we are doing bacterial live cell fluorescence imaging with out fixing the cells in glass slides. but the cells where moving in mounted glycerol or PBS when its viewed in different fields (Bright field and fluorescent field). how to rectify the problem with out killing the cells.
This method uses a thin layer of warmed gelatin:
http://www.chem.agilent.com/Library/slidepresentation/Public/Sample%20Preparation_Immobilization%20and%20Imaging%20Dave%20Allison_032708.pdf
It looks like they are specifically using it for AFM, but there's no reason I can see that you can't use it for fluorescence, unless the gelatin is autofluorescent. It will be important to make sure the layer is very thin if you want to transmit light through it, though. Or, alternatively, to image the top of the gelatin layer by inverting onto a glass slide, for example.
I woulld suggest you to use cell adhesives such as cell tack, ..
Here is the ref. http://www.seahorsebio.com/resources/tech-writing/protocol-non-adherent.pdf
Please let me know if it works for you
Cheers
Hi,
I do time-lapse microscopy on yeast cells. To tethered them to our glass wells, we coat the wells with Concanavalin A, and it works very well for yeasts.
Good luck !
There is a slide called subbed slides. Try it. U can make your own slides using gelatin and chrome alum. Good luck
Jordan Robin Yaron Sir,
I will not be able to use Gelatin coz i have gone through the fluorescence datas of gelatin which shows a wide range of fluorescence show I think using gelatin will not preferable. not only that the compound is a protein so i am afraid about some kind of interactions...
Alexander Lorenz sir,
That was really a great idea I will simply say What I have done coz some others are also doing this let it be useful for them also...
Initially we have done heat fixing to fix the cells in the slide. but after viewing through microscope it clearly found some cell wall disruption and cell bursting because of heat..
Once we have tried this 1% agaros methodology to afix them it was found to be minimal movement of cells and living cells we can view with out having any trouble.. great to see ur reply sir...
Delphine Aymoz mam
Concanavalin A technique my sir also advised to me but I faced some limitation in purchase so i have not tried this technique.. I feel it will work better coz after that i seen various paper saying about the technique...
Venkata Suresh Reddy Vajrala sir
I have not tried with any other adhesives coz of lack of availability.. thanks for the reply..
Murugadas sir,
wer are using normal glass slides with coverslips (bluestar).. to reduce the purchase we have not gone the one..
Thanks all for your valuable suggestion...
I really respect it and thought abt it..
For imaging on inverted microscope:
1) Melt 3% low-melting-temp agarose (e.g. SeaPlaque GTG) in your media of choice at 70 C (~30-60 minutes); reduce to 50 C and use any time
2) Make a 'slab' of the gel that's ~1-mm thick between slide and coverslips; there are various ways to do this; set ~60 minutes
3) Remove one coverslip, add ~0.5-1 uL of your cell suspension, wait ~5 minutes or dry more quickly with air.
4) Replace coverslip (with one removed before or new one), invert onto microscope stage.
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