**Sang-Hyuk Lee [1], Yohai Roichman [1], Gi-Ra Yi [2],
Shin-Hyun Kim [3], Seung-Man Yang [3], Alfons van Blaaderen [4]
Peter van Oostrum [4] and David G. Grier [1]**

[1] *Department of Physics and Center for Soft Matter
Research, New York University, New York, NY 10003*

[2] *Korea Basic Research Institute, Seoul 136-713, Korea*

[3] *National Creative Research Initiative Center for
Integrated Optofluidic Systems and Department of Chemical and
Biomolecular Engineering, Korea Advanced Institute of
Science and Technology, Daejeon, 307-701 Korea*

[4] *Soft Condensed Matter, Debye Institute, Utrecht
University, 3508 TA Utrecht, The Netherlands*

**Date:** September 27, 2007

We use digital holographic microscopy
and Mie scattering theory to
simultaneously characterize and track individual colloidal particles.
Each holographic snapshot provides enough information
to measure a colloidal sphere's
radius and refractive index to within 1%,
and simultaneously to measure its three-dimensional
position with nanometer in-plane precision and 10 nanometer
axial resolution.

In addition to their ubiquity in
natural and industrial processes,
colloidal particles
have come to be prized as building blocks
for photonic and optoelectronic devices,
as probes for biological and macromolecular processes,
and as model systems for fundamental studies of many-body
physics.
Many of these existing and emerging applications
would benefit from more effective methods for tracking colloidal
particles' motions in three dimensions.
Others require better ways to measure particles' sizes
and to characterize their optical properties, particularly
if these measurements
can be performed on individual particles *in situ*.

This Article demonstrates that images obtained with in-line holographic microscopy (2,1) can be interpreted with Lorenz-Mie theory (3,4) to obtain exceptionally precise measurements of individual colloidal spheres' dimensions and optical properties (5,6) while simultaneously tracking their three dimensional motions with nanometer-scale spatial resolution at video rates (7). This method works over the entire range of particle sizes and compositions for which Mie scattering theory applies, and requires only a single calibration of the optical train's magnification. Unlike other light scattering techniques for measuring particle size (8) or refractive index, holographic particle analysis can be applied directly to individual particles in heterogeneous samples and also is compatible (2) with scanned (9) and holographic (10) optical trapping.

Analyzing these digitized holograms yields the particle's three-dimensional position, , its radius, , and its index of refraction, . We assume that the incident field, , is uniformly polarized in the direction and varies slowly enough over the size of the particle to be treated as a plane wave propagating along the direction. Its amplitude at position in the plane of the particle is thus the same as its amplitude in the focal plane, . The wave propagates along the direction with wave number , where is the light's wavelength in vacuum and is the refractive index of the medium. For pure water at , at .

The particle at scatters a portion of the incident field into a highly structured outgoing wave, , where accounts for variations in the illumination, and where is the Lorenz-Mie scattering function (11,3,4), which depends on , , and . The scattered field generally covers a large enough area at the focal plane that the interference pattern,

is dominated by long-wavelength variations in . The resulting distortions have been characterized (12), but were not corrected in previous analyses of (5,6,13,12,7). Fortunately, can be measured in an empty field of view, and the in-line hologram can be normalized to obtain the undistorted image

on the plane . If we further assume that the phase of the collimated incident beam varies slowly over the field of view, the normalized image is related to the calculated Mie scattering pattern, , in the plane by

Equation (4) can be fit to measured holograms by treating the particle's three-dimensional position, its radius and its refractive index as free parameters. Previous studies fit non-normalized holograms to phenomenological models (17,16,15,6,13,14) or Mie scattering theory (18) for some of these quantities, but never all five. Because errors in the adjustable parameters are strongly correlated, failing to optimize them all simultaneously yields inaccurate results. Fitting instead to the full Lorenz-Mie theory (11,3,4,12,20,19) provides more information with greater precision.

Numerical fits to digitized and normalized holographic images were performed with the Levenberg-Marquardt nonlinear least-squares minimization algorithm (21,23,22) using the camera's measured signal-to-noise ratio to estimate single-pixel errors. The deviates for all of the fits we report are of order unity, so that the calculated uncertainties in the fit parameters accurately reflect their precision (21).

Because the laser's wavelength and the medium's refractive index are both known, the only instrumental calibration is the overall magnification. This contrasts with other three-dimensional particle tracking techniques (25,26,13,24,2,1), which require independent calibrations for each type of particle, particularly to track particles in depth.

The image in Fig. 2(a) shows the normalized hologram, , for a polystyrene sulfate sphere dispersed in water at height above the focal plane. This sphere was obtained from a commercial sample with a nominal diameter of (Bangs Labs, Lot PS04N/6064). The camera's electronic shutter was set for an exposure time of 0.25 msec to minimize blurring due to Brownian motion (27). After normalizing the raw 8-bit digitized images, each pixel contains roughly 5 significant bits of information. The numerical fit to faithfully reproduces not just the position of the interference fringes, but also their magnitudes. The quality of the fit may be judged from the azimuthal average; the solid curve is an angular average about the center of , the dashed curves indicate the standard deviations of the average, and the discrete points are obtained from the fit.

The fit value for the radius, , falls in the sample's specified range, which reflected a lower bound of obtained with a Beckman Z2 Coulter Counter and an upper bound of obtained by analytical centrifugation. Agreement between the quoted and measured particle size suggests that the present measurement's accuracy is comparable to its precision. In that case, both precision and accuracy surpass results previously obtained (6) through analysis of . The trajectory-averaged value for the refractive index, , also is consistent with the properties of polystyrene colloid inferred from light scattering measurements on bulk dispersions (28).

