Holographic particle characterization uses in-line holographic microscopy and the Lorenz-Mie theory of light scattering to measure the diameter and the refractive index of individual colloidal particles in their native dispersions. This wealth of information has proved invaluable in fields as diverse as soft-matter physics, biopharmaceuticals, wastewater management, and food science but so far has been available only for dispersions in transparent media. Here, we demonstrate that holographic characterization can yield precise and accurate results even when the particles of interest are dispersed in turbid media. By elucidating how multiple light scattering contributes to image formation in holographic microscopy, we establish the range conditions under which holographic characterization can reliably probe turbid samples. We validate the technique with measurements on model colloidal spheres dispersed in commercial nanoparticle slurries.

Detecting and characterizing colloidal particles dispersed in turbid media is a long-standing challenge in many application areas. Optically dense slurries of oxide nanoparticles, for example, are widely used as polishing and lapping agents for photonics and microelectronics. Large-particle contaminants (LPCs) are highly undesirable, even at part-per-billion concentrations, because of their adverse effect on surface quality.1,2 Efforts to avoid processing failures by detecting LPCs are hampered by a lack of suitable measurement techniques. Direct imaging, laser occultation, and light-scattering techniques, for example, are ruled out both by the slurries' turbidity3–5 and also by the lack of contrast between the nanoparticles and the larger contaminants.6 Conventional particle counters are clogged and fouled by slurry particles at full concentration.7 Addressing these problems by dilution is impractical both because of the large volume of fluid that then would have to be analyzed to amass a statistical sample of particle characteristics and also because dilution can influence processes that create oversized particles. Here, we demonstrate that in-line holographic video microscopy8 can identify micrometer-scale inclusions in commercial nanoparticle slurries at full concentration and yield accurate information on these impurities' size distribution and composition.

Our measurement system, depicted schematically in Fig. 1(a), illuminates the sample with the collimated beam from a diode laser operating at a vacuum wavelength of λ = 532 nm (Thorlabs, CPS532). Light scattered by a colloidal particle interferes with the remainder of the beam in the focal plane of a microscope objective lens (Nikon, CFI 40 × Plan Fluor, numerical aperture 0.75). The objective lens and its matched tube lens relay the interference pattern to a video camera (Allied Vision, Mako U130). The camera then records the pattern's intensity with an effective magnification of 120 nm/pixel on a 1280 pixel × 1024 pixel grid, which corresponds to a 154 μm × 123 μm field of view. Because the illumination is collimated, the depth of the observation volume is set by the path length of the microfluidic channel through which the sample flows.

FIG. 1.

Holographic characterization of colloidal particles in a nanoparticle slurry. (a) The slurry flows through a microfluidic channel where it is illuminated by a collimated laser. Light scattered by particles in the slurry is collected, along with the unscattered portion of the beam, by an objective lens, and is relayed by a tube lens to a video camera. (b) A normalized video frame, b ( r ) , including holograms of three silica spheres dispersed in a nanoparticle slurry. The square region of interest surrounding one holographic feature measures 15 μm on a side in the microscope's focal plane. (c) Hologram of a silica sphere from (b). (d) Fit to Eq. (10b). (e) Residuals showing interference between first-order scattered field and multiply scattered light. (f) Radial profile of fit hologram from (d) superimposed on the experimental profile from (c) to illustrate quality of fit. (g) Scatter plot of the diameter, dp, and refractive index, np, of individual colloidal particles, colored by the relative probability density ρ(dp.np).

FIG. 1.

Holographic characterization of colloidal particles in a nanoparticle slurry. (a) The slurry flows through a microfluidic channel where it is illuminated by a collimated laser. Light scattered by particles in the slurry is collected, along with the unscattered portion of the beam, by an objective lens, and is relayed by a tube lens to a video camera. (b) A normalized video frame, b ( r ) , including holograms of three silica spheres dispersed in a nanoparticle slurry. The square region of interest surrounding one holographic feature measures 15 μm on a side in the microscope's focal plane. (c) Hologram of a silica sphere from (b). (d) Fit to Eq. (10b). (e) Residuals showing interference between first-order scattered field and multiply scattered light. (f) Radial profile of fit hologram from (d) superimposed on the experimental profile from (c) to illustrate quality of fit. (g) Scatter plot of the diameter, dp, and refractive index, np, of individual colloidal particles, colored by the relative probability density ρ(dp.np).

