Bioinspired, Cholesteric Liquid-Crystal Reflectors with Time-Controlled Coexisting Chiral and Achiral Structures

: The twisted structures of the chitin-based cuticle of beetles confer on them specific optical characteristics. Intrigued by the observation of Bragg gratings with a depth-dependent periodicity in the cuticle of Chrysina beetles, we determine the experimental conditions leading to their transcription into cholesteric liquid crystal oligomers. We correlate the optical properties of reflectors thus produced with their internal morphology, as observed by transmission electron microscopy. With the use of a single parameter, the thermal annealing time, the reflection color is made time-tunable. Different spectral bands and reflection colors from golden yellow to NIR are available, and irreversibility of the final color is reached at the end. On the basis of the design concept and these properties, these hybrid chiral-achiral materials inspire the fabrication of smart reflective labels. When encapsulated in the package of a product to be kept in cold conditions, the label records the history of the product conservation. Two kinds of information based on color changes are recorded: qualitative information reporting that the product was kept outside of the specified storage temperature and

properties of reflectors thus produced with their internal morphology, as observed by transmission electron microscopy.With the use of a single parameter, the thermal annealing time, the reflection color is made time-tunable.Different spectral bands and reflection colors from golden yellow to NIR are available, and irreversibility of the final color is reached at the end.On the basis of the design concept and these properties, these hybrid chiral-achiral materials inspire the fabrication of smart reflective labels.When encapsulated in the package of a product to be kept in cold conditions, the label records the history of the product conservation.Two kinds of information based on color changes are recorded: qualitative information reporting that the product was kept outside of the specified storage temperature and

INTRODUCTION
Main features of cholesteric liquid crystals.Cholesteric liquid crystals (CLCs) are chiral materials that occupy a prominent place in the field of photonic crystals and optical materials, generally speaking, because of their helical molecular organization and specific properties [1][2][3][4] .
First, CLCs exhibit double selectivity in wavelength and polarization.A cholesteric material may selectively reflect light, and the mean reflection wavelength 0 is related to the helical pitch p (periodicity, which represents a 360° rotation of the orientation of the rod-like molecules) by Bragg's law: 0 = npcos where n is the average refractive index and  is the angle between the light propagation direction and the helical axis.Second, unlike common mirrors, which reverse the polarization of the light they reflect, the cholesteric planar texture reflects circularly polarized light with the same handedness as the twist of the helix, while light with the other polarization (50% of the ambient unpolarized light at the selected wavelength) is transmitted and possesses the inverse handedness.This rule is known as the polarizationselectivity rule, and up to 50% of the ambient unpolarized light at the selected wavelength can be reflected.Third, an exceptional optical rotation may occur, up to 10 3 to 10 5 °/mm in the visible spectrum, compared with typical values between 0.01 and 100 °/mm for a chiral liquid (sugar water) or crystal (quartz).The wavelength regions for this optical activity are separated by the Bragg band and have opposite signs of rotation.A properly oriented slab of cholesteric material is thus a multifunctional material by acting as a selective filter, a vibrant-color reflector, a polarizer and an optical rotator.