Comparable precision in measuring a single particle's refractive index has been achieved by analyzing a colloidal particle's dynamics in an optical trap (29). This method only can be applied to particles with comparatively small refractive indexes, however, because particles with relative refractive indexes greater than are difficult to trap. Holographic characterization, by contrast, requires only a single holographic snapshot rather than an extensive time series, does not require optical trapping, and so does not require separate calibration of the trap, and is effective over a wider range of particle sizes and refractive indexes.

The corresponding data in Fig. 2(b) were obtained for a 1.45 diameter TiO sphere at above the focal plane. This sample was synthesized from titanium tetraethoxide and was heat-treated to increase its density (31). Strong forward scattering by such high-index particles gives rise to imaging artifacts unless the medium is index matched to the cover slip. Dispersing the particle in immersion oil ( ) eliminates these artifacts, but introduces spherical aberration for the lens we used, which must be corrected (32) to obtain reliable results. The fit diameter of and refractive index of are consistent with results obtained by electron microscopy and bulk light scattering, respectively. This result is noteworthy because no other single-particle characterization method works for such high refractive indexes.

The data in Fig. 2(c) show results for a nominally 5 silica sphere (Bangs Labs, Lot SS05N/4364) dispersed in water at above the focal plane. The fit refractive index, , is appropriate for porous silica and the diameter, agrees with the value obtained for this sample with a Beckman Z2 Coulter Counter. We have successfully applied holographic characterization to colloidal spheres as small as 100 nm in diameter and as large as 10 . Unlike model-based analytical methods, fitting to the exact Lorenz-Mie scattering theory is robust and reliable over a far wider range of particle sizes, provided that care is taken to maintain numerical stability in calculating (3,20,19).

The same fits resolve the particle's position with a precision of 1 nm in-plane and 10 nm along the optical axis. Comparable nanometer-scale tracking resolution can be obtained with conventional illumination, but requires detailed calibrations for each particle (30). Another benefit of holographic imaging is its very large depth of focus compared with conventional microscopy. Our system provides useful data over a range of more than 100 , which contrasts with the useful depth of focus using conventional illumination (30).

Holographic video microscopy lends itself to three-dimensional particle tracking, as the data in Fig. 3 demonstrate for a colloidal silica sphere (Bangs Labs, Lot SS04N/5252) dispersed in water. This particle was lifted 30 above the focal plane with an optical tweezer, and then released and allowed to sediment. The images in Fig. 3(a) and (c) show the particle near the beginning of its trajectory and near the end. Fits to Eq. (4) are shown in Figs. 3(b) and (d).

The particle's measured trajectory in 1/30 s intervals during 15 s of its descent is plotted in Fig. 3(e). Its vertical position , Fig. 3(f), displays fluctuations about a uniform sedimentation speed, . This provides an estimate for the particle's density through , where is the density of water and is its viscosity at , and where is the acceleration due to gravity. The fit value for the particle's radius, at , remained constant as the particle settled. This value is consistent with the manufacturer's specified radius of , measured with a Beckman Z2 Coulter Counter. Accordingly, we obtain , which is a few percent smaller than the manufacturer's rating for the sample. However, the fit value for the refractive index, , also is 1.5% below the rated value, suggesting that the particle is indeed less dense than specified.

The mean-square displacements, , of the components of the particle's position provide additional consistency checks. As the data in Fig. 3(g) show, fluctuations in the trajectory's individual Cartesian components agree with each other, and all three display linear Einstein-Smoluchowsky scaling, , with a diffusion coefficient . This is consistent with the anticipated Stokes-Einstein value, , where is Boltzmann's constant. The offsets obtained from linear fits to also are consistent (27) with no worse than 1 nm accuracy for in-plane positions and 10 nm for axial positions throughout the trajectory. The optical characterization of the particle's properties thus is consistent with the particle's measured dynamics.

The techniques we have described are readily extended for particles and media whose refractive indexes have large imaginary components. Extensions for core-shell particles (3) and particles with more complex shapes (11,3), such as cylindrical nanowires, similarly should be feasible.

We have demonstrated that a single snapshot from an in-line holographic microscope can be used to measure a colloidal sphere's position and size with nanometer-scale resolution, and its refractive index with precision typically surpassing 1 percent.

A video stream of such images therefore constitutes a powerful six-dimensional microscopy for soft-matter and biological systems. Holographic particle tracking is ideal for three-dimensional microrheology, for measuring colloidal interactions and as force probes for biophysics. The methods we have described can be applied to tracking large numbers of particles in the field of view simultaneously for highly parallel measurements. Real-time single-particle characterization and tracking of large particle ensembles will be invaluable in such applications as holographic assembly of photonic devices (34,33). Applied to more highly structured samples such as biological cells and colloidal heterostructures, they could be used as a basis for cytometric analysis or combinatorial synthesis (35).

**Acknowledgments**- This work was supported by the National Science Foundation under Grant Number DMR-0606415. SHL acknowledges support of the Kessler Family Foundation. KSH and SMY have been supported by the NCRI Center for Integrated Optofluidic Systems of MOST/KOSEF. We are grateful to Ahmet Demirörs for synthesizing the 1.4 diameter TiO particles.
**Competing Interests**- The authors have applied for patents on methods and applications described in this article.
**Correspondence**- Correspondence and requests for materials should be addressed to D.G.G. (email: david.grier@nyu.edu).