Close modal

The camera's exposure time of 50 μs is short enough to avoid artifacts due to motion blurring9,10 at flow speeds up to 2.4 mm s−1. At the instrument's maximum flow speed of 1.5 mm s−1, the camera's recording rate of 50 frames/s is fast enough to observe and characterize each particle several times during its transit of the observation volume. We track the moving particles using a maximum likelihood algorithm11 and report trajectory-averaged values for each particle's characterization data.9 

When applied to clear samples, this instrument produces in-line holograms that can be interpreted with the Lorenz-Mie theory of light scattering12,13 to measure the size, shape, and optical properties of individual colloidal particles as they travel down the microfluidic channel.8,14–21 Here, we demonstrate that the same approach can be used to characterize micrometer-scale inclusions in turbid nanoparticle slurries.

An ideal hologram for holographic particle characterization8,9,22 results from the superposition of the collimated illumination with the light scattered out of the beam by an illuminated particle. When the particle is embedded in a nanoparticle slurry, both the illumination and the scattered wave are attenuated by multiple light scattering. The resulting diffuse wave furthermore contributes time-dependent speckle to the recorded hologram. These processes have not been considered in previous studies.

Figure 2 schematically depicts the imaging geometry. Positions are described in Cartesian coordinates relative to the center of the microscope's focal plane, which we denote as the “image plane.” We model the incident field as a monochromatic plane wave at frequency ω, propagating along z ̂ and linearly polarized along x ̂ :

E 0 ( r , t ) = u 0 ( r ) e κ 2 ( L z ) e i k z e i ω t x ̂ .
(1)

Here, u 0 ( r ) is the illumination's amplitude at position r within the observation volume, and k = 2πnm/λ is the wavenumber of light with vacuum wavelength λ = 2πc/ω propagating through a medium of refractive index nm. Scattering by nanoparticles attenuates the incident field according to the Beer-Lambert law with a penetration depth, κ−1, that depends on the concentration of slurry particles and on their light-scattering characteristics.12,23,24 In this formulation, the image plane is located at z = 0. Light enters the slurry at the top of the channel, which is located in the plane z = L. Dispersed nanoparticles influence the medium's refractive index, nm, by an amount that can be estimated with the effective medium theory.25,26 In practice, we measure nm with an Abbe refractometer (Edmund Optics).

FIG. 2.

Collimated laser light, E 0 ( r ) , illuminates a colloidal sphere at position r p within a slurry of nanoparticles. The recorded hologram of this scene results from interference between the incident wave, light scattered by the sphere, E 1 ( r ) , and the diffuse wave scattered by slurry particles, E d ( r ) .

FIG. 2.

Collimated laser light, E 0 ( r ) , illuminates a colloidal sphere at position r p within a slurry of nanoparticles. The recorded hologram of this scene results from interference between the incident wave, light scattered by the sphere, E 1 ( r ) , and the diffuse wave scattered by slurry particles, E d ( r ) .

Close modal

First-order scattering by a particle centered at r p = ( x p , y p , z p ) has the form

E 1 ( r , t ) = E 0 ( r p , t ) f s ( k ( r r p ) ) e κ 2 ( z p z ) ,
(2)

where f s ( k r ) is the Lorenz-Mie scattering function12,13,27 that describes how a sphere of diameter dp and refractive index np scatters the incident plane wave. Multiple scattering by slurry particles, not shown in Fig. 2, attenuates the first-order field by an amount that is modeled by the exponential dependence in Eq. (2).

