1. Introduction

Security printing and hidden images play a vital role in protecting against counterfeiting, ensuring the authenticity of documents and products, and enhancing security measures in various industries, including fashion, luxury goods, electronics, pharmaceuticals, automotive parts, and more. The most severe global problem is pharmaceutic drug counterfeiting [1,2]. One the most important examples of hidden images include banknotes with hidden symbols or patterns, passports and ID cards, product labels revealing buried logos or security marks, official documents containing light-sensitive fibers, and more.

The physics and technology behind hidden images rely on two closely intertwined yet contrasting optical phenomena: fluorescence and photobleaching. Fluorescent hidden images are created using special inks that emit visible light when exposed to ultraviolet (UV) light [3] or nanomaterials and among them cadmium-containing quantum dots or perovskite-based composites. [4,5,6]

This optical encoding is developed using fluorescent materials of different origins, such as quantum dots, carbon dots, organic dyes, lanthanide nanocrystals, DNA (or RNA). [7] Although these fluorescent materials exhibit high quantum yield and tunable excitation and emission wavelengths, they are subjected to strong photodamage. Their intrinsic photobleaching effect leads a low photostability and significant noise-limiting FL images application. [2,8]

The once conventional perception of the photobleaching effect as a restriction in fluorescence imaging has shifted, as it has emerged as a valuable asset for hidden imaging applications in encoding microcarriers by spatial selective photobleaching. [9] The photobleaching effect was also applied in fluorescence microscopy for super-resolution imaging. [10] In this technique, a small region of a sample is illuminated with a high-intensity laser, causing the fluorophores within that region to undergo photobleaching. By imaging the sample before and after photobleaching, it is possible to determine the precise location of each fluorophore within the region, allowing super-resolution imaging beyond the diffraction limit of light. In addition, photobleaching can be used for hidden imaging by selectively bleaching a fluorophore in a hidden location, such as within a complex sample or living tissue. This technique can reveal the location of the hidden fluorophore and provide information about the structure or function of the tissue. [2,9,10]

In this work, we revealed the photobleaching effect in a new class of FL nanomaterials, peptide FL nanodots, [11,12] and applied it for high-resolution hidden imaging. In the context of the present study, the term “nanodot” refers to nanoscale (1-100 nanometers) peptide structures with a roughly spherical shape. Recording of the hidden encoding images was performed with the developed by us thin polyvinyl alcohol (PVA) polymer films incorporating a densely arranged array of FL peptide nanodots. This new technology based on irreversible photo-induced damage allows to tailor highly stable photo-bleached patterns and barcodes as well hidden images for long-term optical memory both for security printing as well for nanoscale imaging of biological tissues, single cells, bacteria, and viruses, as well as in-vitro and in-vivo monitoring of functional biosystems. The developed FL bionanodots [11,12] are originally biocompatible and can be used in these biology-related applications.

2. Fluorescent peptide nanodots

Self-assembly is the fundamental mechanism in biological and bioinspired materials that enables biological elementary entities to interact with one another, forming supramolecular structures of various origins. The elementary building blocks of these biostructures are nanometer-scale particles. These bio-nanodots were first observed in bioinspired short peptides composed of two phenylalanine residues, self-assembling into bioinspired diphenylalanine nanotubes and in biological human insulin. [13] It has been discovered that the self-assembly process of nanodots into micrometer tubes and the disassembly process of the tubes into the dots are reversible, rendering the peptide dots as independent nanotechnological units. These biological building blocks-bio-nanodots, self-assembling into various proteins and peptide structures, demonstrate significant crystallinity and are regarded as promising components for various technological applications. [10] They can be integrated into bio-electronic devices. One of these applications suggests employing these structures as ultrasmall charge-storage elements in non-volatile memory devices. [14]

This paper considers nanoscale optical labeling using FL bio nanodots for encrypting technology, a promising bioimaging tool. The state of the art in FL bioimaging relies on using naturally biocompatible genetically encoded fluorescent proteins, the green fluorescent protein, and its homologs. [15] The fluorescence emission spectra of such proteins depend on the dipole-allowed relaxation paths of their excited electrons, which are determined by the proteins molecular composition and electronic structure.

