Nanophotonic granules populate the interior of cephalopod chromatophores, contributing to their visible color by selectively absorbing and scattering light. Inspired by the performance of these granules, we fabricated nanostructured aerosols by nebulizing a pigment solution extracted from native squid chromatophores. We determined their optical extinction using cavity ring-down spectroscopy and show how extinction cross section is dependent on both particle concentration and size. This work not only advances the fundamental knowledge of the optical properties of chromatophore pigments but also serves as a proof-of-concept method that can be adapted to develop coatings derived from these pigmentary aerosols.

Nanostructured materials that exhibit size and shape dependent optical properties for sensing, visualization, and photo-protection are observed in many natural systems. For instance, the bird-of-paradise, Parotia lawesii, can selectively reflect light across the visible spectrum to attract mates due to constructive interference propagated from the ordered layers of melanin nanoparticles confined within their feather barbules.1 Opals, which are historically regarded as good luck stones, similarly reflect visible light; however, they do so using a tightly packed arrangement of silicon dioxide nanoparticles.2 In cephalopods, protein-based nanostructured granules populate the chromatophore and leucophore (cuttlefish and octopus only) organs in their dermal tissue and contribute to pigmentary color and diffuse scattering, respectively.3–7 While many of the components integral to the optical function of these organs have been identified, the relationship between chemical composition and structural morphology remains largely unknown. This is especially true for the chromatophore granules, where proteins and pigments are both associated with granule structure and function;5,6 yet, it is still unclear how they coordinate together within a nanoparticle.

Recently, we reported an important role of the chromatophore pigments xanthommatin and decarboxylated (DC) xanthommatin in maintaining the structure and optical function of the chromatophore granules.6 When pigments are selectively removed, granule diameter reduces by ∼70%, and the remaining solution is devoid of color. To better understand the structure dependent function of these pigments, we first set out to manufacture pure pigment nanoparticles. We adapted a protocol commonly used to generate environmental aerosols and measure their optical extinction.8–11 In many ways, aerosols can be described as nanoparticles present in the gas phase, of which a major fraction are known to contain organic carbon.12,13 Because the squid pigments contain xanthommatin and DC xanthommatin, both rich in carbon, we asked whether it is also feasible to generate aerosols using a solution of pigments. We describe a method for producing and collecting select sizes of pigment aerosols and show how experimentally measured optical extinction is dependent on both particle concentration and size.

Chromatophore pigments are extracted from squid Doryteuthis peallei using previously described methods.6,14 The extracted solution is dried in a vacuum desiccator overnight and used to make a 1.00% (w/w) solution in 0.20M hydrochloric acid (HCl). The acidic pigment solution is then placed in the intake tube of a Collison-type atomizer (Fig. 1), where a 3.0 L/min stream of N2 dispersed the solution into solvated aerosol droplets producing a log normal distribution of sizes (Fig. S1 of the supplementary material).15 To mitigate the effects associated with solvent inclusions during aerosol formation, we employed two diffusion dryers in series prior to analysis. The dryers are effective at achieving a low relative humidity (<9%, Fig. S1). Once dried, the aerosol stream is directed through a differential mobility analyzer (DMA, TSI model 3080L) which separated the particle diameters based on their electrical mobility. In the DMA, the charged particles are entrained in a flow under an electric field where particle geometry and charge are combined to produce an aerosol stream nearly monodisperse (∼±10%) in diameter.16 

FIG. 1.

Atomization scheme and experimental setup for the optical measurement of squid pigment aerosols. The pigment solution is drawn into the atomization block where it is nebulized under N2. The particles pass through two diffusion dryers and are size separated by the differential mobility analyzer (DMA). The particles are then measured optically by cavity ring-down (CRD) spectroscopy and are counted by the condensation particle counter (CPC). The ratio of B (optical path length) to A (sample path length) in this experiment is 1.17.

FIG. 1.

Atomization scheme and experimental setup for the optical measurement of squid pigment aerosols. The pigment solution is drawn into the atomization block where it is nebulized under N2. The particles pass through two diffusion dryers and are size separated by the differential mobility analyzer (DMA). The particles are then measured optically by cavity ring-down (CRD) spectroscopy and are counted by the condensation particle counter (CPC). The ratio of B (optical path length) to A (sample path length) in this experiment is 1.17.

