Structural Colors from Amyloid‐Based Liquid Crystals

The helical periodicity and layered structure in cholesteric liquid crystals (CLCs) may be tuned to generate structural color according to the Bragg's law of diffraction. A wide range of natural‐based materials such as condensed DNA, collagen, chitin, cellulose, and chiral biopolymers exhibit cholesteric phases with left‐handed helixes and ensued structural colors. Here, the possibility of using amyloid CLCs is reported to prepare films with iridescent color reflection and opposite handedness. Right‐handed CLCs assembled by left‐handed amyloid fibrils are dried into layered structures with variable pitch controlled by the addition of glucose. Circularly polarized light with the same handedness of amyloid CLCs helix is reflected in the Bragg regime. Varying the drying speed leads to the switching between films with a rainbow‐like color gradient and large area uniform color. It is confirmed that the origin of the colors derives from the layered structures of the amyloid CLCs, given the negligeable birefringence of the films, calculated from optical rotatory dispersion. These findings provide a facile approach to constructing biosourced cholesteric materials and introduce an original class of proteinaceous materials for the generation of structural colors from right‐handed circularly polarized light.


Introduction
Many -sheet forming polypeptides assemble into amyloid fibrils, known in relation to the development of neurodegenerative diseases and showing a well-defined chirality at the single fibril level. [1]The process is driven by hydrogen bonding leading to chemically stable and mechanically rigid proteinaceous filament with a large aspect ratio. [2]Up-concentrating amyloid fibrils resulting in filamentous alignment with longrange orientational ordering, i.e., phase transition from isotropic to liquid crystalline phases, [3][4] including cholesteric liquid crystals (CLCs) with helical twisting of fibrils, also known as chiral nematic phases.This complex behavior, mostly driven by entropy, most often include chirality inversion, in analogy with peptides, [5] cellulose [6] and other filamentous building blocks. [7]he ordered state of the fibrils may lead to interesting optical properties. Additionally, taking advantage of the birefringence of CLCs, researchers have unveiled the mechanism of emergence and growth dynamic of disordered branching patterns in chiral liquid crystals, which may direct large-scale spatial organization of CLCs without defects. [10]18][19][20][21] Under certain conditions, naturally occurring or artificially synthesized biosourced colloidal systems made of rod-like particles, such as DNA, virus, collagen, etc., are routinely observed in the formation of left-handed helix cholesteric phases; [16,[22][23][24][25][26][27][28][29][30][31] however, they are rarely reported to show structural color in the visible spectrum since decreasing the helical pitch down to length scales approaching the visible light wavelength remains challenging.Cellulose-based photonic CLCs, as an exception, reflect remarkable iridescent light that arises from hierarchical nanostructures by self-assembly, which, however, is always left-handed. [16]myloid-based CLCs assemble into right-handed helical structures starting from amyloid fibrils of left-handedness, [32] differently from the greatest majority of natural bio-filamentous systems, where right-handed filaments end up in left-handed  [35] image of amyloid fibrils liquid crystals.The patterns are results of the birefringence originating from the collective orientation of the fibrils and show a few defects.The scale bar is 100 μm.g) Microscope image of a bulk cholesteric phase, where the average pitch is 11.6 ± 0.8 μm.
CLCs.This unique property allows for the production of righthanded iridescent light from the cholesteric structure, which is, however, yet to be demonstrated experimentally.
Here we report colored dry films prepared from amyloid CLC medium stabilized by glucose which exhibit distinctive righthandedness chirality.The films feature highly ordered and layered architectures that can be tuned for reflecting single-color right-handed circularly polarized light (i.e., the same handedness of the amyloid CLCs template) in visible light spectra.We also show possibilities for optimizing the color distribution with the coffee-ring effect via fast assembly under vacuum condition, which avoids branching patterns formation and paves the way for scalable production of the colored films.The average refractive index n is calculated via measuring the reflected wavelength () and pitch (P) by a spectrometer and scanning electron microscope (SEM), respectively.Moreover, quantifying birefringence with the help of de Vries theory enables analysis of reflected light components that derive from the microstructure of the films.This novel material expands the scope of the existing bio-colloidal systems featuring structured color, which is still dominantly lefthanded circularly polarized, and provides new directions for distinctive optical and photonic functional applications, such as chiral reflectors and structural color matrices.

