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