3)Formatreferstohowmanypixelswillbecapturedwhenyouacquireanimage.A512x512pixelformatwill
giveyougoodresolutionatareasonablespeed.Ifyouselect1024x1024,youwillhavebetterresolutionbut
itwilltakelongertoimageandbeamuchbiggerfilesize.
4)Speedreferstothespeedofthescanheadasthelaserspassovereachpixel.Theslowerthescanspeed,the
betteryoursignal-to-noiseratiobecomes.However,slowerscanspeedsincreasethepixeldwelltimewhich
canincreasephotobleachingofyoursample.Thescanspeeddefaultsto400Hz,butbyincreasingthespeed
to600Hz,youlimitbleachingandtheimagequalityisusuallycomparable.Either400or600Hzaregoodscan
speedstoselect.
5)LineAveragingisanothermeanstocleanupthesignal-to-noiseinyourimage.Youwanttohaveline
averagingatatleast4(8isrecommended)toacquirethebestimagewithminimalbackground.Thehigher
theaveraging,thelongeryourscanwilltakebuttheimagequalityshouldimprovesignificantly.Keepinmind
thatwhenscanninginLivemode,thelineaveragingisnotincorporated.OnlywhenyouCaptureanimage
doesthelineaveragingbecomeactive.Note:Frameaveragingisanoptionandaverageseachscannedframe
separately,accumulatingtheaveragingasitaddsthenumberofscansindicated.It’samatterofpreferenceas
towhichaveragingtechniqueyouuse,butwegenerallyrecommendlineaveraging.
6)Pinhole-thisisthesizeoftheopeninginwhichthelaser(s)passthrough.Fortrueconfocalmicroscopy,you
alwayswanttochooseAiry1.WhenyouhitAiry1,theredsliderwilladjustthepinholetotheappropriate
diameterbasedontheobjectiveandlaserwavelengthsyouareusing.However,ifyoursignalisweak,youcan
openupthepinholetoallowmorelaserlightthroughtothesampleandyouwillbeabletodetectastronger
signal.Keepinmindthatthemoreopenapinholeis,thecloseryouareapproachingbasicfluorescence
microscopyandlosingthe“confocalness”ofyourscan.Ifyourgoalistodetermineco-localizationofmultiple
fluorophores,allyourchannelsneedtobesettoAiry1.
7)Ifyouselectedseq(thesequentialbutton,see#2)youcanseeyourseparatelightpathsequenceshere.
Eachsequencecorrespondswiththeproperexcitationsettingsandemissiongatingtovisualizeasingledyein
yoursample.
8)Allowsyoutochangeobjectivesfromthesoftwarescreeninsteadofthetouchpad(seepage12).
9)InitiateaLivescaninordertopreviewyourimageandsetthebrightness(Gain)andcontrast(Offset)levels
accordingly(seepage17).
10)Afteryouhavesetyourimagingsettingsappropriately,hitCaptureImagetoactuallycollectthepicture.By
capturingtheimage,youareincorporatingalltheaveragingsettingsintothefinalpicture.Allcapturedimages
arestoredunderthe“Projects”tab.IfyouaremerelydoingaLivescan,thoseimagesarenotstored.