Cholesteric LCs from Amyloid Fibrils
Lysozyme amyloid fibrils were used as a model system to prepare lyotropic amyloid CLCs (Figure 1a,b).Up-concentrating shortened amyloid fibril suspensions lead to a phase transition from the isotropic to the nematic, as suggested by Onsager theory. [33]his process is driven by entropy, where the evolution of the newly forming phase starts with the nucleation of droplets, also referred to as tactoids, exhibiting distinct bright birefringence. It usually takes several weeks to form a macroscopic aggregate because of the difference in concentration of amyloid fibrils between isotropic and anisotropic phases, particularly at relatively low concentrations. [35]Then, a bulk cholesteric phase is achieved after full phase separation, which is affected by the equilibration times, sample compositions, and properties.
Here we prepare concentrated rod-like lysozyme amyloid fibrils (3.6 wt%), with an average length of <L avg > = 349.9± 45.3 nm and height of <h avg > = 4.3 ± 1.1 nm (Figure 1c,d).Interestingly, we have found that the higher fibril concentration enables faster sedimentation to form a bulk cholesteric phase within several hours.Given the parameters, the amyloid fibrils bulk solution shows colorful light scattering (Figure 1e). [36]onfined in the capillary tube, the amyloid fibrils suspension shows topological line defects as disclination and dislocation under a polarizing optical microscope (POM).In the meantime, the micrographs reveal that the inherent chirality of the amyloid fibrils leads to a uniform cholesteric phase with a pitch ≈11.6 μm (Figure 1f,g).Examining the chirality of amyloid CLCs using a previously reported method, [6] we confirmed the righthandedness by rotating a tactoid in between crossed polarizers (see detailed explanation in Figure S1, Supporting Information).It is worth stressing again that left-handed single amyloid fibrils assemble into right-handed CLCs, which are rarely found in natural biocolloid liquid crystalline systems.This periodic helical structure is expected to give rise to the intrinsic optical property of selective reflection of light referred to as Bragg reflection. [23]hich may result in colored reflections when the periodicity approaches the range of dimensions typical of the wavelength of visible light.

Iridescent Color from Dried Protein Cholesteric Films
Drying an isotropic amyloid droplet on the glass usually produces a nematic phase near and parallel to the contact line of the droplet edge, where liquid crystalline tactoids can be observed within the whole drying stages (details can be found in ref. [34]).Although the cholesteric tactoids and their corresponding colloidal deposition patterns in dried thin film have been well investigated in previous works, [34] light reflection in the visible spectra from amyloid CLCs has yet to be reported.This is because of the mismatch between the cholesteric pitch of the CLCs and the wavelength of visible light, according to Bragg's law.For instance, in liquid phase, the pitch of CLCs is in the order of 10 microns, while the pitch in the dried films may be either too large or too small compared to the visible wavelength.Here, D-(+)-glucose, as a nonionic additive, is introduced in the amyloid CLCs for tuning the cholesteric pitch, serving as an independent plasticizer and spacer without further reactions with amyloid fibrils. [37]igure 2a shows a schematic illustration of the preparation of amyloid CLC films doped with glucose (prepared at a mass ratio ≈0.46:1 glucose:amyloid fibrils) by slowly evaporating water from the suspensions.At ambient conditions, the deposition process behaves similarly to the colloidal deposition patterns from pure amyloid CLCs. [34]We examine the drying process under a light microscope in the reflection mode with crossed polarizers, and find birefringence patterns dominating the region between the outer edge and central region of the radius of the film during the solvent evaporation process until the formation of the film (Video S1, Supporting Information shows the drying process and is illustrated in Figure S2a, Supporting Information).On the drying edge, as the birefringence patterns disappear, red, green, and blue reflected light appear sequentially, corresponding to a decrease in wavelength.This edge stops at approximately half of the film radius as the drying process finishes.Crossed polarizers are used to reduce the impact of the background reflected light whose polarization state is not changed by the sample.Without the polarizers, the completely dried film shows a weak color