Dear Roman,

1- If you use an illumination for the bright field imaging that do not excite your fluorophore (e.g.: 633 nm if you use a dye that is typically excited at 450 nm), you will be able to image using phase contrast without photobleaching your sample.

2- Recording fluorescence images without significantly photobleaching your sample can be done by exposing your sample to low light intensity and for a short time duration every time you want to record an image.

Cheers,

Mathieu

It is all about intensity and exposure times, as well as how good of a fluorophore you are using.  You should be able to use bright field white light for phase contrast without significantly affecting the intensity of your fluorophore, provide you keep exposure intensity low and of a short duration.

Additionally, if you are looking at it over the course of two weeks, there will probably be turnover of your protein, which will help to counter any bleaching that does occur.  

Mathieu A Bennet, I didn't realize that phase contrast could be done using just one wavelength (633 nm in your example).   I thought that it is typically done using *white* light?

Timothy S Jarvela,  could you please explain what you mean by "turnover of your protein"?  Thanks!

Dear Roman,

phase contrast can be done using a monochromatic light source. If you use a commercial system, this has probably be designed to work best with green light, i.e. this is the region of the spectrum where you will get the best contrast using phase contrast.

Thank you, Mathieu!

I am not sure what you are actually imaging.  But I assume if you are looking at something for two weeks, its probably a fluorescently tagged protein expressed in either cells or an organism, so correct me if I am wrong.  In that case, it is highly likely that you will have new protein production throughout those two weeks, unless the protein is under an inducible promoter or developmentally regulated or something like that. So with that in mind protein turnover would be production of new fluorophore protein as old fluorophore protein gets degraded naturally.

If I am incorrect, it is probably useful in the future to go into a bit more detail in your original question so people understand how to help you better.

I would recommend you to take the fluorescent image first, and the phase image afterwards, just to be sure that you don´t bleach any faint signals from your fluorophore.  Also, using another wavelenght to image the phase contrast, as suggested, is a good idea, specially considering the long term of your experiment. 

Hi,

I agree of the others wrote and I would add this,

Generally for DIC and Phase as well as Darkfield, a tungsten lamp is used. If you take a look at the spectrum  you will notice that it have its highest intensity around the red light. Thus I would recommend using a red filter. This way, there is enough light for phase imaging but not for exciting the fluorophore

It is also possible to use HBO 100 Mercury arc lamps which I would not recommend.

In Our lab we did image phase and fluorescent simultaneously which I think it is the best practice but it all depends on your experiment. But you could give it a try if your fluorescent signal is strong enough.