Abstract: A method for optical microscopy, including using a first laser beam to excite dye particles in a sample region with light having a first wavelength. A second laser beam with a second wavelength based on the emission spectrum of the excited particles is used to de-excite the excited particles. The first and second beams have first and second respective intensity distributions which are spatially different when co-aligned; the second profile has a minimum where the first has a maximum. The region is once concurrently illuminated with the first and second beams, and an emission signal is detected. For each scanning point, the region is illuminated also with a pulse of the second laser beam or continuously prior to or after illuminating the region of the sample concurrently with both lasers. The illumination with only the second laser beam defines a background signal that is subtracted from the emission signal.

Abstract: A method for optical microscopy, including using a first laser beam to excite dye particles in a sample region with light having a first wavelength. A second laser beam with a second wavelength based on the emission spectrum of the excited particles is used to de-excite the excited particles. The first and second beams have first and second respective intensity distributions which are spatially different when co-aligned; the second profile has a minimum where the first has a maximum. The region is once concurrently illuminated with the first and second beams, and an emission signal is detected. For each scanning point, the region is illuminated also with a pulse of the second laser beam or continuously prior to or after illuminating the region of the sample concurrently with both lasers. The illumination with only the second laser beam defines a background signal that is subtracted from the emission signal.