biostudies-literatureStachelek PBiotechnology and Biological Sciences Research CouncilEngineering and Physical Sciences Research Council553https://www.ebi.ac.uk/biostudies/studies/S-EPMC8795401biostudies-literatureUnknown13(1)The molecular machinery of life is founded on chiral building blocks, but no experimental technique is currently available to distinguish or monitor chiral systems in live cell bio-imaging studies. Luminescent chiral molecules encode a unique optical fingerprint within emitted circularly polarized light (CPL) carrying information about the molecular environment, conformation, and binding state. Here, we present a CPL Laser Scanning Confocal Microscope (CPL-LSCM) capable of simultaneous chiroptical contrast based live-cell imaging of endogenous and engineered CPL-active cellular probes. Further, we demonstrate that CPL-active probes can be activated using two-photon excitation, with complete CPL spectrum recovery. The combination of these two milestone results empowers the multidisciplinaryNature communicationsCircularly polarised luminescence laser scanning confocal microscopy to study live cell chiral molecular interactions.PMC8795401EP/I010319/1BB/T009268/1BB/T009268/2BB/S017615/1EP/P025013/1Stachelek PMacKenzie LPal RParker DfalseCircularly polarised luminescence laser scanning confocal microscopy to study live cell chiral molecular interactions.The molecular machinery of life is founded on chiral building blocks, but no experimental technique is currently available to distinguish or monitor chiral systems in live cell bio-imaging studies. Luminescent chiral molecules encode a unique optical fingerprint within emitted circularly polarized light (CPL) carrying information about the molecular environment, conformation, and binding state. Here, we present a CPL Laser Scanning Confocal Microscope (CPL-LSCM) capable of simultaneous chiroptical contrast based live-cell imaging of endogenous and engineered CPL-active cellular probes. Further, we demonstrate that CPL-active probes can be activated using two-photon excitation, with complete CPL spectrum recovery. The combination of these two milestone results empowers the multidisciplinary2022-01-01T00:00:00Z2022 Jan2026-07-16T16:06:29.814Z2025-04-05T13:23:40.106ZS-EPMC87954013508704710.1038/s41467-022-28220-z