Ratiometric 4Pi single-molecule localization with optimal resolution and color assignment.

4Pi single-molecule localization microscopy (4Pi-SMLM) with two opposing objectives achieves sub-10 nm isotropic 3D resolution when as few as 250 photons are collected by each objective. Here, we develop a new ratiometric multi-color imaging strategy for 4Pi-SMLM that employs the intrinsic multi-phase interference intensity without increasing the complexity of the system and achieves both optimal 3D resolution and color separation. By partially linking the photon parameters between channels with an interference difference of π during global fitting of the multi-channel 4Pi single-molecule data, we show via simulated data that the loss of localization precision is minimal compared with the theoretical minimum uncertainty, the Cramer-Rao lower bound.

Compact single-shot radial shearing interferometer with random phase shift.

We propose a compact phase-shifting radial shearing interferometer based on a pixelated micro-retarder array (MRA). The MRA is made of a thin birefringence plate, and is composed of identical units that have pixels of four different thicknesses. We demonstrate that pixelated phase delay between two shearing beams can be introduced by applying the fast axis of birefringence plate in the horizontal or vertical orientation. We also present an approach to extract the wavefront under test with random phase shift, which dramatically reduces the machining difficulty of the MRA. The feasibility and accuracy of the proposed method are further validated through our numerical analysis. With the advantages of vibration immunity, simultaneous phase stepping, and broad spectral range, the presented interferometer is expected to be of potential use in a variety of applications, such as the detection of moving objects and dynamic processes.