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.

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