ring consisting of red and green reflections that are barely discernable (Figure 2b).When polarizers are in use, we observe an iridescent rainbow-like ring at the visible wavelength, while the birefringence patterns coincide with color regions at four different diagonal directions outside the circular rainbow ring, as shown in Figure 2b.Meanwhile, analyzing individual RGB colors of the photos (along the direction of the red arrow in Figure 2c,d) in both light conditions confirms the interference from the background light, as the color differentiation is not readily apparent under bright light conditions.The dark area in the center, however, consists of no cholesteric structures since the high viscosity of the suspension quenches the fibrils and increases the energy barrier for reorienting amyloid fibrils (Figure 2b). [34] During the drying process, the CLC tactoids are transported to the droplet edge by a strong capillary outflow heading toward the contact line.This leads to a condensed layered structure close to the film edge reflecting blue light and loosening layers toward the film center until the red light reflection disappears.Interestingly, the rainbow-like region moves outward when increasing the mass ratio between glucose and amyloid CLCs (Figure S3, Supporting Information).However, when the ratio is over ∼1.0, these colors are no longer observed (Figure S3, Supporting Information).The excessive amount of glucose may have caused pitch of the CLC structure to increase beyond the visible wavelength range, making it unsuitable for reflecting visible light.We confirmed this phenomenon by observing it also in other protein-based amyloid CLCs, such as -lactoglobulin (BLG, Figure S4, Supporting Information).Given that the average membrane thickness exceeds ∼75 microns, it is unlike that these colors arise due to thin-film interference (Figure 2e). [40]

Color Components Derived from Glucose-Amyloid CLC Film
When we rotate the film under crossed polarizers, the color patterns distribution within the film remains unchanged.This excludes the possibility that rainbow-like colors come from birefringence (Figure 3, reflection mode) as reasoned below.In general, when a linearly polarized incident light, considered as a combination of left-and right-handed circular polarized components, passes through a CLC film along the helical axis, the component of the light possessing the same handedness of CLCs is reflected, while the component with the opposite handedness is transmitted. [23]Therefore, due to the right-handedness nature of the amyloid CLCs, the reflected should also be right-handed (Figure S1, Supporting Information).When the reflected circularly polarized light passes through the second linear polarizer, perpendicular to the previous one, the strength of the signal will stay the same.On the other hand, birefringence patterns change as the sample is rotated because the patterns depend on the collective fibrils orientations with respect to the polarizers.There-fore, the different and contrasting behavior of the two types of reflected light can be used to tell each other apart.
Additionally, when color filters are inserted in the light path to produce single-color illumination, the areas with the corresponding color patterns demonstrate consistent color intensity compared to the sample under full white light spectrum, and show no dependence on the sample orientation, as shown in Figure 3   precisely aligning with the filtered light patterns in all cases (right panel in Figure 3).As a control experiment, films prepared separately with only glucose or only amyloid cholesteric liquid crystal do not produce such angle-independent rainbow color patterns (Figures S5 and S6, Supporting Information).
To observe the effect of the handedness of the illuminating light on the color reflection, we replace the linear polarizer inserted in the incident light path of microscope with left and righthanded circular polarizers.When illuminating the film with the right-handed polarized incident light, bright colors are observed (Figure S7, Supporting Information).In contrast, color reflections are barely visible when the chirality of incident light is inversed to left-handedness.A control experiment made with films based on cellulose CLC, known to reflect left-handed circularly polarized light, features -as expected-opposite behavior (Figure S7, Supporting Information).These observations suggest that the glucose-doped amyloid CLC film has indeed formed chiral nematic structures capable of reflecting right-handed circular light in the visible range.