Insect cuticles as cholesteric reflectors.
Light reflection in the animal kingdom is diverse and ancient 27 .The earliest known reflectors, diffraction gratings, are 515 million years (Ma) old.Fossil beetle specimens ranging from 15-47 Ma in age still reflect light 28 because structural coloration is generally permanent and does not bleach like pigment colors.CLC structures are omnipresent in living matter, and many studies on this topic have focused on the insect cuticle 29 .
The cuticle covers the insect body and consists of three main layers-epicuticle, exocuticle and endocuticle-secreted by a single layer of epidermal cells 30 .Both the exocuticle and endocuticle involve twisted CLC organization of chitin macromolecules, which causes a spatially varying index of refraction 31 .Chitin macromolecules form fibrils that wrap with proteins, and these fibril-protein associations assemble into fibers.Fibers assemble into bundles, which arrange parallel to each other to form pseudolayers.Finally, the pseudolayers assemble into a cholesteric structure, often referred to as the twisted plywood model from Bouligand 32,33 (Figure 1a).A continuous twist occurs along the axis perpendicular to the fibers, which interpenetrate from one pseudolayer to the next (this is why the term pseudolayer is preferred over layer).A 360° rotation of the fiber orientation defines the helical pitch.When the structure is observed by optical or electron microscopy in a direction perpendicular to the helical axis, a fingerprint texture composed of parallel stripes may appear (as in Figure 1c).The distance between two identical stripes is related to the half-pitch of the twisted organization, not to the full pitch, because a 360° rotation of the molecular orientation defines the pitch (Figure 1a).
In the search for biomimetic or bioinspired materials and devices, the structures and optical properties of insect cuticles are a first-rank source of inspiration for material scientists 34,35 .
Motivation of the research.The motivation of our research arises from the observation of electron microscopy images of cuticular structures in scarab beetles from the genus Chrysina, which show vibrant reflections from bright green to metallic silver-gold, including broadband reflections 36 .Here, we refer to the multiband-green and silver-cuticle of Chrysina gloriosa (Figure 1b) and the golden yellow cuticle of Chrysina aurigans (Figure 1f).Both cuticles of C.
Accepted version in: ACS Applied Materials & Interfaces 2021, 13, 30118-30126.6 gloriosa 37,38 and C. aurigans [39][40][41][42] are broadband reflectors from the beginning of the visible spectrum to the NIR spectrum.To our knowledge, the nature of the organization in the border zone (localized by pink frames in Figures 1c, d, e and g) between the end of the twisted region (fingerprint texture) and the epidermis has not been discussed in the literature.Continuity of the variation in the helical pitch in the thickness direction of the cuticle and of the orientation of the helical axis in the case of C. gloriosa 43 remarkably occurs.It could thus be assumed that the end of the twisted structure does not occur abruptly but instead follows a structural gradient, as suggested by the image in Figure 1e showing the lowest stripes of the endocuticle of C. gloriosa: the stripes' contrast gradually fades, and then, the stripes disappear by being overwhelmed in a nonpatterned area.These incomplete lines embedded in a nonpatterned matrix could correspond to a frustrated CLC structure-prevented from complete twistingcoexisting with an unwound helix.Then, these lines are followed by an area without a pattern over a small thickness, which could correspond to a fully unwound helix, an area for which the helical pitch diverges, i.e., an achiral nematic LC (NLC) structure forced by geometric conditions that prevent the free development of a large-pitch helical structure.In this case, it would ensure a soft end to the twist of the chitin molecules.From the top to the bottom, the transverse structure of the chitinous part could thus consist of the coexistence of a pitch-gradient structure followed by a frustrated twisted structure and then a fully unwound helix, with the whole architecture being realized without the requirement of interfaces.
Our goal is not to answer stimulating questions on the morphogenesis of cuticular reflectors but instead to take inspiration from the above observations to design photonic materials with a controlled balance between chiral and achiral structures inside a single material exhibiting no discontinuity or interface-as in nature.We present a simple and effective method to fabricate bandgap-tunable reflective materials whose versatility of the characteristics of the Bragg band does not depend on temperature or external parameters but is underlain by this bioinspired approach.An optimal use of resources and a reduced number of stages during the fabrication procedure are targeted, in the spirit of eco-design as in nature.We aim to investigate the reflectance and transmittance properties of the bioinspired cholesteric materials in close relation to their transverse structure, as observed by TEM.Finally, a concrete use of materials based on the design of hybrid (chiral-achiral) structures in the field of time-temperature indicators is presented and discussed.