Close modal

The separated pigment aerosols are then collected on straight-through pore polycarbonate membrane filters (Whatman, 200 nm pore size) using an in-line filter holder17 with a custom-designed bypass system that allowed the filters to be changed without ceasing aerosol flow [Fig. 2(a)]. Once collected on the filters, the size-selected aerosols are analyzed using scanning electron microscopy (SEM, Tescan Lyra3-GMU), and diameters are measured using ImageJ software.18 The average measured diameters of the particles exhibited some variance to the selected DMA size [Fig. 2(b)]. For instance, we observed that for particle sizes up to 240 nm, the measured diameters trended larger than the ones specified by the DMA, and that for diameters greater than 300 nm, the measured values trended smaller. These variances are likely a result of the charging effects, where larger, multiply charged particles pass through the DMA with the same electrical mobility as the singly charged particles, effectively increasing the average measured diameter.19,20 This is more likely to occur at the smaller sizes which are closer to the peak of the particle distribution (Fig. S1 of the supplementary material). Beyond this point, the aerosols exhibited a smaller physical diameter than those specified with the DMA. We also observed the emergence of a heterogenous surface topology (e.g., non-spherical) and the appearance of collapsed structures. These findings suggested that either the pigments are not capable of forming homogenous nanospheres, especially at the larger diameters, or that the solvent gets trapped in the forming aerosols. If it is the latter, then the spherical aerosols are likely to deform as the solvent inclusions dry under the high vacuum of the SEM, reducing the overall sizes of the selected particles [Fig. 2(b), inset].

FIG. 2.

(a) Aerosol collection setup. (b) Bar graph of average measured diameters from SEM micrographs plotted against the DMA selected diameters. Error bars indicate standard error, where N = 82–135. The inset includes representative SEM micrographs of pigment aerosols (scale bar = 500 nm).

FIG. 2.

(a) Aerosol collection setup. (b) Bar graph of average measured diameters from SEM micrographs plotted against the DMA selected diameters. Error bars indicate standard error, where N = 82–135. The inset includes representative SEM micrographs of pigment aerosols (scale bar = 500 nm).

Close modal

Because many nanostructured materials have optical properties that are dependent on size, we next measured the effects of size on optical extinction. Extinction cross sections are extrapolated from the measured optical extinction of particles at increasing number concentrations using our custom-built cavity ring-down (CRD) cell. Within the CRD, generated aerosols are exposed to 20 Hz pulsed 532 nm light within a ∼70 cm cell from a neodymium-doped yttrium aluminum garnet (Nd:YAG) laser source (Quantel USA, Ultra). Highly reflective concave mirrors (R > 0.999) on either end of the cell reflects the light multiple times to increase the effective path length to ∼30 km, albeit with an exponential decay of intensity. During this process, a small amount of light leaks out of the cell opposite to the source, and its intensity is measured by a photomultiplier tube (PMT).9,21 In a particle-free CRD cell, light intensity decays at a time constant τ0; whereas, the cell that is filled with an aerosol stream absorbs and scatters more light, decaying with a shorter time, τ. These decay times are measured and used to determine the extinction of the sample with units of inverse length, αext, per Eq. (1),

αext=RLc1τ1τ0,
(1)

where c is the speed of light and RL is the ratio between optical path length, B, and sample path length, A (Fig. 1; RL = 1.17 in this case).9,21,22 A condensation particle counter (CPC, TSI model 3775, ±10%) is then used to determine the number concentration (N, particles/cm3) of the aerosol stream that passed through the CRD. The CPC makes a measurement once every second, and these measurements are averaged over the same range of time used for the CRD (typically 60-80 s). Based on the measurements from the CPC and CRD, the extinction cross sections (σext, cm2/particle) are calculated [Eq. (2)],

σext=αextN.
(2)

Three number concentrations varying in magnitude are achieved for the seven diameters (216–545 nm) selected by the DMA using increasing atomization pump speeds, and their optical extinctions are measured using the CRD. As expected, the concentration-dependent extinctions are found to increase linearly with particle number concentration, indicating that the generated pigment aerosols are indeed responsible for extinguishing light. The size dependent extinction cross sections are calculated from the slope of the linear least squares fit (illustrated in Fig. 3; values in Table I).11,23 In each case, the error in the slope is propagated due to the high linearity (R2) portrayed by the regression at each aerosol size.

FIG. 3.

Optical extinction of squid pigment particles based on CRD measurements versus number concentration as counted by the CPC. Symbols indicate the DMA size selected diameter. Y-error propagated from error on τ values. X-error is simple standard deviation of average number concentration (1σ, N = 81 concentration measurements).

FIG. 3.

Optical extinction of squid pigment particles based on CRD measurements versus number concentration as counted by the CPC. Symbols indicate the DMA size selected diameter. Y-error propagated from error on τ values. X-error is simple standard deviation of average number concentration (1σ, N = 81 concentration measurements).

Close modal
TABLE I.