Amyloid CLC Film with Uniform Color
Varying the sugar content and drying conditions are used to achieve a uniform color distribution over a large film area.We observe an increase of the average pitch of the CLCs when glucose is introduced in amyloid fibril suspensions.Three suspensions of glucose-amyloid fibrils (AF-B, AF-G, and AF-R) are prepared, producing films reflecting blue, green, and red light.Even at a low mass ratio of approximately 0.074:1 (glucose: amyloid fibrils), a noteworthy increase of approximately 3 μm is observed in the average pitch of AF-B solution (B standing for Blue), compared to its original value of approximately 11 μm.An additional increase in pitch is also observed when introducing more glucose in both AF-G (mass ratio ≈ 0.63, G standing for Green) and AF-R (mass ratio ≈ 1.11, R standing for Red) solutions (Figure 4a; and illustrated in Figure 4b).This observed increase in pitch values suggests a direct influence of the sugar content on the inter fibrils distance and twisting power of the amyloid CLCs.
Interestingly, accelerated solvent evaporation at a 50 mbar vacuum condition results in the contact line pinned at its initial position, as illustrated in Figure S2b, Supporting Information.Subsequently, solid film formation is initiated from the central location.As the drying progresses, the surface tension squeezes amyloid CLC solution outward to the edge, ultimately leading to the formation of a flat film with uniform thickness deposited on the glass (Figure 4c and Figure S2b, Supporting Information).It is important to note that the solution accumulates at the edges during this process and forms a thick deposition exhibiting birefringent patterns (Figure 4c and Figure S8, Supporting Information).
Around the central region, the films display uniform colors (blue, green, or red), that are confirmed by the RGB intensity analysis (Figure 4d,e).However, a slight fluctuation in color reflections could be observed.For instance, the AF-R film mainly reflects wavelength at 645 nm resulting in red color.However, the co-presence of the green (548 and 578 nm) and blue (438 nm) is observed within the same spectral profile (Figure 4f).This is because the pitch may be unevenly distributed across the colored region after a fast drying process.The phenomenon is quite common as it can be observed in BLG CLC films as well (Figure S10, Supporting Information).
The changes of the color of the film, i.e., the reflected spectrum, correspond to the structural change of the CLC layers in the dried films.This can be observed by cross-sectional SEM images showing an increase in pitch values of the dried films (from 330 nm to 550 nm) with increasing glucose concentration (Figure 4g).Varying the thickness of the film, however, does not change the cholesteric structures as suggested by a control experiment showing consistent blue color reflections from films made with the same mixture (AF-B) but different amounts (Figure S9, Supporting Information).This observation further excludes the effect from the thin-film interference in generating colors.From this, the refractive index, at normal incidence, can be determined by dividing  0 , the wavelength of maximum reflection, with P, as stated by Bragg's law.Assuming an even distribution of the pitch in the layered film, the average refractive index can be expressed as n =  0 /P, and is roughly computed to be ≈1.18,≈1.23, and ≈1.32 for CLC films prepared with AF-R, AF-G, and AF-B, respectively.Additionally, the theory of de Vries allows for determining the strength of birefringence (Δn) of amyloid CLC film as a function of wavelength () from optical rotatory dispersion (ORD) measurements.The proposed theory is limited to the perpendicular incidence, where the waves propagate in the liquid crystals and are circularly polarized along the optic axis in the reflection region (′ < 1 ± /2).Outside the reflection region, the rotatory power is then [41] Equation ( 1) can be simplified to [42] r = (360∕P) where ′ = / 0 and  = Δn/n (relative birefringence of the single layer).By measuring P and the ORD of the amyloid CLC films outside the region of total reflection (′ = 1), it is possible to study Δn (, T), where T is the temperature.Since we confine ourselves at room temperature, the effect of T is neglected.Thus, or In the present work, the pitch of the dried CLC films is within the range 330 nm < P < 550 nm and the wavelength considered is 300 nm <  < 800 nm.Therefore, the condition P ≫  or ′ ≪ 1 cannot be satisfied, while ′ >  holds true because the birefringence Δn is small.By measuring r by the ORD and using Eqs.