Minimizing the Optical Illusion of Nanoparticles in Single Cells Using Four-Dimensional Cuboid Multiangle Illumination-Based Light-Sheet Super-Resolution Imaging.

Abstract

Although light-sheet-based super-resolution microscopy is an excellent detection technique for biological samples because of minimal photodamage, uneven light paths due to solid-angle illumination limits it, resulting in an optical illusion. Furthermore, the optical illusion limits the observations of individual molecules in diffraction. In this study, a four-dimensional cuboid multiangle illumination-based light-sheet super-resolution (4D CMLS) imaging system was developed to minimize optical illusions in cells. The lab-built 4D CMLS imaging system was integrated with total internal reflection fluorescence and a differential interference contrast microscope. A specially designed rotatable cuboid prism simply overcame the optical illusion by rotating a specimen on the prism to change the direction of light coming from an illumination lens. 4D CMLS reconstructed images of nanoparticles of different sizes were acquired in multi-illumination angles of 0°, 90°, 180°, and 270°. Additionally, a 4D multiangle illumination-based algorithm was created to select the optimal illumination angle by combining three-dimensional super-resolution imaging with multiangle observation, even in the presence of obstacles. The 4D CMLS imaging method demonstrates the in-depth 4D observation of samples at an optimum angle that can be used in various applications, such as single-molecule and subcellular organelle observations in single cells at subdiffraction limit resolutions that describe the scenario of nature.