The ViewSizer® 3000 offers the ability to visualize nanoparticle colloids without requiring calibration standards or knowledge of any particle material properties, such as refractive index. It was developed by MANTA – the Most Advanced Nanoparticle Tracking Analysis – and offers the user an unprecedented ability to count and size highly polydispersed samples, such as milk, sea water, or blood plasma. View recorded webinars: http://bit.ly/particlewebinars

1. Improved Visualization, Counting and Sizing
of Polydisperse Nanoparticle Colloids
using ViewSizer® 3000
Jan “Kuba” Tatarkiewicz, PhD
VP Engineering
MANTA Instruments, Inc.

2. Established technologies
• Flow Cytometry (FC)
• Transmission Electron Microscopy (TEM)
• Static Light Scattering (SLS)
• Dynamic Light Scattering (DLS)
• conventional Nanoparticle Tracking Analysis (cNTA)

3. Unmet needs
• Accurate & reproducible measurement of:
 particle number concentration
 particle size distribution
 particle kinetic processes
• and visualization of highly polydispersed colloids

4. Visualization of Brownian motion
microscope + camera
light sheet
scattered light
light sheet thickness
light source
investigated volume

5. Problem
>6 orders of
magnitude in
scattered light
intensity (Mie)
445 nm laser on polystyrene beads in water
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
Diameter [nm]
Scatteringcross-section[nm2]

6. Six orders of magnitude
• DLS – large particles skew results
(small ones not detected)
• DLS – experimental complications that users overlook
(concentration-dependent results)
• cNTA – different sized particles can’t be seen simultaneously
(highly irregular images for large particles, dim for smallest)
• cNTA – interrogated volume depends on particles sizes and their
refractive indices (similar to FC problem when sizing)

7. Problem is well known
“Sample polydispersity affects the ability to track and therefore analyze
different size fractions in the particle number-size distribution. […]
In a polydisperse sample large particles scatter a lot more than small
particles making it difficult to detect or track small size particles.”

8. 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
Diameter [nm]
Scatteringcross-section[nm2]
MANTA solution US patent 9645070
(Multispectral Advanced Nanoparticle Tracking Analysis)

9. Sample of three color video

10. Size determination (Einstein 1905, Langevin 1908)
• Mean Squared Distance (MSD in 2D, N frames, jumps of n):
• Diffusion coefficient D (optimized least-square fit of MSD vs. n):
• Hence diameter:
MSD(n) =
1
N -n
(xi+n
i=1
N-n
å - xi )2
+(yi+n - yi )2
MSD n( )= 4·Dt·D( )·n
𝑑 =
𝑘 𝐵 𝑇
3𝜋𝜂𝐷

11. Light sheet thickness
Intensity
Thickness
“Top hat”
Gaussian

12. Concentration (counts per volume)
• Observed volume depends on intensity of scattered light
• Calibration of interrogated volume is done using standards
(various sizes and refractive indices) → lookup table
• Volume factor is calculated from average intensity of
scattered light for each tracked particle (takes laser power,
camera exposure & gain into account)
• Density of particle size distribution (PSD) is calculated with
variable volume factor for each size bin

13. Volume factor US patent 9857283
Volume
Diameter
Intensity
d0
I0
V0
Lookup surface
0
1
2
3
4
5
6
7
0
200
400
600
800
1000
0
200
400
600
800
1000
1200
1400
1600
Volume[nL]
Diameter [nm]
Mie
 [nm
2 ]
Silica
Polystyrene
Silver

14. ViewSizer® 3000
cuvette w/insert
US patent 9541490

15. *Sample material dependent
Specifications
Range of particle sizes measured* 10 nm to 15 µm
Minimum sample volume 0.4 mL
Typical sample concentration 5 x 106 to 1 x 108 particles/mL
Sample temperature range (controlled) 10 °C to 50 °C, ± 0.1 °C (-15 °C to 110 °C available)
Dimensions 55 cm W x 66 cm D x 35 cm H
Weight 27 kg
Operational Environment 15 °C to 30 °C with < 85% RH

16. NIST exploratory poly-standard
Diameter [nm]
50 100 200 500 1000
DensityofPSD[counts/mL/nm]
1.0e+3
1.0e+4
1.0e+5
1.0e+6
1.0e+7

17. Gold mixes: DLS vs. MANTA

18. 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]
DensityofPSD[counts/mL/nm]
cNTA
MANTA
3x DLS
TEM

19. Whole vs. fat-free milk
DA,B alpha DA,B,α Reject?
0.2335 0.050 0.0338 yes
Diameter [nm]
0 200 400 600 800 1000
Cumulative
0.0
0.2
0.4
0.6
0.8
1.0
whole milk
fat-free milk
How to compare two distributions
with unknown shapes (no theory)?
Use the so called nonparametric tests
like Kolmogorov-Smirnow statistics:
dav=256 nm, SD=145 nm, CV=0.57
dav=163 nm, SD=68 nm, CV=0.42