The same scattering processes that attenuate the incident and first-order fields also create a diffuse field

E d ( r , t ) = a ( r , t ) e i ϕ ( r , t ) e i ω t ϵ ̂ ( r , t ) ,
(3)

whose amplitude, a ( r , t ) , phase, ϕ ( r , t ) , and polarization, ϵ ̂ ( r , t ) , vary randomly in position and time. Treating the diffuse scattered light as a speckle field, the joint probability density for the amplitude and phase in the imaging plane is a Rayleigh distribution28 

P ( a , ϕ ) = a π σ 2 ( L ) exp ( a 2 σ 2 ( L ) ) ,
(4)

whose standard deviation, σ(L), depends on the thickness L of the turbid medium but not on position or time. From this, the speckle field's average intensity in the image plane is

| E d ( r , t ) | 2 A = | E d ( r , t ) | 2 T = σ 2 ( L ) ,
(5)

where the subscripts A and T refer to averages over the field of view in a single snapshot and over time at a particular location, respectively.

If no particles are in the field of view, the instantaneous intensity in the image plane is I 0 ( r , t ) = | E 0 ( r , t ) E d ( r , t ) | 2 . Its time average

(6)
I 0 ( r ) | E 0 ( r , t ) | 2 T + | E d ( r , t ) | 2 T ,
(6a)
= u 0 2 ( r ) e κ L + σ 2 ( L ) ,
(6b)
constitutes the background on which holograms of particles are superposed. In practice, we compute the background as a running average over T = 0.4 s. This window substantially exceeds the longest correlation time in a speckle field, which arises from single scattering by the slurry particles and typically is measured in milliseconds.

The hologram of a colloidal particle at position r p ( t ) within this light field has the form

(7)
I ( r ) = | E 0 ( r , t ) + E 1 ( r , t ) + E d ( r , t ) | 2 ,
(7a)
u 0 2 ( r ) e κ L | x ̂ + e i k z p f s ( k ( r r p ) ) | 2 + σ 2 ( L ) .
(7b)
Equation (7b) follows from Eq. (7a) in the approximation u 0 ( r p ) u 0 ( r ) , which has been justified in previous studies.8,22 It omits interference between coherent and diffuse waves, whose time average should vanish. It also neglects spatial variations in the speckle field, which we treat as noise in the fitting process.

Because σ2(L) is the variance of the speckle field's amplitude, the additive offset in Eqs. (6b) and (7b) also is an estimate for speckle's contribution to the noise in a recorded hologram. The slurry's principal influence, therefore, is to suppress the hologram's signal-to-noise ratio, s = u 0 2 ( r ) e κ L / σ 2 ( L ) . Requiring s ≫ 1 for successful particle detection and analysis establishes whether or not a turbid medium is amenable to holographic particle characterization. The noise level can be obtained from the spatial variance of the background intensity

(8)
Δ 2 = | I 0 ( r , t ) I 0 ( r ) | 2 A , T ,
(8a)
= σ 2 ( L ) [ 2 u 0 2 ( r ) A e κ L + σ 2 ( L ) ] ,
(8b)
which yields

σ 2 ( L ) = I 0 ( r ) A [ 1 1 Δ 2 I 0 ( r ) A 2 ] .
(8c)

The signal-to-noise ratio for holographic imaging in a turbid medium therefore can be estimated entirely from internal information as

s = I 0 ( r ) A σ 2 ( L ) 1.
(9)

The foregoing analysis suggests that any influence of the turbid medium on a recorded hologram can be mitigated by subtracting the diffuse contribution and normalizing by the background8,22

(10)
b ( r ) I ( r ) σ 2 ( L ) I 0 ( r ) σ 2 ( L ) .
(10a)
The image in Fig. 1(b) was normalized in this way after taking care to subtract the camera's measured dark count from both the recorded intensity pattern and the background. Referring to Eq. (7b), a normalized hologram can be modeled as

b ( r ) | x ̂ + e i k z p f s ( k ( r r p ) ) | 2 ,
(10b)

which does not depend on properties of the slurry beyond its overall refractive index.

