Improved localization accuracy in double-helix point spread function super-resolution fluorescence microscopy using selective-plane illumination

Recently, three-dimensional (3D) super resolution imaging of cellular structures in thick samples has been enabled with the wide-field super-resolution fluorescence microscopy based on double helix point spread function (DH-PSF). However, when the sample is Epi-illuminated, much background fluorescence from those excited molecules out-of-focus will reduce the signal-to-noise ratio (SNR) of the image in-focus. In this paper, we resort to a selective-plane illumination strategy, which has been used for tissue-level imaging and single molecule tracking, to eliminate out-of-focus background and to improve SNR and the localization accuracy of the standard DH-PSF super-resolution imaging in thick samples. We present a novel super-resolution microscopy that combine selective-plane illumination and DH-PSF. The setup utilizes a well-defined laser light sheet which theoretical thickness is 1.7μm (FWHM) at 640nm excitation wavelength. The image SNR of DH-PSF microscopy between selective-plane illumination and Epi-illumination are compared. As we expect, the SNR of the DH-PSF microscopy based selective-plane illumination is increased remarkably. So, 3D localization precision of DH-PSF would be improved significantly. We demonstrate its capabilities by studying 3D localizing of single fluorescent particles. These features will provide high thick samples compatibility for future biomedical applications.