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4Pi MINFLUX arrangement maximizes spatio-temporal localization precision of fluorescence emitter

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Rickert,  Julian D.
Optical Nanoscopy, Max Planck Institute for Medical Research, Max Planck Society;
Max Planck School Matter to Life, Max Planck Schools, Max Planck Society;

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Held,  Marcus O.
Optical Nanoscopy, Max Planck Institute for Medical Research, Max Planck Society;

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Engelhardt,  Johann
Optical Nanoscopy, Max Planck Institute for Medical Research, Max Planck Society;

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Hell,  Stefan W.       
Optical Nanoscopy, Max Planck Institute for Medical Research, Max Planck Society;

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Citation

Rickert, J. D., Held, M. O., Engelhardt, J., & Hell, S. W. (2024). 4Pi MINFLUX arrangement maximizes spatio-temporal localization precision of fluorescence emitter. PNAS, 121(11): e2318870121, pp. 1-7. doi:10.1073/pnas.2318870121.


Cite as: https://hdl.handle.net/21.11116/0000-000E-A19B-D
Abstract
We introduce MINFLUX localization with interferometric illumination through opposing objective lenses for maximizing the attainable precision in 3D-localization of single inelastic scatterers, such as fluorophores. Our 4Pi optical configuration employs three sequentially tilted counter-propagating beam pairs for illumination, each providing a narrow interference minimum of illumination intensity at the focal point. The localization precision is additionally improved by adding the inelastically scattered or fluorescence photons collected through both objective lenses. Our 4Pi configuration yields the currently highest precision per detected photon among all localization schemes. Tracking gold nanoparticles as non-blinking inelastic scatterers rendered a position uncertainty <0.4 nm3 in volume at a localization frequency of 2.9 kHz. We harnessed the record spatio-temporal precision of our 4Pi MINFLUX approach to examine the diffusion of single fluorophores and fluorescent nanobeads in solutions of sucrose in water, revealing local heterogeneities at the nanoscale. Our results show the applicability of 4Pi MINFLUX to study molecular nano-environments of diffusion and its potential for quantifying rapid movements of molecules in cells and other material composites.