Zeiss ELYRA - Fiducial Markers for Localization Microscopy; Purpose of Fiducial Markers; Fiducial Marker and Coverslip Order Information

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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uorescentbeads(e.g.TetraspekbeadsfromInvitrogen)orgoldnanoparticles,

dependantonthelaserpower,asducialmarkers.Choosethebeadsaccordingtotheuorophores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ducialmarkersintheeld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eld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xed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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