20. Micellisation of a polymer
If number of particles retained, diameter/volume increases -> growth by adding polymer
Kinematykapolimeryzacji miceli – współpraca dr. Nia Bell, UCSD
5 min 45 min25 min
DensityofPSD[counts/mL/nm]

21. Neat proteins
Sample 1 Sample 2

22. Viscosity of proteins
𝜂 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 =
𝑑𝑖𝑛 𝑝𝑟𝑜𝑡𝑒𝑖𝑛
𝑑𝑖𝑛 𝑤𝑎𝑡𝑒𝑟
∗ 𝜂 𝑤𝑎𝑡𝑒𝑟
203 nm PSL in water and proteins
Diameter [nm]
0 500 1000 1500 2000
DensityofPSD[counts/mL/nm]
0.0
1.0e+5
2.0e+5
3.0e+5
4.0e+5
5.0e+5
6.0e+5
7.0e+5
in water
in protein 1
in protein 2

23. Neat proteins PSD
Instrument Info
Serial Number=007
Calibration Constant=225.6 [nm/pixel]
Software Version=1.5.0.4016,
1.0.5 WeekBuild 3716
Interrogation Volume=2.5E-06 [mL]
Sample Info
Sample ID=Protein1
Sample Description=neat
Sample 1 Sample 2
Report something from fridge
Measurement date: 9/15/2017
Measurement time: 13:49:01
Measurement type: NTA
Operator: JB
Instrument information
Serial number: 023
Software version: 1.8.0.2517,
1.0.7 WeekBuild 2317
Calibration constant: 195.79 [nm/pixel]

24. Lysozyme heated to 60 °C
before after

25. Fluorescence
0.0E+00
5.0E+04
1.0E+05
1.5E+05
2.0E+05
2.5E+05
0 100 200 300 400 500 600 700 800 900 1,000
DensityofPSD[counts/mL/nm]
Diameter [nm]
Gravimetric mix:
B 44%
G 33%
R 23%
FL measured:
B 36%
G 49%
R 15%
Mix of three types of carboxylate fluorescent beads (all nominally 500 nm diameter, stained with Fluoresbrite®)
MANTA

26. Vesicles stained with fluorophore
Fluorophore DilC18(3) Ex=520÷551 nm, Em=569÷620 nm
1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindocarbocyanine-5,5'-Disulfonic Acid
0.00E+00
5.00E+05
1.00E+06
1.50E+06
2.00E+06
2.50E+06
3.00E+06
3.50E+06
0 200 400 600 800 1000
DensityofPSD[counts/mL/nm] Diameter [nm]
MANTA
FL

27. Vesicles processed differently
DA,B alpha DA,B,α Reject?
0.0647 0.050 0.0297 yes
Vesicles stained with DilC18(3)
Diameter [nm]
0 200 400 600 800
Cumulative
0.0
0.2
0.4
0.6
0.8
1.0
MANTA
FL dav=278 nm, SD=166 nm, CV=0.60
dav=264 nm, SD=169 nm, CV=0.64

28. Nanoparticles dissolution rate

29. Nanoparticles dissolution rate II
0.00
0.20
0.40
0.60
0.80
1.00
1.20
0:00:00 1:12:00 2:24:00
#particles
0.00
0.20
0.40
0.60
0.80
1.00
1.20
0:00:00 1:12:00 2:24:00
density
0.00
0.20
0.40
0.60
0.80
1.00
1.20
0 50 100 150 200
Relativeintensity
Time [minutes]

30. Settling rate for large particles
𝑑 =
18 ∗ 𝑣 ∗ 𝜂
𝑔 ∗ 𝜌 − 𝜌0

31. Protein crystallization rate
0
5
10
15
20
25
30
35
40
0 50 100 150 200 250 300 350 400 450 500
Intensity[a.u.]
Time [minutes]
Brownian sizing
Sinking sizing
PBS 2
PBS 1
Crystallization
Dissolution
Wittbold & Tatarkiewicz 2017, BioProcess International 15 (3)

32. polystyrene, also with PEG coating, silica, silver, gold, 316L stainless,
sand/dirt, clay, CaO, YAG, SiO2, carbon, PMMA, LiMnO
sea water, fresh water, rain water, tap water, acetone, wine, urine,
blood plasma, milk, ammonia, jet A-1 fuel
small molecule APIs, protein aggregates, silicon oil, protein crystals,
liposomes, exosomes, vesicles, micelles, α-lactalbumin, rolled DNA,
RNA, viruses, bacteriophages, emulsions, polymeric API carriers,
self-adjuvanted proteins
Successfully tested samples

33. Key benefits of MANTA
 Individual particle method, not ensemble average
 Accurate density of PSD for polydisperse samples
 Concentration measured, not estimated
 Absolute method (no calibration w/standards needed)
 Particles and processes visualization

34. Other benefits
 Kinetic processes (time constants)
 Temperature range and ramp rates
 Real time agitation
 Real time reagent addition
 Multiple tests w/o changing sample

35. Customers love ViewSizer® 3000
“I can’t do my
research without
MANTA”

36. Thank you
Jan “Kuba” Tatarkiewicz PhD
VP Engineering
MANTA Instruments, Inc.