Re: Flat glass bottom multiwell plates

[darren.thomson]

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Hi Sylvie,

Thanks for raising such a critical discussion.

We just went through this issue also. We're trying to tile-scan entire wells of 96-well plates but end up with varied focal planes for each tile in the mosaic (Leica SP8x MATRIX lingo).

When we scan in reflection of the surface (Ibidi polymer) in XZ and move around the well, the line representing the substrate's focal plane moves up and down and even slants at an angle. This uneveness was later confirmed by simply running a finger on the 'glass' and feeling clear ripples/bumps on the glass.

There are clear outward bumps in every well of the Ibidi u-plate 96-well polymer plates. We also noticed the same for Greiner BioOne Sensor 96-well glass bottomed plates. I was surprised that such trusted suppliers had these bumps.

We don't have the luxury of hardware based AF, so we compensate for this somewhat with MATRIX's drift-compensation (a mini software-AF).

I think the simple 'running the finger down the row of wells' test works pretty well! Can you confirm if the Cellvis 96-well plates have this feature?

Many thanks,

Darren Thomson
University of Manchester

[darren.thomson]

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(sorry it removed the original discussion; now below my comment)

--------


Hi Sylvie,

Thanks for raising such a critical discussion.

We just went through this issue also. We're trying to tile-scan entire wells of 96-well plates but end up with varied focal planes for each tile in the mosaic (Leica SP8x MATRIX lingo).

When we scan in reflection of the surface (Ibidi polymer) in XZ and move around the well, the line representing the substrate's focal plane moves up and down and even slants at an angle. This uneveness was later confirmed by simply running a finger on the 'glass' and feeling clear ripples/bumps on the glass.

There are clear outward bumps in every well of the Ibidi u-plate 96-well polymer plates. We also noticed the same for Greiner BioOne Sensor 96-well glass bottomed plates. I was surprised that such trusted suppliers had these bumps.

We don't have the luxury of hardware based AF, so we compensate for this somewhat with MATRIX's drift-compensation (a mini software-AF).

I think the simple 'running the finger down the row of wells' test works pretty well! Can you confirm if the Cellvis 96-well plates have this feature?

Many thanks,

Darren Thomson
University of Manchester

-----------------
Dear listers
 
A while ago I asked the list about the flatness of glass bottom multiwell plates since we have been having issues with that.
 
I thought I should let you know the result of our test. Here are two references that seem to comply to these criteria:
-              They do not kill the cells (unfortunately, this is not a joke!)
-              They do not leak (no joke either! Sigh…)
-              They can be completely imaged with our Nikon 20x air, NA 0.75, WD 1mm objectives (meaning there is a low ‘skirt’ = rim at the edge of the plate. A high skirt raises the plate so that it becomes impossible to image the wells at the edges of the plate as the objective bumps into the insert edge)
-              They come from a company that will send you a free sample to try
 
1- Perkin Elmer #6005430. Skirt height: 1mm. Expensive: US$30/plate (sold in packs of 40)
2- CellVis #P96-1.5H-N. These seem to be flat. The skirt is 0.5 mm They have a glass bottom of thickness 1.5H (170 +/- 5um). Cheaper: US$10/plate (sold in packs of 20)
 
If you try these plates and find that anything above is not true, or if you know of other plates that fulfil these criteria, please let me know.
 
Hope that helps your day be slightly brighter... :(
 
Med vänlig hälsning / Best regards
 
Sylvie
 
@@@@@@@@@@@@@@@@@@@@@@@@
Sylvie Le Guyader, PhD
Live Cell Imaging Facility Manager
Karolinska Institutet- Bionut Dpt
Hälsovägen 7,
Novum, G lift, floor 6
14157 Huddinge
Sweden
mobile: [phone]
office: [phone]
LCI website

[Sylvie Le Guyader]

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Hi Darren



We have had really good user feedback about the Zell Kontakt glass bottom plates.<http://www.zell-kontakt.de/imaging-plate-cg.html> They seem to be very flat. They have a very low skirt so are safer when one images the outer wells with a low WD objective. And they get the added bonus that they are not outrageously expensive! :)



Med vänlig hälsning / Best regards



Sylvie



@@@@@@@@@@@@@@@@@@@@@@@@

Sylvie Le Guyader, PhD

Live Cell Imaging Facility Manager

Karolinska Institutet- Bionut Dpt

Hälsovägen 7,

Novum, G lift, floor 6

14157 Huddinge

Sweden

mobile: [phone]

office: [phone]

LCI website







-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Darren Thomson
Sent: den 4 december 2017 15:02
To: [hidden email]
Subject: Re: Flat glass bottom multiwell plates



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Hi Sylvie,



Thanks for raising such a critical discussion.



We just went through this issue also. We're trying to tile-scan entire wells of 96-well plates but end up with varied focal planes for each tile in the mosaic (Leica SP8x MATRIX lingo).



When we scan in reflection of the surface (Ibidi polymer) in XZ and move around the well, the line representing the substrate's focal plane moves up and down and even slants at an angle. This uneveness was later confirmed by simply running a finger on the 'glass' and feeling clear ripples/bumps on the glass.



There are clear outward bumps in every well of the Ibidi u-plate 96-well polymer plates. We also noticed the same for Greiner BioOne Sensor 96-well glass bottomed plates. I was surprised that such trusted suppliers had these bumps.



We don't have the luxury of hardware based AF, so we compensate for this somewhat with MATRIX's drift-compensation (a mini software-AF).



I think the simple 'running the finger down the row of wells' test works pretty well! Can you confirm if the Cellvis 96-well plates have this feature?



Many thanks,



Darren Thomson

University of Manchester