Many microscopy methods achieve special capabilities by recording data, separated in time and subsequently processing this data. Confocal microscopy generates optical sections which look like the object having been cut by a sharp scalpel into slices which are only 1/100th as thick as a human hair. In a confocal microscope this is achieved by scanning a focused beam and a localized detection, needing a about 1 Million measured values separated in time. By using structured illumination, sections of similar quality can be obtained but needing only three subsequent exposures recorded by a camera. The images then need to be fused by a calculation to remove the out-of-focus light. In both methods one can think about the necessary sectioning information being encoded in time. At first, with only three exposures the minimum in the number of exposures seemed to have been reached.

In this publication a method is described which allows to obtain optical sections requiring only a single exposure. The aforementioned structured illumination method is used but the information is now not encoded in time but in the orientation of electric field of the light (“polarization”). The scientists used 2 cameras and 4 polarization filters combined with a special polarized illumination. Since the researchers modulate the direction of polarization spatially they can record images containing the necessary sectioning information simultaneously. These are then used to compute an optical section free of out-of-focus light.

In this publication the scientists describe the polarization coded structured illumination microscopy (“PicoSIM”) method and show PicoSIM images of optically sectioned muscle fibers.

But nature cannot always be fooled at will. For the method to work well, the fluorescent molecules must be randomly oriented. This is the case for most samples, but not all.

The publication presents experiments performed at Leibniz IPHT by two PhD students (S. Shukla Mukherjee and Daniel Appelt). The PicoSIM method can be extended to other imaging modes (e. g. lightsheet microscopy, high resolution structured illumination, Raman microscopy) and can be made almost arbitrarily fast using femtosecond lasers. But the drawback of requiring non-oriented molecules remains as well as its reduced susceptibility to noise.