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What does it take to accurately measureconcentration of nanoparticles in colloids
Jan “Kuba” Tatarkiewicz, PhDVP Engineering
MANTA Instruments, Inc.
Beginnings
NASA sponsored ICESCAPE Projecttwo cruises on USCGC Healy
2010 - 2011
Open circles: Stations from 2011 cruise with NanoSight data
-50
-50
-50
-50
-500
-2000
-2000
8
15
1836
34
5
333213
ALASKASIBERIA
CHUKCHISEA
BEAUFORTSEA
Results
Fig. 2. Average calibration function determined Particle diameter d [ µm ]
0.0 0.2 0.4 0.6 0.8
Cal
ibra
tion
func
tion
0
1
2
3 power function-like PSD of mixturenearly flat PSD of mixturefitted calibration function
Final multiplicative cali-bration function (Fig. 2) determined by averaging data from different expe-
Particle diameter d [ µm ]
This is a very strange function...
•
Particle diameter d [ µm ]
0.1 1 10 100
Den
sity
of P
SD
[ c
m-3µm
-1 ]
10-1
102
105
108
CoulterNanoSight
2 m, station #15
27 m, station #15
147 m, station #15
J. J. Tatarkiewicz, R. A. Reynolds, and D. Stramski Counting and sizingof colloidal particles in the Arctic Ocean 2012 Ocean Sci Meeting A0412
2nd generation NTA
• Multispectral Advanced Nanoparticle Tracking Analysis
• NSF grant for MRI #1126870, 2012-2014
• MANTA Instruments, Inc. founded in 2014
• US patents granted up to now:
• 9541490, 9645070, 9857283, 9909972
Visualization
microscope + camera
light sheet
scattered light
light sheet thickness
light source
investigated volume
1.0E-05
1.0E-04
1.0E-03
1.0E-02
1.0E-01
1.0E+00
1.0E+01
1.0E+02
1.0E+03
1.0E+04
10 100 1,000
Scat
terin
g cr
oss-
sect
ion
[nm
²]
Diameter [nm]
MANTA
Mie calculations for 445 nm, 520 nm and 635 nm polarized laser beams scattering on PSL in water (n=1.337), objective NA=0.28, 80°÷100° integration
MSD(n) = 1N − n
(xi+ni=1
N−n
∑ − xi )2 + (yi+n − yi )
2
MSD n( ) = 4·Δt·D( )·n
!" =$%&3()*
Sizes• Mean Squared Distance MSD (2D, N frames track, n frames lag*):
• Diffusion coefficient D (least-squares fit of MSD as a function of n):
• Hence hydrodynamic diameter:
* ergodicity: assembly average ≡ time average
Statistics• Cramér-Rao statistics decides length of each track used for
optimal MSD fitting• X. Michalet and A.J. Berglund Optimal diffusion coefficient
estimation in SPT, Phys Rev E85, 061916 (2012)
• Binning diameters with different schemes (like equal or logarithmic widths) into density of particle-size distribution (PSD) with variable investigated volume (explained later)
• Statistical parameters of PSD (average size, standard deviation)
Mode?