We test this result by using Eqs. (8) and (10) to measure the properties of well-characterized colloidal silica spheres (Bangs Laboratories, Catalog Number SS04N, Lot Number 5303) dispersed in an undiluted commercial slurry of 70 nm-diameter silica particles at 27% volume fraction (Eminess Technologies, Ultra-Sol 2EX). The slurry's measured refractive index is nm = 1.36 ± 0.01, which is nearly 3% higher than that of plain water. It appears opaque white and has a measured turbidity of 647 NTU. Its measured penetration depth29 at the imaging wavelength is κ−1 ≈ 1 mm, which substantially exceeds the optical path length L = 25 μm in our microfluidic channel. With the laser intensity adjusted to provide a mean background of I 0 ( r ) = 80 on the camera's eight-bit range, the measured diffuse offset is σ2(L) = 0.1, which corresponds to a signal-to-noise ratio of s = 800. Indeed, the micrometer-scale silica spheres contribute distinct bulls-eye features to the normalized hologram in Fig. 1(b). We detect these features using an image-analysis filter that emphasizes centers of rotational symmetry30 and then select pixels from the surrounding region for analysis. Figure 1(c) shows a typical feature detected in this way.

The image in Fig. 1(d) results from the nonlinear least-squares fit of Eq. (7b) to the data in Fig. 1(c). The difference between the measured and fit holograms is plotted in Fig. 1(e). The particle's diameter obtained from this fit, dp = 1.498 ± 0.012 μm, is consistent with the manufacturer's specification; the refractive index, np = 1.424 ± 0.005, is consistent with values obtained for the same sample of silica spheres dispersed in pure water. The quality of the fit may be judged from the radial profile, plotted in Fig. 1(f), which tracks the experimental data to within measurement noise, which is indicated by the shaded region.

Figure 1(g) shows a compilation of 875 single-particle measurements of the radii and refractive indexes of silica spheres from the same monodisperse sample. Each data point in the plot reflects the measured properties of a single sphere and is colored by the relative probability ρ(dp, np) of measurements in the (dp, np) plane. The population-averaged diameter, dp = 1.50 ± 0.01 μm, agrees with the manufacturer's specification. The mean refractive index, np = 1.425 ± 0.010, is slightly smaller than the value 1.485 for bulk silica at the imaging wavelength31 and is consistent with the spheres having a 3% porosity, as has been discussed elsewhere.14 The data for this plot were acquired in 10 min.

Figure 3 presents a more stringent test. The data in Fig. 3(a) show characterization results obtained with Eqs. (8) and (10) for a mixture of four different types of colloidal spheres, all dispersed in the same silica nanoparticle slurry. The individual populations of colloidal spheres were obtained from Bangs Laboratories and include polystyrene spheres with dp = 2.5 μm (Catalog No. NT18N, lot 10497), polystyrene spheres with dp = 1.5 μm (Catalog No. NT16N, lot 12035), silica spheres with dp = 2.2 μm (Catalog No. SS04N, lot 6911), and silica spheres with dp = 1.4 μm (Catalog No. SS04N, lot 5303). Particles were analyzed in random order as the slurry flowed down the channel. The four clusters of data points in Fig. 3(a) demonstrate successful differentiation of silica spheres with a refractive index of np = 1.43 from polystyrene spheres with a refractive index of np = 1.60. Smaller silica spheres with nominal diameters of dp = 1.4 μm also are clearly resolved from larger spheres with dp = 2.2 μm. The polystyrene spheres similarly are clearly distinguished into two groups with diameters of 1.4 μm and 2.5 μm.

FIG. 3.

(a) Particle-resolved characterization data for a mixture of four types of colloidal spheres dispersed in Ultra-Sol 2EX nanoparticle slurry. Each of the 1413 data point represents the diameter, dp, and refractive index, np, of a single particle. The four populations are clearly distinguishable as clusters of data points. The number of particles in each cluster is indicated. (b) One-, two-, and three-standard-deviation confidence intervals for the data in cluster A superimposed on reference results obtained for the same particles dispersed in water. Plot symbols in (a) and (b) show reference values for the four populations measured in water.

FIG. 3.

(a) Particle-resolved characterization data for a mixture of four types of colloidal spheres dispersed in Ultra-Sol 2EX nanoparticle slurry. Each of the 1413 data point represents the diameter, dp, and refractive index, np, of a single particle. The four populations are clearly distinguishable as clusters of data points. The number of particles in each cluster is indicated. (b) One-, two-, and three-standard-deviation confidence intervals for the data in cluster A superimposed on reference results obtained for the same particles dispersed in water. Plot symbols in (a) and (b) show reference values for the four populations measured in water.

