Dear Brijesh,

It depends on the sample you want to visualize! If you have a rather thick sample with light scattering properties 2 photon microscopy is your way to go.  Using 1 photon confocal microscopy on samples like this will give you worse results because of the shorter wavelength excitation light used here compared to the long wavelenghts and thus less scattering with the 2 photon approach. Also if you use the 2 photon approach you have less damage outside the confocal volume like bleaching or phototoxic effects.

As i do not know your sample properties i can't give you a definit answer if you will see anything at all or if you have a weak/strong loss of signal...

Greetings,

Simon

I'll be analyzing plant cells. I'll try first with cell wall. If it doesn't work, then will try with plant protoplast.

To add to what has been said

It depends on the application.

1. 2-photon approach requires higher excitation intensity.

2. 2-photon gives you lower resolution (since higher wavelength used) compared to 1-photon approach.

3. Both have different selection rules.

4. For fluorescence anisotropy measurements, the limits of anisotropy change.

Just check, this change can lead to some complications depending on what you are studying.