Preparationofgeneralusebeadslides

‐ Usegoodquality,cleanslidesandno.1.5,22x22mmcoverslipswithanominalthicknessof170µm(160to190µm).

‐ TreatcoverslipwithpolyL‐lysinesolutionbyapplying200µLtocoverslipandspreadingitwithpipettetip.Leavefor10sandremovebywashingwithdistilledwater,tiltcoverslipandleavetodry.

‐ UseNileRedbeads0.2µmforassessingmicroscopealignmentorRed/Greenchannelregistration.

‐ Vortexbeadsandmakesolutionsof200µlat1:500to1:1000forNileRedalignmenttestslides.

‐ Whenpreparingbeadsyoucanavoidclumpingbysonicating.Sonicateasampleofstockfor3min,vortexanddiluteandvortexagain.

‐ Apply100µLpercoverslip(orcoverlipandslideforatwolayerpreparation)spreadingthebeadsasmuchaspossible.Leavetosettlefor30mintoadheretothepolylysineandthenremoveasmuchliquidaspossiblewithapipette.Leaveanother5minwhenadryfilmshouldhaveformedaroundtheedgeandthenremoveremainingliquidbytiltingthecoverslipandsuckingthesolutionoffwithtissuepaper.

‐ Leavetodrycompletelyinatiltedposition.‐ Mountonglassslidewithapprox.11‐15µlofmountante.g.Vectashield,

glycerolorProlongGold.Formationofairbubblesasyouplacethecoversliparebestpreventedbyplacinganeedleunderonesideofthecoverslipandslowlywithdrawingitasyoulowerintoposition.

‐ Doublesealtheslidewithnailvarnish.ForProlongGold,cureasappropriate,usuallyovernightatroomtemp.beforesealing.

PreparationofPSFbeadslides

‐ Usegoodquality,cleanslidesandno.1.5,22x22mmcoverslipswithanominalthicknessof170µm(160to190µm).

‐ WashthecoverslipsthoroughlyinethanolandtheninMilliQwaterinacleanplasticPetridish.Handleonlywithcleantweezers.

‐ Useredbeads0.1µm(0.11µm)andyellowgreenbeads0.1µm(0.1µm)forPSFmeasurementsatdilutionswheresinglebeadcanbefoundinafieldofview.

‐ Vortexbeadsandmakedilutesolutionsof200µlinMilliQH2O:1:10,000orbelowwillbegoodforPSFmeasurements.Applyupto100ul,spreadwiththeGilsontipandallowtoairdryinacleandustfreeenvironment(e.g.acupboard)

‐ Alternatively,Spread1µLofneat100or170nmPSFbeadsolutionasthinlyaspossibleonthecoverslipusingthesideofa200ulGilsontip,andallowittodryinacleandustfreeenvironment.Thisgivesamixtureofsparsespreadbeadsandbrightpuddlesofdensebeadswhichcanassistinfindingthefocalplane.

‐ Mountonglassslidewithapprox.13µlofmountante.g.Vectashield,glycerolorProlongGold.Formationofairbubblesasyouplacethecoversliparebestpreventedbyplacinganeedleunderonesideofthecoverslipandslowlywithdrawingitasyoulowerintoposition.

‐ Doublesealtheslidewithnailvarnish.ForProlongGold,cureasappropriate,usuallyovernightatroomtemp.beforesealing.

‐ IdeallyforPSFmeasurementsthereshouldbesingleisolatedbeadswithafieldofatleast256x256pixels.TomeasureaPSFthereshouldbenocontaminationofthesinglebeadimagewithAiryringsfromnearbybeads.