RESULTS
Formulation of LC mixtures.To formulate the CLC and NLC films in relation to our research objectives, we used LC photopolymerizable and crosslinkable oligomers with a siloxane backbone (Figures 2a and b) 44 .They consist of a cyclic siloxane ring to which two types of side chains are attached via aliphatic spacers: an achiral mesogen and a chiral cholesterol-bearing mesogen (Figure 2a) or only achiral mesogens (Figure 2b).When formed into a thin film with a planar texture (the helical axis is perpendicular to the film), the reflection  The thickness of the CLC films (14.0±1.0 m) was chosen to be almost twice the thickness of the NLC (7.5±0.5 m).Spacers 12.5 m thick were inserted between the plates.After closing the cell at room temperature, the open surfaces of the layers were in contact.The cell was then placed on a heating stage at 60°C.To describe the time-dependent optical and structural properties of the system, five scenarios are presented in which the annealing time was varied from 20 s to 50 min.Placing two LC substances between a pair of transparent substrates into contact to generate a concentration gradient allows us to obtain a qualitative overview of some optical textures present in the mixture 45 .Figure 3b displays an optical micrograph showing a concentration gradient between the CLC and NLC compounds in the x-y plane of the substrates.
The purpose is to find the conditions for generating a temporal concentration gradient between the layers of chiral and achiral materials in the z-direction perpendicular to the layers; this event has to occur at a thickness several dozen times thinner than the height of the region imaged in Figure 3b.
When the targeted time is reached, the soft material is polymerized and crosslinked with UV light (see the Experimental section).The optical properties of the material are thus fixed into an elastomeric layer, which can be cut in a transverse direction in 150-nm-thick slices to allow TEM imaging of its internal structure.The reflectance is displayed between 0 and 50% with regard to the polarization-selectivity rule (see Introduction); the signal level of the spectra is 8-10% due to the reflectance of the cover glass plate (which was not taken into account in the baseline).The mean wavelength of the Bragg band allocated to the golden yellow CLC film is 585 nm.The bandgap limits MIN and MAX (Figure 5), determined at the half-height of the reflection peak, are 555-620 nm (CLC film), 560-620 nm (20 s), 560-615 nm (1 min) and 560-625 nm (2 min), which means that the diffusion between the CLC and NLC films up to 2 min is not sufficient to significantly modify the position of the Bragg band inherent to the CLC film.A clear-cut bandgap shift is visible at 20 min in Figure 4, with a mean reflection peak at 650 nm (red color).Some light reflection is present above 700 nm (the signal level does not return to 10%): as shown in the following section by TEM, the 20-min sample exhibits a pitch gradient; however, the number of helix turns ascribed to the twist in this spectral region is too low to allow a significant contribution to the Bragg band from this part of the pitch gradient.The reflectance depends on the number of pitches (i.e., the number of helix turns), which means that the twist needs to develop within a minimal film thickness to enable the appearance of Bragg reflections that are detectable under standard spectrophotometry conditions 46 .Annealing the cell for 50 min gives rise to an enlarged Bragg band with MIN and MAX equal to 690 nm and 915 nm, from dark red to near-IR.The bandgap  = MAX -MIN varies from 65 to 220 nm from the pristine golden film to the 50-min red film (inset in Figure 5).The present set of annealing times thus allows the fabrication of different cells with a structural color ranging from golden yellow to the near-IR for a single material with the same chemical composition from one cell to another, the difference only lying in the time.An NLC is usually considered a CLC with an infinite pitch, and the pitch of a CLC can be augmented by adding a quantity of NLC 47,48 .Here, spatially directed dissolution of CLC and NLC layers occurs in the transverse direction inside a single material, similar to the result of the design of a gradient-structure material.From 0 s to 50 min, MAX undergoes a stronger amplitude increase than doesMIN: 295 nm vs. 135 nm (Figure 5).It may be expected that this graded stretching of the Bragg band from its red part is the consequence of the graded dissolution between a constant-pitch cholesteric LC and an infinite-pitch cholesteric LC.
Transmission spectra and textures are given in Figure S1.Negative peaks occur in spectral bands comparable to those of the positive peaks assigned to the reflection.Transmission investigations mainly provide additional information about the evolution of the transmittance with time (details are given in the caption of Figure S1).

TEM investigations of the internal structure of different CLC-NLC films. By taking
advantage of the solid nature of the material, TEM allows us to correlate the temporal behavior of reflection properties with the internal structure of CLC-NLC films.TEM imaging of cholesteric polysiloxane-based oligomers reveals the typical fingerprint texture 49 , similar to observations made by optical microscopy (Figure 3b).However, the periodic bright-and-dark contrast in TEM is the result of several processes, including mainly mass loss during irradiation acting as selective etching 50 .Figure 6 shows the TEM textures of cross-sections accompanied by the variation in the half-pitch (distance between two bright stripes) along the depth.Different regimes in the thermal diffusion between the chiral and achiral layers are revealed.
Let us first remark that the sample thickness may differ from one case of annealing to another (Table S1), in particular because the film laterally spreads out when the annealing time increases.For this reason, we choose for the abscissa a normalized range (depth over the total thickness of the sample) to display the pitch variation for comparison from sample to sample of the pitch variation in the whole volume of the sample.The zero abscissa is chosen as the top of the CLC film.At a short annealing time (20 s), the whole set of bioinspired, target structures is visible (Figure 6a).From the CLC layer (at the left side of the image) to the NLC layer, the following structures successively occur: a fingerprint texture related to a constant pitch of approximately 360 nm, a frustrated fingerprint texture that materializes as deformed and larger bright-and-dark cholesteric fingers, an unwound cholesteric helix that materialize as a bright halo without stripes and the pure NLC (for reference, the TEM image of the NLC material alone is shown in Figure S2a).The term cholesteric fingers is often used in the case of strong frustration of the helical structure when the stripes are separated by homeotropic regions, which correspond to local unwinding of the helix with molecules perpendicular to the surfaces of the CLC film 51,52 ; the helix is unwound when the film thickness is close to the pitch or above it.At 1 min (Figure 6b), the fingerprint texture with a near-constant pitch is followed by a region corresponding to a CLC-NLC gradient in which the helix is frustrated; this event occurs over a thicker thickness compared to the same kind of region at 20 s (this is the result of progressive diffusion).The textural pitch progressively increases and gives rise to umbilical structures for which the direction of the stripes is no longer parallel to the film surfaces.The NLC layer disappears due to it being consumed in the course of rapid thermal diffusion.The situation at 2 min (Figure 2c) is quite similar; the curve shows that the stripe periodicity is lowered compared to that at 1 min.A third situation is shown at 20 min (Figure 6d): the sample no longer includes frustration of the helix but instead a pitch gradient from 430 nm to 1000 nm, from approximately 32% of the thickness to the end of the film.Finally, a cholesteric structure with a nearly constant 540-nm pitch is obtained at 50 min (Figure 6e).The pitch value is 1.5 times larger than that inherent to the CLC film, similar to the result of full mixing of CLC and NLC layers.
To summarize, the annealing process presents four stages: (a) the coexistence of a constant-pitch helix, a frustrated helix, a (nematic-like) unwound helix and a nematic structure made from the pure NLC compound; (b-c) the coexistence of a constant-pitch helix followed by a frustrated helix; (d) the coexistence of a constant-pitch helix (over approximately one-third of the thickness) followed by a pitch-gradient helix; and (e) a quasi-constant-pitch helix.

