Fiducial Markers for Localization Microscopy
Fiducial Markers (FM) are used to:
1) correct for small drifts (tens of nanometer range) during the course of an experiment
2) to align channels in multicolor experiments
3) to correct for chromatic aberrations in multicolor experiments
4) serve as a reference for the software autofocus
For
PALM imaging
we recommend fluorescent beads (e.g. Tetraspek beads from Invitrogen) or gold nanoparticles,
dependant on the laser power, as fiducial markers. Choose the beads according to the fluorophores used in your
experiment.
For
dSTORM imaging
we recommend nanoparticles (gold colloids) of which the photoluminescence persists through
the entire measurement and which should be immobilized on the coverslip.
Ideally one has about 1-3 fiducial markers in the field of view. You can either:
I) fix fiducial markers on coverslip before seeding of cells:
• sonicate FM solution in an ultrasound bath for at least 5 min
• add FM (100nm, BBI 1:1000 diluted) to dH2O solution
• vortex FM solution
• coat cover slips with Poly-L-lysine (PLL) for adhesion of FM
• add FM Mix solution onto PLL coated cover slips
• check density (1-3 FM in field of view)
• prepare sample on the slide
II) add fiducial markers to sample before experiment
• sonicate FM solution
• add 2-5µl FM solution into 2ml dH2O and vortex several minutes
• apply onto fixed cells
• wait ~1h for FM to settle
Note
Only dissolve FM in water (no salt solution), otherwise they will aggregate and not stick to the coverslip.
Fiducial Marker Order Info
• BB International [www.british-biocell.co.uk] & [http://www.bbigold.com]
Gold Colloid 80 nm (Cat. No. GM.GC 80) and 100 nm (Cat. No. GM.GC 100)
• Microspheres-Nanospheres (http://www.microspheres-nanospheres.com/)
40 nm (Cat. No. 790122-010) and 80 nm (Cat. No. 790120-010) nanospheres Au particles
• Nanopartz [www.nanopartz.com]
Nanorods 550 (Cat. No. 30-25-550) and 600 (Cat. No. 30-25-600)
• Tetraspek Beads [www.invitrogen.com]
Fiducial coverslips
• Hestzig LLC (http://www.hestzig.com)
• Fiducial coverslips 500 ± 50 nm spectral range at two densities (Cat. No. 550-30AuF & 550-100AuF);
• Fiducial coverslips 600 ± 100 nm spectral range at two densities (Cat. No. 600-30AuF & 600-100AuF)
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