I do not have access to a confocal microscope.
Can I localize gene-gfp fusion in onion epidermal cell(transformed by Agrobacterium mediated transformation) using a inverted fluorescence microscope (Nikon Eclipse Ti-U)?
Please give some suggestions as to the excitation/emission filters.
when I am using blue excitation and green emission my entire field is appearing green.
Hi Ayan! My experience with Nikon Ti-U predicts that it has no confocal properties! However there are several ways to get over this as I know from my personal experience;
1-Use UV excitation in conjugation with a blue filter ( wear protective spectacles).
2-Move the condenser all the way to the top and then bring it down gradually to see when the image is crisp.
3-The field appearing green is an indication that the radiation is diffusing in the medium... You can solve this by using a higher than blue excitation in conjugation with a blue or red emission.
4-One technical problem with GFP is that although it can be tracked easily it gives of a very diffused light in various mediums. In fact once in my project on detecting the T-DNA in Agrobacterium I had a lot of trouble separately identifying the GFP... until I got the olympus BX-53.
Hope this helps...
Please ask for if you want...
Thanks
Sid
Ayan:
You need to know something about the gene-GFP fusion and where you expect to see it. If it is a cytoplasmic protein, then a diffuse green inside the cells is expected. If the areas between your cells are also green there are problems. The first thing to try it to make sure you use a fluorescence-approved mounting media; nail polish is known to kill GFP fluorescence, and some mounting media have high background fluorescence that could hide your signal. Similarly, make sure your immersion oil is okay for fluorescence (if you are using oil immersion lenses). Next, check and make sure your laser or mercury lamp bulb is properly aligned. Finally, make sure your filters are still in good condition (no scratches or central burn marks from long use); I am using a bandpass excitation filter 450-490 nm, dichroic mirror 510 nm cutoff, and a longpass emission filter 515 nm (Chroma technologies). If you still have high background you might want or need a neutral density filter to reduce the intensity of your excitation wavelength.
The reason for the green background might be that u are using an intense excitation. Reduce the intensity and the exposure time. Hopefully you will get a good image. Make sure that the other components of your cell do not fluoresce in the range u are using.
:)
Amit
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