Abstract
Two-photon direct laser writing is an additive fabrication process that utilizes two-photon absorption of tightly focused femtosecond laser pulses to implement spatially controlled polymerization of a liquid-phase photoresist. Two-photon direct laser writing is capable of nanofabricating arbitrary three-dimensional structures with nanometer accuracy. Here, we explore direct laser writing for high-resolution optical microscopy by fabricating unique 3D optical fiducials for single-molecule tracking and 3D single-molecule localization microscopy. By having control over the position and three-dimensional architecture of the fiducials, we improve axial discrimination and demonstrate isotropic subnanometer 3D focusing (<0.8 nm) over tens of micrometers using a standard inverted microscope. We perform 3D single-molecule acquisitions over cellular volumes, unsupervised data acquisition and live-cell single-particle tracking with nanometer accuracy.
Highlights
Two-photon direct laser writing is an additive fabrication process that utilizes two-photon absorption of tightly focused femtosecond laser pulses to implement spatially controlled polymerization of a liquid-phase photoresist
We demonstrate the versatility of our approach by acquiring 3D single-molecule acquisitions and live-cell single-particle tracking with nanometer accuracy
We presented a solution for lateral and axial focusing at depth with a ~20-fold improvement over current focusing methods
Summary
Two-photon direct laser writing is an additive fabrication process that utilizes two-photon absorption of tightly focused femtosecond laser pulses to implement spatially controlled polymerization of a liquid-phase photoresist. Real-time 3D focus-locking performed with high-accuracy (~1 nm) maximizes the photon collection from individual fluorescent events and has shown a > 10-fold improvement in the localization precision over standard methods without active stabilization[9]. A focusing method that has live-cell compatibility, absolute 3D positioning across micrometer ranges and an accuracy better than the photon-limited localization precision of any previously reported single-molecule method is highly desirable. We address these focusing limitations by using twophoton direct-laser writing to control the position and threedimensional architecture of the fiducials in the sample. We demonstrate the versatility of our approach by acquiring 3D single-molecule acquisitions and live-cell single-particle tracking with nanometer accuracy
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