RichardCavicchi,DeanRipple,JasonKing,CaylaColle7#
BiomolecularMeasurementDivision,NIST#WestVirginiaWesleyanCollege
ESTIMATIONOFPROTEINAGGREGATEDENSITYUSINGSEDIMENTATIONCOMBINEDWITHMICRO-PARTICLE
TRACKING
1. PossibleimprovementstoFlowMicroscopyAnalysis
a. Shapeeffect
b. Newimageprocessingalgorithm
2. AggregateDensitymeasurementsbasedonsedimentaMon
3. NewReferenceMaterials
a. Fluoropolymeraggregatesimulants
b. Othermaterialsunderdevelopment
Outline
LightobscuraMon FlowImaging
ElectricalSensingZone(Coulter)
SubvisibleParCcleSizingMethods
ΔR~volume
(ESZ)
(FI)(LO) areaΔI~area
ResonanceMass ΔM
ParMcletrackingD(relatedtodhydrodynamic)
Measureddiameter,concentraConforpolystyrenebeads.
Goodagreement
ForProteinaggregates,methodsdon’tagree…
Ripple,D.C.andZ.S.Hu(2016)PharmaceuMcalResearch33(3):653-672.
Proteinaggregatesvaryinsize,shape,intensity…
VariableThresholdMethodforimprovedParCcleBoundaryDetecCon
CanwegetimprovedboundarydetecMonwithoutlosingnearlytransparentparMcles?
Newalgorithm,breakanalysisintotwoparts:a) FindparMcleswithasinglelowthresholdb) EvaluateparMcleboundariesusinga
thresholdappropriatetotheintensityofeachindividualparMcle
eba
d
c
VariableThresholdMethodforimprovedParCcleBoundaryDetecCon
Result:
(e)
(b)(a)
(d)
(c)
Strategyforfragments
a. Connectfragments
b. Drawtheconvexhull
c.andd.Perform“AND”operaMonwithoriginalthesholdedarea
e. ResulMngboundarysuperimposedonimage
Collagewithoverlays
ImplementedinImageJ/FIJI,opensourcesoeware
ResultstableofanalyzedparCcles
CollagestripsofsamebeadsanalyzedwithsinglethresholdandVariableThreshold(redbackground)
10µm
2µm
10µm
5µm
20µm
(ImagesobtainedwithFlowcam10x)
TestofVariableThresholdMethod
10µm
ECD13.4µm
SEMimage
VariableThreshold
10µm
VariableThreshold
TestofVariableThresholdMethodproteinaggregates
PossibleimprovementstoFlowMicroscopyAnalysis
a. Shapeeffect,canuseperimetertoimproveequivalentcirculardiametercalculaMonforelongatedparMcles.
b. ImageprocessingalgorithmyieldsMghterboundariesforintenseparMcles,withoutlosingcountsonnearlytransparentparMcles.
i)be7eranalysisforsampleswithvaryingparMcleproperMes
ii)sameinstrumentseingsforcalibraMonandoperaMon
iii)maybeusefulforimprovedclassificaMonofparMcles
Whatisthedensityofaproteinaggregateandwhyisitimportant?
Differentmethodscharacterizedifferentaspectsofsize:Flowimaging:spaMalextent(A~V)ElectricalSensingZone:liquidexcludedvolume(V’)ResonanceMass:MParMcleTracking:VhTocompareparMclecountsaboveacertainsize,orsizedistribuMonfromthesemethods,adensityvalueforproteinaggregatesisneeded.
ρ =VM
Forasphere
SedimentaCon/ParCcletracking
2gR29µv
Δρ= µviscoscityvvelocityg9.8m/s2
Rhydrodynamicradiusofsphere
ToreduceconvecMon,useRectangularglasscapillary50µmor100µminsidethicknessx1mm,sealedatbothends
MeasuringRfromimage,andmeasuringvbyfollowingimages,candeduceΔρ
AggregatesfromNISTMaB
100µm
Forasphere
BrownianMoCon-MicroparCcleTrackingAnalysis
2Dt<x2>= Ddiffusionconstant
Rhydrodynamicradiusofsphere6πµR
kBTD =
2gR29µv
Δρ=
SedimentaCon:StokesLaw
Whereµ isviscoscity,gisgravitaMonconstant,visthevelocity(iniMal–finalverMcalposiMon/Mme),andRisparMcleradiusfromimageorBrowniantrajectoryanalysis(below)
<x2>ismeansquaredisplacement,calculatedfromparMcletrajectory(seeD.Ernst&J.Kohler,Phys.Chem.Chem.Phys.,2013,15,845)
Whatisthedensityofaproteinaggregate?
100µm
3mmPolystyrenebeadsinwater
100µm
3mmPolystyrenebeadsinwater,backgroundsubtracted
100µm
3mmPolystyrenebeadsinwater,withtracks
ParMclesanalyzedbycustomImageJpluginTracksdeterminedusingTrackmate
Beadtrajectories-MicroparCcletrackinganalysis
1µm 2µm
5µm 10µm
Polystyrenebeadsinwater
3µm
Results1µmbead
Results2µmbead
Results5µmbead
Results10µmbead
Beads:ImageDiametervsTrackingDiameter
MSDImage1 54.2431 66.78522 51.9681 69.55425 43.1026 41.079310 53.313 43.664
Beads:DensitybyMSD&Image(aaerdiamcorrecCon)
Beads:ImageDiametervsTrackingDiameter
MSDImage1 54.2431 66.78522 51.9681 69.55425 43.1026 41.079310 53.313 43.664
Beads:DensitybyMSD&Image
100µm
AggregatesfromNISTMaBwithtracks
ParMclesanalyzedbycustomImageJpluginTracksdeterminedusingTrackmate
DensityvsTrackingDiameter
Average
Avgdensity~1040kg/m3Aggregatedensityincreaseswithdecreasingsize
Conclusions• Improvementsinanalysiscanprovidebe7eraccuracyinparMclesizingforFlowimaging:variablethresholdpluginforImageJ/FIJI
• ParMcledensitycriMcalforrelaMngcountsfromdifferentinstruments
• MicroparMcletrackinggivesusefuldimensionsupto~5µm• ResultsbasedonsedimentaMonof1µm-7µmparMclesindicatesadensityof1040kg/m3,withdensityincreasingwithdecreasingsize.Thisdensityislowerthangenerallyassumedintheliterature.
20µm
Silicabeadsn=1.33fluid
Silicabeadsn=1.43fluid
VariableThreshold
6µm
4µm
4µm
6µm
TestofVariableThresholdMethod
5µm
Proteinaggregateatdifferentfocaldistances