DISCUSSION
The association of LC layers was realized in the literature by using cholesteric layers with the goal of broadening the characteristics of the Bragg band 3 , mainly the wavelength band.Two cases were considered with regard to the discontinuous or continuous (interface-free) nature of the final architecture: stacking of layers leading to a multilayer system 53,54 and thermal diffusion between a pair of layers with distinct Bragg bands, in which diffusion aims to broaden the bandgap by covering the pristine bandgaps and the wavelengths in between 55,56 .Our system differs from the latter case by involving a chiral layer along with an achiral layer and a diffusion process leading to different cases of narrow-band Bragg reflectors.This last characteristic is relevant for applications aimed at the field of time-temperature indicators that we discuss below.
We searched for an experimental protocol in which the reflection band was tunable by controlled interpenetration of cholesteric and nematic layers.The first layer defines the left limit of the Bragg band and is not intended to stay fixed in the course of the annealing process.The role of the second layer is to allow different scenarios for the behavior of the Bragg band that are not achievable with two CLC layers: increasing the helical pitch up to an infinite pitch, realizing a variable-pitch twisted structure, and finally realizing a constant-pitch CLC.
Time-temperature indicators record the thermal history 57 .Our design inspired us to realize such smart devices on the basis of the graded, irreversible evolution with temperature and time of the label color (i.e., the pristine bilayer of the present study encapsulated in a plastic matrix or carrier).Usual temperature-variable cholesteric labels (LC thermometers 58 ) do not have this dual property with added irreversibility, which is of paramount importance for this kind of application.We may name these indicators cholesteric cookies by analogy with the web cookies designed to record a user's browsing activity.When tagged on the package of a food product or medicine-such as vaccines to be stored and transported under ultracold conditions 59 -to be kept in a precise low temperature range, the color of the cholesteric indicator is frozen because the diffusion between layers is forbidden (in our case, the glassy transition temperature of LC oligomers under study is approximately 0°C).When the temperature of the product goes beyond a critical value defined in relation to the product specifications, the label color changes, and this color evolves with time.These color changes reveal to the potential user that the thermal chain has been broken and, in addition, could give an indication of the time since the temperature exceeded the storage temperature range of the product.The user would have at her/his disposal a color chart giving the relationship between color, temperature and time.The fact that the label will never return to its initial color, regardless of what the handler does, is fundamentally based on the physical interdiffusion between chiral and achiral materials.In addition to a temperature-time tunability stage, an irreversibility stage is necessary.Formulation of the properties of the label layers (chemical composition, thickness) in their native state in relation to the temperature-color variation desired by the manufacturer is required.

CONCLUSIONS
We have designed wavelength-selective cholesteric reflectors by determining the experimental conditions leading to a fine balance between helical, distorted helical and unwound helical structures in the bulk of an interface-free polymer film.Four scenarios were described: (i) constant-pitch helix-frustrated helix-unwound helix (forced nematic structure)-stable nematic structure; (ii) constant-pitch helix-frustrated helix; (iii) pitch-gradient helix; and (iv) monotonic-pitch helix.Obtaining one scenario or another scenario at a constant temperature depends on a single parameter-time.Different spectral bands and reflection colors from golden yellow to NIR are available, and irreversibility of the final color is reached at the end.
The combination of these properties precisely inspired us to use the concept of chiral-achiral hybrid structures for the manufacturing of cholesteric labels.When encapsulated in the package of a product to be kept in cold conditions, the smart label records the history of the product conservation.Two kinds of information based on color changes are recorded: qualitative information reporting that the product was kept outside of the specified storage temperature and quantitative information giving an indication of the time elapsed since the temperature exceeded the storage temperature of the product.