The size-selected and experimentally measured particle diameters and calculated extinction cross sections of the squid pigment aerosols.

DMA selectedSEM measured sizes withN of SEM measuredExtinction cross section withR2 of linear
sizes (nm)standard error (nm)particleserror from regression (cm2/particle)regression fit
216 240 ± 10 100 9.58 ± 0.09 × 10−10 0.9999 
240 258 ± 8 119 1.09 ± 0.04 × 10−9 0.9983 
300 289 ± 5 121 1.77 ± 0.01 × 10−9 1.0000 
351 334 ± 7 115 2.40 ± 0.06 × 10−9 0.9994 
396 375 ± 5 135 3.44 ± 0.06 × 10−9 0.9997 
480 422 ± 11 82 6.66 ± 0.07 × 10−9 0.9999 
545 489 ± 10 96 9.22 ± 0.21 × 10−9 0.9995 
DMA selectedSEM measured sizes withN of SEM measuredExtinction cross section withR2 of linear
sizes (nm)standard error (nm)particleserror from regression (cm2/particle)regression fit
216 240 ± 10 100 9.58 ± 0.09 × 10−10 0.9999 
240 258 ± 8 119 1.09 ± 0.04 × 10−9 0.9983 
300 289 ± 5 121 1.77 ± 0.01 × 10−9 1.0000 
351 334 ± 7 115 2.40 ± 0.06 × 10−9 0.9994 
396 375 ± 5 135 3.44 ± 0.06 × 10−9 0.9997 
480 422 ± 11 82 6.66 ± 0.07 × 10−9 0.9999 
545 489 ± 10 96 9.22 ± 0.21 × 10−9 0.9995 

We observed an increasing extinction cross section that is dependent on the geometric cross-sectional area of the aerosols. For example, the extinction for a given number of 300 nm particles (solid upside-down triangles) is equivalent to approximately twice that number of 216 nm particles (solid circles) per unit volume, indicating that the larger particles extinguished more light than the smaller particles. Number concentrations also appeared to decrease significantly for the larger (480 and 545 nm) diameter particles under the same atomization conditions, indicating that high concentrations of pigment particles in these diameter ranges are less likely to be generated at these pump speeds (Fig. 3). This observation is also in agreement with the particle distribution, as shown in Fig. S1 of the supplementary material, where diameters over 400 nm are at the tail end of the curve. Collectively, these data indicate that pigment particles have a predictable optical extinction behavior that is highly tunable over a wide range of sizes and number concentrations.

We also compared our experimental data to the extinction cross sections calculated using Mie theory at a wavelength of 589 nm from our previous report (Table SI of the supplementary material).7 We observed 5%–30% differences in extinction cross sections between the two data sets, where the largest errors occurred at sizes that reached the highest optical extinctions (240-396 nm). These variances are not all too surprising given the differences in reference wavelengths used in the two studies and the heterogeneity persisting in the pigment aerosol structures. Despite these discrepancies, we observed good agreement between theoretically extrapolated extinction cross sections and the measured values here (illustrated in Fig. S2 of the supplementary material), suggesting that the pigment assembled as these nanostructures is responsible for the optical extinction. Future work will involve diagnosing the differences between the selected DMA diameters and the SEM measured diameters, as this may also impact the comparisons between the theoretical and experimental data.

In this report, we show that we can successfully aerosolize squid pigments and evaluate their number concentration and size-dependent optical properties. Even though these measurements do not account for the full makeup of the native chromatophore granules, the pigment is a major component,6 and, therefore, a dominant contributor to the optical behavior. By exploiting their ability to assemble into nanostructures in the gas phase, we also show a new method for processing these pigments that can conceivably be scaled up for spray-on coatings. We show that particle diameters can be separated by their electronic mobility, illustrating a control over selected size parameters that makes this process highly tunable. Overall these properties, along with the pigment refractive index at a specific wavelength or range of wavelengths, are important considerations in manufacturing pigment particles for future materials or optical applications.

See supplementary material for reference (Figs. S1 and S2 and Table SI).

We acknowledge Mr. Tyler Galpin and Ms. Jillian Morang for assistance with the CRD experiments and Mr. Brent Lawson for assistance with the collection of aerosols. We would also like to acknowledge the University of New Hampshire for the use of their facilities, including the University Instrumentation Center for their assistance with SEM sample preparation. This work was supported by the University of New Hampshire, Department of Chemistry and College of Engineering and Physical Sciences (S.R.D. and M.E.G.), Northeastern University, Department of Chemistry and Chemical Biology (L.F.D.), and the Army Research Office (No. W911NF-16-1-0455, L.F.D. and M.E.G.).

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Supplementary Material