Report ABT-308_after_30min+Measurement date: 2/16/2018Measurement time: 10:10:12Measurement type: NTAOperator: QT
Instrument informationSerial number: 024Software version: 1.8.0.3417,1.0.7 WeekBuild 2617Calibration constant: 195.74 [nm/pixel]
Sample informationSample ID: ABT-308_after_30min+Sample description: Vial 5, 350 rpm between videosDiluent: Custom 2.7 [cP]Diluent impurities concentration: 0 [particles/mL]Dilution: 1 [times]
Processing informationDetection threshold type: PolydisperseDetection threshold: 2AutoThreshold: EnabledFeature radius: 35 [pixels]Tracking starts on: 1 [frame]Drift correction: 0 [%]
Recording informationFrame rate: 30 [fps]Exposure: 15 [ms]Gain: 26 [dB]Stir time: 2 [seconds]Wait time: 1 [seconds]Laser power:Blue: 0 [mW]Green: 16 [mW]Red: 12 [mW]Frames per video: 300Videos recorded: 25
ResultsIntegrated from 30 nm to 1900 nmConcentration (raw): 6.944E+06 [particles/mL]Concentration w/o impurities: 6.944E+06 [particles/mL]Total counts: 434Average size: 387 nmSD of size: 304 nmCV of size: 0.78D10: 87 nmD50: 322 nmD90: 806 nm
Report ABT-308_after_30min+Measurement date: 2/16/2018Measurement time: 10:10:12Measurement type: NTAOperator: QT
Instrument informationSerial number: 024Software version: 1.8.0.3417,1.0.7 WeekBuild 2617Calibration constant: 195.74 [nm/pixel]
Sample informationSample ID: ABT-308_after_30min+Sample description: Vial 5, 350 rpm between videosDiluent: Custom 2.7 [cP]Diluent impurities concentration: 0 [particles/mL]Dilution: 1 [times]
Processing informationDetection threshold type: PolydisperseDetection threshold: 2AutoThreshold: EnabledFeature radius: 35 [pixels]Tracking starts on: 1 [frame]Drift correction: 0 [%]
Recording informationFrame rate: 30 [fps]Exposure: 15 [ms]Gain: 26 [dB]Stir time: 2 [seconds]Wait time: 1 [seconds]Laser power:Blue: 0 [mW]Green: 16 [mW]Red: 12 [mW]Frames per video: 300Videos recorded: 25
ResultsIntegrated from 30 nm to 1900 nmConcentration (raw): 6.88E+06 [particles/mL]Concentration w/o impurities: 6.88E+06 [particles/mL]Total counts: 430Average size: 391 nmSD of size: 303 nmCV of size: 0.77D10: 90 nmD50: 310 nmD90: 810 nm
Report ABT-308_after_30min+Measurement date: 2/16/2018Measurement time: 10:10:12Measurement type: NTAOperator: QT
Instrument informationSerial number: 024Software version: 1.8.0.3417,1.0.7 WeekBuild 2617Calibration constant: 195.74 [nm/pixel]
Sample informationSample ID: ABT-308_after_30min+Sample description: Vial 5, 350 rpm between videosDiluent: Custom 2.7 [cP]Diluent impurities concentration: 0 [particles/mL]Dilution: 1 [times]
Processing informationDetection threshold type: PolydisperseDetection threshold: 2AutoThreshold: EnabledFeature radius: 35 [pixels]Tracking starts on: 1 [frame]Drift correction: 0 [%]
Recording informationFrame rate: 30 [fps]Exposure: 15 [ms]Gain: 26 [dB]Stir time: 2 [seconds]Wait time: 1 [seconds]Laser power:Blue: 0 [mW]Green: 16 [mW]Red: 12 [mW]Frames per video: 300Videos recorded: 25
ResultsIntegrated from 30 nm to 1900 nmConcentration (raw): 6.992E+06 [particles/mL]Concentration w/o impurities: 6.992E+06 [particles/mL]Total counts: 437Average size: 386 nmSD of size: 304 nmCV of size: 0.79D10: 84 nmD50: 324 nmD90: 797 nm
Report ABT-308_after_30min+Measurement date: 2/16/2018Measurement time: 10:10:12Measurement type: NTAOperator: QT
Instrument informationSerial number: 024Software version: 1.8.0.3417,1.0.7 WeekBuild 2617Calibration constant: 195.74 [nm/pixel]
