The dynamic light scattering (DLS) technique is extensively used in industrial and academic research applications to access the motion spectrum of microscopic objects that help determine the material’s particle size and viscoelastic properties. However, such measurements cannot be determined when the samples do not scatter enough light due to low concentrations of scatterers or small scattering cross sections.
Study: Cavity-Amplified Scattering Spectroscopy Reveals the Dynamics of Proteins and Nanoparticles in Quasi-transparent and Miniature Samples. Image Credit: foxaon1987/Shutterstock.com
An article published in the journal ACS Nano presented a convenient approach to overcome the above problem by amplifying the light scattering efficiency. The samples with weak scattering were placed inside a Lambertian cavity. On injecting a laser light into the Lambertian cavity, it produced a three-dimensional (3D) isotropic and homogeneous light field.
Here the photon scattering path length in the sample was effectively elongated, increasing its magnitude by 2-3 orders and leading to a significant increase in sensitivity. Compared to conventional techniques, the current approach increased the sensitivity by 104-folds.
This technique was applied in miniaturized microfluidics for light scattering and to access short-time dynamics of samples with low turbidity. The sensitivity gain in the present light scattering method was determined by measuring the diffusion coefficient and particle size.
Additionally, the highly sensitive cavity-amplified scattering spectroscopy method (CASS) was presented as an efficient approach to studying the problems of short-time dynamics, including the Brownian motion’s ballistic limit, the dielectric dynamics of liquids, or the internal dynamics of proteins.
Light Scattering Techniques to Study Dynamics of Proteins and Nanoparticles
DLS is a robust technique to access the dynamics of materials in various fields, which allows the measurement of colloidal particle’s thermal motion, providing an overview of size distribution. However, the DLS works under the provision that the samples scatter individual photons only once on average. However, diffusing wave spectroscopy (DWS) overcomes this provision by extending each photon’s scattering many times.
The structure of proteins is generally determined by the amino acid sequence. Proteins are known to be dynamical entities, performing their functions as an ensemble of diverse conformations rather than a single static structure.
Protein dynamics is a highly complex phenomenon comprising numerous contributions from motions with different mechanisms of action and happening with diverse timescales and amplitudes that highly depend on the system and the local environment.
Nanoparticles are tiny objects, less than 100 nanometers across any dimension. Nanoparticles dispersed in solution tend to sediment, diffuse, and aggregate as functions of intrinsic and system properties. Once injected into the solution, the process dominating the dynamics of nanoparticles is the diffusion process. Nanoparticles tend to move from zones of high concentration to low concentration. After a certain time, an unstable equilibrium is achieved, and nanoparticles are randomly but homogeneously distributed in the medium.
CASS Toward the Study of Dynamics of Proteins and Nanoparticles
In the present study, the CASS method was adopted to elongate the sample’s optical path length by 102 to 103 times, and the sensitivity limit of DLS and DWS was also improved by 104-fold. The amplification in optical path length and sensitivity was achieved by placing the samples inside a Lambertian cavity of high albedo and injected with laser light of high coherence.
The walls of the Lambertian cavity reflected the light and forced it back through the sample multiple times, resulting in a long photon path that remained shorter than the coherence length of the laser. Since the light from the walls was reflected by Lambertian scattering, the entire sample volume was immersed into, and probed by, an isotopic, homogenous, and coherent optical field.
Moreover, due to the static nature of the scattered light, the resulting 3D interference pattern fluctuated for only dynamic samples. Hence, the CASS setup was efficient in studying the short-time dynamics of dilute samples, whose concentration was lower than the samples in traditional DLS measurement conditions.
It was observed that the CASS setup could measure the dynamics of proteins with a low concentration range of 0.1−1 milligram per milliliter. The CASS measurement of globular protein lysozyme resulted in a 6% error for a 10-fold lower concentration and a 16% error for a 100-fold lower concentration.
However, the high-dilution experiments made with gold nanoparticles with diameters 5, 10, 20, and 30 nanometers did not show sensitivity enhancement for the nanoparticles due to a smaller absorption length.
To summarize, Lambertian cavities allowed the probing of the entire sample volume (Vsa) multiple times through light scattering. Here the mean path length (lsa) of light that traveled across the sample was considered as the length instead of the sample size.
According to the eponym theorem, lsa was given as the ratio; 4Vsa/Σsa, even in the multiple scattering processes. Thus, the sample’s total path length of the sample was given as; g04Vsa/Σsa, where g0 refers to the amplification gain.
In addition, for the cavity of volume Vc and surface area Σc, the probability of a scatterer (with scattering cross-section as σsc) to scatter along a random chord was given as 4σsc/Σc. The same geometric argument was applied to light absorption, and the probability of a single absorber (with scattering cross-section as σa) to intersect with a single chord was proportional to the surface occupancy ratio (σa/Σc).
The enhanced sensitivity of the CASS system could be advantageous in detecting sub-angstrom motions of scattered particles in any samples and can be applied in microfluidics, environmental control, nephelometry, and in the characterization of pharmaceutical samples.
Graciani, G., King, J.T., Amblard, F. (2022) Cavity-Amplified Scattering Spectroscopy Reveals the Dynamics of Proteins and Nanoparticles in Quasi-transparent and Miniature Samples. ACS Nano. https://doi.org/10.1021/acsnano.2c06471
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