Scattered light fluorescence microscopy in three dimensions

“Recently, we have proposed a method to image fluorescent structures behind turbid layers at diffraction limited resolution using wave-front shaping and the memory effect. However, this was limited to a raster scanning of the wave-front shaped focus to a two dimensional plane. In applications, it can however be of great importance to be able to scan a three dimensional volume. Here we show that this can be implemented in the same setup. This is achieved by the addition of a parabolic phase shift to the shaped wave-front. Via the memory effect, this phase shift leads to a shift of the interference based focus in the z-direction, thus opening the possibility of three dimensional imaging using scattered light fluorescence microscopy. Here, we show an example of such a three dimensional image of fluorescent nano-beads taken behind a turbid layer more than 10 mean free paths thick. Finally, we discuss the differences of the scanning in the z-direction with that in the x–y plane and the corresponding possibilities and limitations of the technique.”