Close modal

Figure 3(b) presents the data from cluster A as nested confidence intervals, with levels at one, two, and three standard deviations. The confidence intervals were computed using robust estimation of the covariance. This representation permits easy comparison with results for the same types of colloidal particles dispersed in water, which also are plotted in Fig. 3(b). Quantitative results for the population-averaged diameters and refractive indexes are tabulated in Table I. In all four cases, the results obtained in slurry agree well with reference values obtained in plain water. Similarly, good results are obtained in channels with L = 50 μm (s = 260) and L = 100 μm (s = 110).

TABLE I.

Holographic characterization results for four different types of colloidal spheres dispersed in water and in Ultra-Sol 2EX slurry for L = 25 μm. Colloidal spheres were obtained from Bangs Laboratories.

Material dp (μm) np
A: Polystyrenea  Water  1.47 ± 0.02  1.600 ± 0.007 
  Slurry  1.44 ± 0.03  1.600 ± 0.008 
B: Polystyreneb  Water  2.50 ± 0.04  1.600 ± 0.002 
  Slurry  2.50 ± 0.03  1.596 ± 0.002 
C: Silicac  Water  1.43 ± 0.05  1.428 ± 0.004 
  Slurry  1.41 ± 0.06  1.434 ± 0.003 
D: Silicad  Water  2.27 ± 0.04  1.428 ± 0.002 
  Slurry  2.22 ± 0.05  1.436 ± 0.002 
Material dp (μm) np
A: Polystyrenea  Water  1.47 ± 0.02  1.600 ± 0.007 
  Slurry  1.44 ± 0.03  1.600 ± 0.008 
B: Polystyreneb  Water  2.50 ± 0.04  1.600 ± 0.002 
  Slurry  2.50 ± 0.03  1.596 ± 0.002 
C: Silicac  Water  1.43 ± 0.05  1.428 ± 0.004 
  Slurry  1.41 ± 0.06  1.434 ± 0.003 
D: Silicad  Water  2.27 ± 0.04  1.428 ± 0.002 
  Slurry  2.22 ± 0.05  1.436 ± 0.002 
a

Catalog No. NT16N (lot 12035).

b

Catalog No. NT18N (lot 10497).

c

Catalog No. SS04N (lot 5303).

d

Catalog No. SS04N (lot 6911).

These results demonstrate that holographic characterization can detect, characterize, differentiate, and count micrometer-scale colloidal particles even when they are dispersed in turbid nanoparticle slurries. The ability of holographic microscopy to distinguish particles of interest from slurry particles relies on a separation of length scales. The nanoparticles in our system are so much smaller than the wavelength of light that they cannot be detected individually in holographic images. They also diffuse so much more rapidly than the larger colloidal spheres that their collective contribution to the light scattering pattern can be averaged without blurring the spheres' holograms. Multiple light scattering by the nanoparticles attenuates the larger particles' scattering patterns and also contributes an additive offset that can be estimated by analyzing background intensity fluctuations. The first-order scattering pattern is superimposed on this background. Under conditions where the holograms' signal-to-noise ratio is not unduly degraded, a particle's scattering pattern may be analyzed using the Lorenz-Mie theory of light scattering. Whether or not a particular slurry is amenable to holographic characterization can be assessed from internal evidence or by performing validation measurements with standard particles. The representative measurements presented in Figs. 1 and 3 confirm that characterization data obtained in turbid media can be comparably precise and accurate to those obtained for the same particles in transparent media. These considerations also account for the previously reported32 success of holographic particle tracking in non-ideal environments such as gelled media.

The ability to characterize colloidal particles dispersed in turbid media creates new opportunities for applications. For example, holographic characterization should be useful for detecting and characterizing micrometer-scale impurities in CMP slurries.1,2 Such real-world applications will benefit from the demonstrated ability of holographic characterization to characterize irregularly shaped objects17–19 and will be discussed elsewhere.

This work was supported primarily by the National Science Foundation through SBIR Award No. IPP-1519057 and in part by NSF Award No. DMR-1305875. We are grateful to Professor Gi-Ra Yi and Professor David Pine for helpful conversations.

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