicine (CABIMER)
Avda. Américo Vespucio s/n
Seville 41092 (SPAIN)
Tlf. 34-954 468223
E-mail: [hidden email]<mailto:[hidden email]>
Web: http://www.cabimer.es/web/es/unidades-apoyo/microscopia

Red Española de Microscopía Óptica Avanzada (REMOA)
http://remoa.wikispaces.com<http://remoa.wikispaces.com/>

[Aryeh Weiss]

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On 21/01/2016 4:08 PM, Paloma Domínguez Giménez wrote:
>
> Dear all,
>
> I contact you because I am experiencing some problems in a migration assay and I wonder whether any of you have use these kind of plates before so you can give an idea of what is going on.
>
> We  are trying to analyze a cell migration assay using Phase contrast illumination with a 10x objective.  The sample is a Cytoo chamber 1 well.
> However, we have observed differences in the illumination depending on the position at the well, as if the illumination were more brilliant in some parts of the plate than others. Phase contrast is properly done at the centre of the well but light changes when moving slightly to the corners of the well. As if the phase rings were not properly aligned.

I wonder if the problem is due to imaging through the meniscus, where
the sloped surface displaces the illumination. This is a problem in
multiwell plates (especially 96 well plates, but I do not know the
geometry of your plate.

--aryeh

> First of all I have checked the Phase rings and they are all correctly aligned. I wonder if we might be experiencing some kind of reflection....
> The plate is black and I wonder if, since we are working on an inverted microscope, light might reflect at the wall of the well and we cannot get phase contrast illumination away from the center of the well.
> If this is so, then the region available for image acquisition is really reduced...
> Most probably the problem will be solved using any kind of fluorescence labeling in the cells, but my researcher is interested on PH contrast illumination.
>
> The plate is this:
> 30-010
>
> CYTOOchamber 1 well
>
> Ext. diameter 35 mm; with lid; compatible with CYTOOchip for Live Cell Imaging; 1 well; Max. imaging area 16 x 16 mm²
>
>
>
> All suggestions are very welcome!
> Thank you in advanced.
> Sincerely,
>
> Paloma Dominguez Gimenez, PhD
> Microscopy Facility
> Andalusian Center for Molecular Biology and Regenerative Medicine (CABIMER)
> Avda. Américo Vespucio s/n
> Seville 41092 (SPAIN)
> Tlf. 34-954 468223
> E-mail: [hidden email]<mailto:[hidden email]>
> Web: http://www.cabimer.es/web/es/unidades-apoyo/microscopia
>
> Red Española de Microscopía Óptica Avanzada (REMOA)
> http://remoa.wikispaces.com<http://remoa.wikispaces.com/>
>

--
Aryeh Weiss
Faculty of Engineering
Bar Ilan University
Ramat Gan 52900 Israel

Ph:  972-3-5317638
FAX: [phone]

[Csúcs Gábor-3]

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Dear Paloma,

I agree with Aryeh, the problem is that in well-plates due to the height of the plate wall you will get proper phase contrast only in the middle of the well. Potential solution(s) could be:
1) Use a condenser with smaller NA (of course this will reduce your resolution)
2) Use a different type of contrast method (e.g. DIC - it the plate bottom is made of glass).

Greetings    Gabor

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Paloma Domínguez Giménez
Sent: Thursday, January 21, 2016 3:09 PM
To: [hidden email]
Subject: Re: cytoo chamber assay

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Dear all,

I contact you because I am experiencing some problems in a migration assay and I wonder whether any of you have use these kind of plates before so you can give an idea of what is going on.

We  are trying to analyze a cell migration assay using Phase contrast illumination with a 10x objective.  The sample is a Cytoo chamber 1 well.
However, we have observed differences in the illumination depending on the position at the well, as if the illumination were more brilliant in some parts of the plate than others. Phase contrast is properly done at the centre of the well but light changes when moving slightly to the corners of the well. As if the phase rings were not properly aligned.

First of all I have checked the Phase rings and they are all correctly aligned. I wonder if we might be experiencing some kind of reflection....
The plate is black and I wonder if, since we are working on an inverted microscope, light might reflect at the wall of the well and we cannot get phase contrast illumination away from the center of the well.
If this is so, then the region available for image acquisition is really reduced...
Most probably the problem will be solved using any kind of fluorescence labeling in the cells, but my researcher is interested on PH contrast illumination.

