Live cell microscopy

[tyler1982]

Hi all!

Has any one ever worked with live cell imaging?

Basically I have to microinject my cells with a fluorescent dye and see what happens.

If anybody has any insight about topics like

- will my cell be alive and healty after the injection?

- could the dye change the behaviour of my cells?

- can plastic petri be used or do I need to seed on glass?

 

Thanks in advance to anyone so kind to help!!

[CharonY]

I need more details to give specific answers (e.g. what cell, what dye, what is the purpose of the experiment, what do you want to see).

 

- cells can be alive, depending on type of cells and type of injection

- dyes can alter the cell, depending on cell type and dye

- depending on size of cells and type of microscope you may be able to use petri dishes, however for transmittance microscopy most petri dishes are too thick.

[tyler1982]

C3H cells (murine embryo fibroblasts). Dye is Lucifer Yellow CH (or any other dye used for Gap Junctions if you know any one else...)

 

Besides, it seems petri dishes plastic has some kind of background noise when it comes to fluorescence. I've done immunofluorescence before, but only on glass coverslips.

 

Purpose of the experiment is to observe the status of the gap junctions while a drug treatment is being done.

 

2 dumb questions:

1) it can only be done on single layered cells, not con multilayered colonies, right?

2) what microinjector do you use?

 

tnx!!

[CharonY]

Most plastics are not suitable for fluorescence measurements. There are exceptions, but glass is preferable. LY is generally believed to be of low toxicity and it is supposed not to interfere with the cell too much if used in the normal range of measurements. Of course, in theory everything you throw at a cell will do something, however it is likely that the effects will (hopefully) not interfere what you want to see.

Microinjection will definitely alter cells, but if carefully done cell integrity is generally preserved (and the way cells may react should ideally have no impact on your experiment). Especially Fibroblasts are rather stable.

 

1) fluorescent images of multilayered cells are generally hard to see well. In theory you can focus on a particular layer (unless it is really thick) however stray fluorescent light from cells beneath and above will make it harder to see properly (if at all). single layers are definitely preferable.

2) I actually use a self-made microfluidic chip to electroporate but I look at cell with quite a different focus so someone's else opinion is probably more useful here.

Edited June 17, 2009 by CharonY

[tyler1982]

Thank you CharonY

I know glass is preferable, but the protocol of this experiment is validated on plastic and to validate another one on glass would me a way too long task

 

Best

Ty