(3), one can therefore calculate Δn (). Figure S11, Supporting Information illustrates the ORD curves of colored films, showing increased rotatory power when wavelength decreases and passes through the reflection region in the color area.Given the constraint that the wavelength of interest needs to be outside of the reflection region, the typical benchmarking measurement at 589 nm is inapplicable here since it falls within the reflection region of AF-R and AF-G films.We choose the wavelength of 350 nm (outside any reflection region of colored films), and the Δn is calculated to be ≈0.0067,≈0.0061, and ≈0.0075 for AF-R, AF-G, and AF-B films, respectively.These values are consistent with our observations that the birefringence of the colored region (located away from the edge) is weak and can be considered negligible.Considering that an ideal cholesteric film only reflects lights of selected wavelengths and that the birefringence patterns may come from defects or simply by ordinary nematic domains, this observation implies the dominant presence of CLC phase within the colored regions and CLC as the main source for color generation.We also note that close to the outer edge of the films, the birefringent patterns are much more pronounced, indicating a strong presence of a nematic phase which may come from the reorientation of the fibrils during the fast drying process at the edges (Figure S8, Supporting Information), e.g. the results from coffee ring drying effects.
Controlling the pitch lies at the center of fabricating cholesteric films with structural color.Our experiments demonstrate the importance of D-(+)-glucose in tuning the pitch of both amyloid CLC solutions and the corresponding dried films.In pristine amyloid CLC solutions, their pitches are usually in micrometer range. [32,43]The evaporation of the solvent in the amyloid CLC solution leads to the decrease of the pitch until the film is formed, but the final pitch length of the resulting film is below the visible wavelength.By adding glucose, the pitch of the dried films can be tuned to fall within the visible light wavelength range.Additionally, the length of the amyloid fibrils can also affect the pitch of the resulted films.For instance, with amyloid CLCs, increased fibril length generally decreases the cholesteric pitch, [35] so that a blue shift of color reflections by longer amyloid CLC films prepared under the same conditions is expected.
The choice of additives can vary among those that do not interact with either the constituting mesogens or the surfaces.Previous reports have shown that glucose is replaceable by glycerol in cellulose CLC systems [44] for preparing the films.D-(+)glucose used here is a chemically inert and surface-inactive additive and its chirality does not affect the chirality of the selfassembled cholesteric liquid crystals.For instance, it has been shown that adding D-(+)-glucose into cellulose CLC solution with left-handedness does not reverse the chirality in the final films. [45]e note that the additional glucose may reduce the mechanical strength, [46] such as the breaking stress, which may relate to a reduction in the intramolecular attraction between amyloid fibrils, as well as to the hydrogen bond formation between the glucose and protein fibrils; nonetheless, for films deposited on solid transparent substrates (quartz, glass, ITO, etc.) and thus targeting optical applications, this is not expected to be a problem.
It is also worth noting that the color patterns close to the film edge, may be affected by the wettability of the substrate surfaces. [47]This is because the formation of the film at interface (substrate-mixture-air) is a result of solute deposition, where there is competition between the receding contact line and the deposition of components from glucose-amyloid CLC mixture.However, from our observations, we do not note a differential change in solute deposition (amyloids vs D-(+)-glucose), in color in the central area of the film, nor at the edges, so that no significant change in color is observed due to the coffee-ring effect.

Conclusions
We have demonstrated a facile approach to prepare large area glucose-amyloid CLC solid films with structural colors tunable by varying the amount of glucose.Drying the amyloid CLCs in ambient condition results in the formation of a film showing rainbow-like reflections and birefringence patterns, while a fastdrying process under vacuum yields films with large areas of uniform color.Optical analysis reveals that these colored reflections originate from the cholesteric layered structure with different colors correlating directly to the layer periodicity and the light circularly polarized with right-handedness, as the reflecting glucoseamyloid CLC solid films.The colors, controlled by the CLC pitch, redshift with increasing amount of glucose.Additionally, birefringence is found to have a negligible effect in the generation of the colors according to the calculation using the de Vries theory.Future works exploring the relation between the material composition, drying conditions, and other functional additives may contribute to the development of optical photonic hybrid materials with predefined properties based on amyloid fibril structural templates.

