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Microscopy images clearest near well centre


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#1 seanspotatobusiness

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Posted 13 January 2014 - 01:34 PM

When I micrograph cells cultured in a Nunc 24-well plate with an inverted phase contrast microscope, the image at the centre of the wells is really clear but towards the periphery, the cells are transparent and difficult to make out. Why is this? Is it avoidable?

 

(click pictures to enlarge)

 

Centre:

cMzZPhfs.jpg

 

Periphery 1:

X4VXDcNs.jpg

 

Periphery 2:

QBGSUyXs.jpg



#2 bob1

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Posted 13 January 2014 - 04:14 PM

This isn't avoidable, it is due to the shape of the wells and how light behaves on interaction with the edge of the well.



#3 CellApplicationsInc

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Posted 14 January 2014 - 09:41 AM

while it is an unavoidable edge artefact, you can try tweaking your phase ring alignment and Koehler illumination a little to adjust it to the edge. The image will never be the same as in the middle, but you may see more detail. However, you need to know what you're doing and how to change everything back, in other words, you need to be a fairly advanced microscope user to do it.



#4 seanspotatobusiness

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Posted 29 January 2014 - 06:51 AM

Thanks for the insight! I don't know how they're manufactured; I suppose it would probably increase costs drastically but it would be great if the well walls were opaque to prevent light passing through them like this.



#5 bob1

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Posted 29 January 2014 - 03:03 PM

it's not the clarity of the material that matters, but rather that the wall is blocking some of the light from reaching the objectives, and this interferes with the way koehler illumination and/or phase contrast works.



#6 seanspotatobusiness

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Posted 30 January 2014 - 03:33 AM

Oh right, okay. Thanks again, bob.






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