Sample informationSample ID: ABT-308_after_30min+Sample description: Vial 5, 350 rpm between videosDiluent: Custom 2.7 [cP]Diluent impurities concentration: 0 [particles/mL]Dilution: 1 [times]
Processing informationDetection threshold type: PolydisperseDetection threshold: 2AutoThreshold: EnabledFeature radius: 35 [pixels]Tracking starts on: 1 [frame]Drift correction: 0 [%]
Recording informationFrame rate: 30 [fps]Exposure: 15 [ms]Gain: 26 [dB]Stir time: 2 [seconds]Wait time: 1 [seconds]Laser power:Blue: 0 [mW]Green: 16 [mW]Red: 12 [mW]Frames per video: 300Videos recorded: 25
ResultsIntegrated from 30 nm to 1900 nmConcentration (raw): 6.992E+06 [particles/mL]Concentration w/o impurities: 6.992E+06 [particles/mL]Total counts: 437Average size: 385 nmSD of size: 304 nmCV of size: 0.79D10: 86 nmD50: 318 nmD90: 784 nm
narrow equal bins wide equal bins logarithmic bins variable bins
Counts• 25 (or more) short videos (300 frames each) recorded*
• Track and count particles detected on 1st frame of each video
• Mixing sample between videos to get different aliquots (magnetic stirrer)
• external fluidics for magnetic materials and low concentration samples – do not use sample flow during recording
• Proper PSD binning (bin widths) for polydispersity
* typically between 100 and 150 particles tracked per video
Volume
How to calibrate volume:• Measure concentrations for standards
of different sizes and made out of different materials (various RIs)
• Determine effective volumes• Create look-up surface of volumes• Extrapolate by using intensity of
individual tracks and applying Mie scattering cross-section formula
Volume
Diameter
Intensity
d
I
V
Lookup surface
! = #$%&'()%*#%+,%-.%*
!/
Histogram
Concentration from 50 nm to 700 nm = area of density of PSD histogram
Dens
ity o
f PSD !" =$ 1
&" ∙ ())
*+, =$!",
"-+∙ &" = $$ 1
())
,
"-+
Concentration• Integrate density of PSD (counts/mL/nm) across sizes of interest,
for example from 50 to 700 nm, to get concentration (counts/mL)
• Instruments are calibrated for optics scaling (nm/pix) and for laser(s) power (mW) (manufacturing variability of active elements)
• For unknown materials, extrapolate investigated volumes by using Mie scattering cross-sections of known test materials
• Use measured data with statistically significant number of counts, do not use fitted distributions (PSD is not an invariant)
NIST exploratory mix
N = 1.02E+13*d -2.07
1.0E+06
1.0E+07
1.0E+08
1.0E+09
1.0E+10
100.0 1000.0
Conc
entra
tion
[cou
nts/
mL]
Diameter [nm]Diameter [nm]
50 100 200 500 1000
Den
sity
of P
SD [c
ount
s/m
L/nm
]
1.0e+3
1.0e+4
1.0e+5
1.0e+6
1.0e+7
!~ 1$%
TEM, DLS & NTA vs. MANTA
α-lactalbumin nanoparticles made as per Arroyo-Maya et al. J Dairy Sci 95, 6204 (2012)
Testowaniecd.
α-lactalbumin nanoparticlesTEMZdjęciedr.NiaBell,UCSD Particle diameter d [ nm ]
0 100 200 300 400 500 600 700 800
Par
ticl
e co
nce
ntr
atio
n N
[ c
m-3
nm
-1 ]
107
108
109
1010
α-lactalbumin nanoparticles
NanoSight
TEM MANTA
DLS
TEM, DLS & cNTA vs. MANTA
α-lactalbumin nanoparticles made as per Arroyo-Maya et al. J. Dairy Sci. (2012) 95, 6204-6214
200 300100 500 600400 800700
107
108
109
1010
0
Particle diameter [nm]De
nsity
of P
SD [c
ount
s/m
L/nm
] NTAMANTA
3x DLS
TEM
Testowaniecd.
α-lactalbumin nanoparticlesTEMZdjęciedr.NiaBell,UCSD Particle diameter d [ nm ]
0 100 200 300 400 500 600 700 800
Par
ticl
e co
nce
ntr
atio
n N
[ c
m-3
nm
-1 ]
107
108
109
1010
α-lactalbumin nanoparticles
NanoSight
TEM MANTA
DLS
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