The plate is this:
30-010

CYTOOchamber 1 well

Ext. diameter 35 mm; with lid; compatible with CYTOOchip for Live Cell Imaging; 1 well; Max. imaging area 16 x 16 mm²



All suggestions are very welcome!
Thank you in advanced.
Sincerely,

Paloma Dominguez Gimenez, PhD
Microscopy Facility
Andalusian Center for Molecular Biology and Regenerative Medicine (CABIMER) Avda. Américo Vespucio s/n Seville 41092 (SPAIN) Tlf. 34-954 468223
E-mail: [hidden email]<mailto:[hidden email]>
Web: http://www.cabimer.es/web/es/unidades-apoyo/microscopia

Red Española de Microscopía Óptica Avanzada (REMOA) http://remoa.wikispaces.com<http://remoa.wikispaces.com/>

[Mark Cannell-2]

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Hi

The bathing solution meniscus and chamber walls can create problems for phase contrast illumination.

HTH

On 21/01/2016, at 2:08 pm, Paloma Domínguez Giménez <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Dear all,
>
> I contact you because I am experiencing some problems in a migration assay and I wonder whether any of you have use these kind of plates before so you can give an idea of what is going on.
>
> We  are trying to analyze a cell migration assay using Phase contrast illumination with a 10x objective.  The sample is a Cytoo chamber 1 well.
> However, we have observed differences in the illumination depending on the position at the well, as if the illumination were more brilliant in some parts of the plate than others. Phase contrast is properly done at the centre of the well but light changes when moving slightly to the corners of the well. As if the phase rings were not properly aligned.
>
> First of all I have checked the Phase rings and they are all correctly aligned. I wonder if we might be experiencing some kind of reflection....
> The plate is black and I wonder if, since we are working on an inverted microscope, light might reflect at the wall of the well and we cannot get phase contrast illumination away from the center of the well.
> If this is so, then the region available for image acquisition is really reduced...
> Most probably the problem will be solved using any kind of fluorescence labeling in the cells, but my researcher is interested on PH contrast illumination.
>
> The plate is this:
> 30-010
>
> CYTOOchamber 1 well
>
> Ext. diameter 35 mm; with lid; compatible with CYTOOchip for Live Cell Imaging; 1 well; Max. imaging area 16 x 16 mm²
>
>
>
> All suggestions are very welcome!
> Thank you in advanced.
> Sincerely,
>
> Paloma Dominguez Gimenez, PhD
> Microscopy Facility
> Andalusian Center for Molecular Biology and Regenerative Medicine (CABIMER)
> Avda. Américo Vespucio s/n
> Seville 41092 (SPAIN)
> Tlf. 34-954 468223
> E-mail: [hidden email]<mailto:[hidden email]>
> Web: http://www.cabimer.es/web/es/unidades-apoyo/microscopia
>
> Red Española de Microscopía Óptica Avanzada (REMOA)
> http://remoa.wikispaces.com<http://remoa.wikispaces.com/>

Mark  B. Cannell Ph.D. FRSNZ FISHR
Professor of Cardiac Cell Biology
School of Physiology &  Pharmacology
Faculty of Biomedical Sciences
University of Bristol
Bristol
BS8 1TD UK

[hidden email]

[jerie]

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Olá Paloma,

the chamber you are using comes with a glass lid. Filling up the chamber
until the top and covering with that lid making sure there are no air
bubbles caught , you will abolish the issue with the meniscus.

You may then run into issues with gas exchange for long term time lapse
recordings, which in turn can be addressed by using suitable transparent
films.

Good success!
Abraços,
Jens

Dr. Jens Rietdorf, visiting scientist @ center for technological
development in health, Rio de Janeiro, Brazil.
http://br.linkedin.com/pub/jens-rietdorf/6/4a3/189/


> The bathing solution meniscus and chamber walls can create problems for
> phase contrast illumination.
>
> the illumination were more brilliant in some parts of the plate than
> others. Phase contrast is properly done at the centre of the well but light
> changes when moving slightly to the corners of the well.
>


> > All suggestions are very welcome!
>