Ratiometric 4Pi single-molecule localization with optimal resolution and color assignment
Published in Optics Letters, 2022
4Pi single-molecule localization microscopy (4Pi-SMLM)with two opposing objectives achieves sub-10 nm isotropic3D resolution when as few as 250 photons are collected byeach objective. Here, we develop a new ratiometric multi-color imaging strategy for 4Pi-SMLM that employs theintrinsic multi-phase interference intensity without increas-ing the complexity of the system and achieves both optimal3D resolution and color separation. By partially linking thephoton parameters between channels with an interferencedifference ofπduring global fitting of the multi-channel 4Pisingle-molecule data, we show via simulated data that theloss of localization precision is minimal compared with thetheoretical minimum uncertainty, the Cramer–Rao lowerbound.