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

Jie Yu; Bo Cao; Heng Li; Bin Yu; Danni Chen; Hanben Niu

doi:10.1117/12.2068945

17 September 2014 Improved localization accuracy in double-helix point spread function super-resolution fluorescence microscopy using selective-plane illumination

Jie Yu, Bo Cao, Heng Li, Bin Yu, Danni Chen, Hanben Niu

Author Affiliations +

Proceedings Volume 9230, Twelfth International Conference on Photonics and Imaging in Biology and Medicine (PIBM 2014); 92300N (2014) https://doi.org/10.1117/12.2068945
Event: Twelfth International Conference on Photonics and Imaging in Biology and Medicine (PIBM 2014), 2014, Wuhan, China

Abstract

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.

Citation Download Citation

Jie Yu, Bo Cao, Heng Li, Bin Yu, Danni Chen, and Hanben Niu "Improved localization accuracy in double-helix point spread function super-resolution fluorescence microscopy using selective-plane illumination ", Proc. SPIE 9230, Twelfth International Conference on Photonics and Imaging in Biology and Medicine (PIBM 2014), 92300N (17 September 2014); https://doi.org/10.1117/12.2068945

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