Experimental Section
Preparation Of Amyloid Fibrils Cholesteric Liquid Crystals: Amyloid fibrils were prepared based on our previously reported method. [32]In brief, either lysozyme or BLG monomer were dissolved in milli-Q water followed by adjusting pH to 2. The solutions were heated at 90 °C (24 h for lysozyme and 5 h for BLG) under proper agitation.After quenching with ice to stop the fibrilization, the lysozyme amyloid fibrils were cut and homogenized by applying mechanical shear force with an immersion mixer for 120 s.The solution was dialyzed with a dialysis membrane (MWCO 100 kDa) for 5 days under pH 2 to remove protein monomers.The high purification rate was assured by daily batch change.
To obtain cholesteric amyloid fibrils liquid crystals, the previously prepared solutions were up-concentrated via reverse osmosis by 6-8 kDa dialysis membrane against 10 wt% polyethylene glycol solution (mol wt: Mr ≈35000, Sigma-Aldrich) in pH 2 milli-Q water.After equilibrium in the fridge, the nematic amyloid fibril solutions were self-assembled into the cholesteric liquid crystal phase.This stabilized sample was further used for fibril morphology characterization, i.e., length and height distribution.
Characterizations Of The Amyloid Fibrils: AFM characterization was conducted as previously reported methods. [2]AFM images were analyzed to obtain the length, height, and contour distribution of the amyloid fibrils by tracing the fibrils using the open-source code FiberApp. [48]We analyzed the liquid crystalline structures under a polarized microscope, where the samples were prepared by injecting 40 μL solution into glass cuvettes (0.2 × 4 × 40 mm 3 , VitroTubes, Vitrocom).After equilibration at room temperature for a few days, the epoxy-sealed cuvettes were examined with an optical microscope.We examined the optical properties with crossed polarizer under transparent mode and the orientation of the amyloid fibrils evolving in the liquid crystal phase by a LC-PolScope universal compensator.
Film Formation And Characterization: To prepare colored films showing the rainbow color ring, 3.6 wt% lysozyme amyloid cholesteric solutions were well mixed with 40 wt% glucose solution to achieve a mass ratio at 0.46:1.At the initial lysozyme concentration, the amyloids are in the CLCs, as observed under crossed polarizers.Then, 65 μL mixture was casted onto the glass slide, followed by drying at ambient conditions for several hours.
To prepare films with various uniform colors, we tuned the mass ratio between glucose and amyloid fibrils, where mixtures named AF-B, AF-G, and AF-R have the mass ratio of 0.074:1, 0.63:1, and 1.11:1, respectively (more solutions with different mass ratio can be prepared accordingly).Then, the films were dried from 65 μL mixture solution at an accelerated speed (dozens of minutes) under vacuum condition (room temperature, 50 mbar).The thickness of the films can be adjusted by controlling casting volume of AF-B solution (50, 65, and 80 μL).The ORD spectra of the amyloid CLC films were collected from 900 nm to 280 nm using a Jasco J-815 CD spectrometer.Films with only amyloid fibrils or only glucose were examined as controls.We measured the thickness of the films by analyzing the stitched microscope images of the cross-section of sample with Fiji Im-ageJ.To confirm the layered structure, we performed SEM cross-sectional analyses of the films, and the corresponding pitch was calculated by Fiji ImageJ.The BLG CLC films were prepared accordingly under the same conditions.
Color Analysis: The colored films were imaged by a light microscope coupled with crossed polarizers in both reflection and transmission modes.To induce the left/right-handed circular polarized light, the two different polarizers were integrated into the incident light path of microscope accordingly.The color filters were implemented to produce illuminating color light accordingly.By rotating the sample within 90 o , we collected microscope images with different light reflections (red, green, and blue that were generated by filters).The visible spectra of the reflected light were collected by OceanOptics USB2000+.The RGB analysis of all the microscope images was conducted by Zen (Version 2.3.64.0)Carl Zeiss Microscopy.

