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3D nano fabrication for single molecule microscopy

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3D nano fabrication for single molecule microscopy

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Wednesday, 16.02.2022

The new focus-locking technique involves growing cell on glass coverslips with specially 3D-printed nanometre particles called 'fiducials', which act as markers to stabilise or 'lock' the microscope as it is recorded. Being able to control the 3D-architecture and positioning of the guide markers at the nanoscale means that microscopists have greater control of the imaging.

 

Authors and Affiliations:

Simão Coelho 1, 2, 3, 4, Jongho Baek 5, 6, 7, James Walsh 5, 6, J Justin Gooding 8, 9, Katharina Gaus 5, 6
1 EMBL Australia Node in Single Molecule Science, School of Medical Sciences, University of New South Wales, Sydney, NSW, Australia.

2 ARC Centre of Excellence in Advanced Molecular Imaging, University of New South Wales, Sydney, NSW, Australia.

3 Structural Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA.

4 Instituto Gulbenkian de Ciência, 2780-156, Oeiras, Portugal.

5 EMBL Australia Node in Single Molecule Science, School of Medical Sciences, University of New South Wales, Sydney, NSW, Australia.

6 ARC Centre of Excellence in Advanced Molecular Imaging, University of New South Wales, Sydney, NSW, Australia.

7 NetTargets, National Nanofab Center, KAIST, Daejeon, Republic of Korea.

8 School of Chemistry and Australian Centre of NanoMedicine, University of New South Wales, Sydney, NSW, Australia.

9 ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, University of New South Wales, Sydney, NSW, Australia. 

 

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.

 

Journal: Nature Communications

 

Link: https://www.nature.com/articles/s41467-022-28219-6

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