Localization-based super-resolution imaging methods, such as the photoactivated localization microscopy (PALM) and the stochastic optical reconstruction microscopy (STORM), have been demonstrated to have the capability of overcoming the diffraction limit in three dimensions. In these methods, the resolution is determined by the localization precision rather than the diffraction limit. For PALM and STORM, it has been shown that engineering the PSF of the imaging system by using the astigmatic point spread function (PSF), double helix PSF (DH PSF), and Airy PSF can enable a high localization precision over a large depth range. Up to now, the DH PSF has been widely used in localization-based super-resolution imaging methods and localization-based single particle tracking methods because of its large imaging depth and simplicity in extracting the emitter’s 3D position. However, anisotropy in the localization process affects the axial precision, making it worse than the transverse precision.
Recently, researchers at Shanghai Institute of Optics and Fines Mechanics (SIOM) of Chinese Academy of Sciences propose a new kind of rotating PSF: the interferometric DH PSF (iDH PSF). As shown schematically in Fig. 1, the two lobes in the coherent DH PSFs collected by the two objective lenses overlap perfectly in the proposed iDH PSF, thus allowing the interference in the azimuthal direction. They show that the axial information is encoded in the azimuthal direction in iDH PSF. The interference between the waves collected by the two opposed objective lenses in 4Pi imaging improves the axial resolution by a factor of 3–7 compared with using a single objective lenses. Therefore, iDH PSF based on interferometric detection schemes shows super-resolution in the azimuthal direction, and side lobes occur in the original lobe position which is similar with the axial super-resolution and axial side bands in 4Pi imaging. Further, they show that localization-based super-resolution imaging using iDH PSF improves the transverse precisions by two-fold, and the axial localization precision by a factor of 4-5 in comparison with using DH PSF. Thus, the anisotropy in localization precisions using DH PSF is greatly improved by using iDH PSF. Moreover, they design an iDH PSF to enable 3D imaging over a 3 μm depth range which is around 3 times of other interferometric localization based super-resolution methods.
The study was published in Scientific Reports
This work is supported by China Postdoctoral Science Foundation, National Natural Science Foundation of China, and the Chinese Academy of Sciences.
Figure 1. Schematic illustration of the setup for generating interferometric rotating PSF. In the interferometric detection scheme, the light fields E1, E2 collected by objective lenses OL1 and OL2 are modulated by conjugate pupil functions T1 and T2 (T1 = T2_) respectively. The beam splitters (BS) and an constant phase retardation plate before BS 4 combines the light fields to produce four phase-shifted interferometric signals I1, I2, I3, I4 on the cameras. DM: Dichroic mirror, BS: beam splitter. Inset: pupil functions (DH-P3) for generating the DH PSF. In the non-interferometric detection scheme, only one objective lens OL 1 is used, and the four beam splitters are removed from the detection path.
Wei Wang & Guohai Situ
Shanghai Institute of Optics and Fine Mechanics