Always image the most unstable fluorophore first. For example, in a sample dual-stained with an Alexa dye and a traditional fluorophore eg. FITC, it is better to image the FITC first because it photo-bleaches easily compared to the more stable fluorophore. This is especially important for dyes in the DAPI range, the high intensity wavelength of light will visibly bleach regions of interest.
High resolution fluorescent images can be obtained with objectives that are not “phase” or “PH” specified–i.e. those with a higher numerical aperture, closer working distance, and an unrestricted optical path because more light can be transmitted back to the camera/ oculars leading to brighter fluorescence and better resolution.