EXPERIMENTAL SECTION
Specimen of Chrysina gloriosa.A male specimen of Chrysina gloriosa was collected at Madera Canyon (Santa Cruz County, Arizona, USA) in July 2014.the photoinitiator Irgacure © 907 and 2.0 wt.% of the thermal polymerization inhibitor BHT were added.A 14±1-m-thick sandwich film of CLC was formed between two treated glass plates and annealed at 80°C.A 7.5±0.5-m-thicksandwich film of SN was formed between two plates and annealed at 100°C.The thickness was measured by the interference method (based on the wave interference due to the internal reflection between the two inner surfaces of the cell substrates 60 ).The dispersion in thickness of the individual CLC and NLC films constituting the five samples was kept as low as possible.Special care was taken to ensure that the NLC films had a monodomain texture (Figure S2b) and exhibited a very high transmission (90%-Figure S3).To remove one plate and obtain open films while avoiding the formation of scratches on the surface of the film, the sandwich cells were kept at low temperature in a refrigerator; one of the plates was sharply removed from the film with the aid of a Handi-Vac ® coverslip pick-up tool (Ted Pella, Inc.).That the free surface of the film remained smooth after the operation was checked by optical microscopy.
Polymerization conditions.The films were placed at 60°C on a heating stage and irradiated with UV light for 10 minutes with a maximum intensity at 365 nm equal to 0.50 mW/cm 2 .
Inclusions of synthetic samples for ultramicrotomy.To obtain free-standing films ready to be embedded in a piece of resin, each sample was placed in a vial of distilled water for 1 day.
Due to the presence of water-soluble PVA layers, the films detached as a block from the plates while maintaining clear-cut surfaces, as verified by TEM transverse views.A small piece of material was embedded in EMbed-812 resin (Electron Microscopy Sciences), which was then cured at 62°C for 2 days.
Accepted version in: ACS Applied Materials & Interfaces 2021, 13, 30118-30126.20   Inclusions of biological samples for ultramicrotomy.The cuticle samples corresponded to the elytra and were removed with a razor blade.A small piece of material was embedded in EMbed-812 resin (Electron Microscopy Sciences), which was then cured at 62°C for 2 days.
Preparation of biological samples for TEM by ultramicrotomy.A diamond knife at ambient temperature was used to cut 80-nm-thick slices (Figure 1c) or 120-nm-thick slices (Figure 1d) with a Leica UCT ultramicrotome.The samples were cut perpendicular to the film surface (cross-sections).Slices were placed on single-slot Formvar-coated copper grids (GS2x1-C3, Gilder Grids Ltd.) and stained with UranyLess (brand mixture of lanthanides from Delta microscopy) for 20 min and with 3% Reynolds lead citrate (from Chromalys) for 5 min.

Preparation of synthetic samples for TEM by ultramicrotomy.
A diamond knife at ambient temperature was used to cut 150-nm-thick ultrathin slices with a Leica UCT ultramicrotome.
The material was cut perpendicular to the film surface (cross-sections).Slices were placed on single-slot Formvar-coated copper grids (GS2x1-C3, Gilder Grids Ltd.).TEM conditions.A Jeol JEM-1400 microscope operating at 80 kV equipped with a Gatan Orius SC1000B CCD camera was used.

Periodicity profiles from TEM images. Quantitative image analysis was performed with
DigitalMicrograph software from Gatan.The intensities of the gray levels on a given surface of the images (typical surface = 8x18 m 2 ) were analyzed using the Profile option.The distance between two consecutive positive or negative peaks corresponded to the distance between two successive stripes of the same contrast in the fingerprint texture.Positive (resp.negative) peaks correspond to bright (resp.dark) stripes because the transmitted light is higher (resp.lower) in

Figure 1 .
Figure 1.Origin of bioinspired reflectors.(a) Transverse cut of the twisted plywood model photoinitiator e and 2.0 wt.% thermal inhibitor f were added.

Figure 3 .
Figure 3. Representation of the structure gradient to target along with the experimental cell

Figure 4 .
Figure 4. Reflection properties of the cells for different annealing times and at normal

Figure 5 .
Figure 5. Bandgap limits of reflection spectra as a function of the annealing time.Inset:

Figure 6 .
Figure 6.Structural analysis of samples.TEM transverse views accompanied by the half-pitch