Figure 1 .
Figure 1.Preparation and characterization of cholesteric liquid crystals from shortened lysozyme amyloid fibrils.a) Schematic illustration of the synthesis of lysozyme amyloid fibrils and the formation of the corresponding cholesteric phase.The amyloid fibrils are obtained from lysozyme, shortened into rigid rods, and concentrated after purification through a dialysis membrane.A rich phase behavior leads to the formation of a cholesteric phase.b) Atomic force microscope (AFM) is used to characterize the distribution of c) length and d) height of lysozyme fibrils.The red lines show the Gaussian function fitting of the distributions.The scale bar is 1 μm.The inset in c is end-to-end distribution of internal contour length, showing the persistence length of lysozyme fibrils.e) Photo of lysozyme amyloid CLCs showing colorful scattering.f) The Polscope[35] image of amyloid fibrils liquid crystals.The patterns are results of the birefringence originating from the collective orientation of the fibrils and show a few defects.The scale bar is 100 μm.g) Microscope image of a bulk cholesteric phase, where the average pitch is 11.6 ± 0.8 μm.

Figure 2 .
Figure 2. Film from amyloid-based liquid crystals showing iridescent color.a) Schematic illustration of film preparation and characterization.The entire film area is controlled at approximately 50 mm 2 .b) Combined microscope images of a film prepared with the mass ratio glucose:fibrils = 0.46:1 taken with reflection mode show that the film displays slight rainbow color under a non-polarized illumination.When crossed polarizers are integrated into the incident and reflected light path of the microscope, an iridescent rainbow ring, and strong birefringence patterns are present.The arrow shows the RGB color intensities under c) non-polarized illumination and d) crossed polarizers, respectively.A fast Fourier transform (FFT) is used to smooth the original data.e) Thickness distribution of the dried lysozyme cholesteric film prepared by slow evaporation.The inserted microscope image shows the cross-section of the film that is artificially colored in red.The area of the colored region is ≈15 mm 2 .
left panel.In contrast, under transmission mode of the microscope, angle-dependent birefringent patterns are exclusively observed,

Figure 3 .
Figure 3. Color pattern analysis in different lighting conditions.Crossed polarizers are used when observing the sample in both the reflection mode and transmission mode of the microscope.illuminate the sample with specific colors, red, green, and blue color filters are inserted before the polarizer.Images are captured in both modes at the same position of the film.The film is horizontally rotated from 0 o to 90 o , as illustrated on the left.During the rotation, the color produced from the cholesteric structure remains the same, while the birefringence patterns depend on the sample orientation.The scale bar in the images is 400 μm.

Figure 4 .
Figure 4. Tuning reflected color by manipulating cholesteric pitch in amyloid CLC films.a) Comparison of the pitch of the amyloid CLC suspension in water, prior to evaporation.pitch of the pristine amyloid CLC suspension increases with increasing the mass ratio between glucose and amyloid fibrils.b) Illustration of the right-handed helix in amyloid CLCs (liquid state).c) Thickness distribution of amyloid CLC film prepared by fast evaporation process in vacuum.The inserted microscope image shows the cross-section of the film that is artificially colored in red.The area of the colored region is approximately 28 mm 2 .d) Microscope images taken with reflection mode show that the film displays uniform colors of blue, green, and red under polarized illumination.The arrows indicate the positions where the RGB color intensities are analyzed in e).f) The visible spectra show a clear shift of the reflection peaks from ∼450 nm (blue color), to 548 nm (green) and to ≈680 nm (red).Measurements with the glass and air are shown as controls.g) The corresponding SEM images of the cross-section of the dried films show layered structures with different pitches, immediately related to the wavelength of the reflected light via Bragg's Law.The scale bar is 0.5 μm.