Another bio-fluorescence effect was discovered in biological [16], amyloid, and bioinspired [17] amyloidogenic peptide/protein structures. We demonstrated that the thermally-induced refolding of the bioinspired ultrashort peptide structures into specific β-sheet secondary architecture unexpectedly results in acquiring of a new biophotonic property of the visible FL. [11,17,18,19] This fold-sensitive FL effect exhibits a wideband tunable spectrum ranging from 400 to 650 nm, with a relatively high quantum yield (QY) of up to 30%. [11] The observed phenomenon in synthetic peptide/protein β-sheet structures, particularly those with amyloidogenic properties, [20] resembles the FL generated by amyloid β-sheet fibrils associated with neurodegenerative diseases such as Alzheimer's and Parkinson's [16,21]. Therefore, our findings confirm a unique and common FL mechanism in amyloid and amyloidogenic synthetic structures, irrespective of their biochemical composition, primary structure, or origin. [20] It should be noted that the described in this paper FL imaging technique is based on the use of inherently non-toxic, bio-compatible peptide nanodots.

This biophotonic effect is utilized to develop a new class of fluorescence biomarkers based on β-sheet peptide/protein supramolecular nanostructures wrapped in amyloid bio-nanodots. [11] The originally non-fluorescent peptide/protein nanodots of various biochemical compositions (aromatic and non-aromatic) have been subjected to thermally-induced refolding into highly fluorescent β-sheet enriched nanostructures. [11,12,20] The photo-physical properties of the individual bio-nanodots have been characterized in two modes: by measurements of FL intensity and energy spectrum of biodots arrays [11] and single-particle nanodots using fluorescence microscopy and spectroscopy techniques. [12] It has been demonstrated that the tunability of the FL emission spectrum of the peptide nanodots in a solution originates at the single emitter level. The study of the FL temporal dynamics of single nanodots revealed a relatively short FL lifetime of 1.27 ± 0.03 ns. [12]

3. Results and discussion

3.1 Thin polymer films with incorporated FL peptide nanodots

In this work, we developed a new FL material for advanced photo-bleaching technology to tailor and read hidden and patterned images. This development involves three consequent stages: fabrication of originally non-fluorescent peptide bio-nanodots, a thermally-induced transformation of these dots to a fluorescent beta-sheet dots structure, and PVA polymer films incorporating the FL dots. Tri-phenylalanine (FFF) peptide nanodots (Atomic Force Microscopy (AFM) image, Fig. 1(a)) were prepared by bottom-up self-assembly approach [10] from FFF-bioorganic solvent provided inhibition of the self-assembly process at the dots-stage. Height distribution of the FFF-nanodots obtained from AFM measurement exhibited the average Z-dimension of the FFF-dots ∼10 nm. [11] At this preliminary stage, these dots do not demonstrate any FL effect in the visible range. At the next stage, the FFF-dots were subjected to thermal treatment in organic ethylene glycol solvent using slow heating to 160 °C for 1 hour. This treatment allows to refold original alpha-helical secondary structure of the FFF-dots to the beta-sheet one. The thermally-induced reconfirmation creates in these beta-sheet bio-nanodots visible FL properties. [11,20] At the last stage, the FL FFF-dots were embedded into PVA film by using spin-coating technology (Fig. 1(b)) resulted in the fabrication of the thin PVA/FL-dots film deposited on a glass substrate (Fig. 1(c)) (the details of the process can be found in the Experimental section).

 

Fig. 1. PVA polymer thin films with embedded tri-phenylalanine beta-sheet peptide nanodots and their photo-bleaching properties. (a) Atomic Force Microscopy images of tri-phenylalanine (FFF) nanodots. (b) scheme of spin-coating technology of polyvinyl alcohol (PVA) polymer films with embedded peptide nanodots. (c) Secondary Electron Microscopy image of cross-section of PVA polymer films with embedded peptide nanodots. (d) Fluorescence (FL) spectra of PVA films with embedded FL peptide nanodots for different excitation intensities: (1) 0.9 W/cm2; (2) 1.2 W/cm²; (3) 2.0 W/cm²; (4) 2.6 W/cm² (5) 3.3 W/cm2. (e) Photo-bleaching in PVA films with embedded FL peptide nanodots: variation of FL spectra measured in 5 min intervals under continuous laser excitation. (f) Photo-bleaching in PVA films with embedded FL peptide nanodots. Integrated FL emission intensity measured from PVA polymer thin films with embedded tri-phenylalanine FL peptide nanodots under different laser excitation intensities: (1) 3.3 W/cm²; (2) 2.6 W/cm²; (3) 2.0 W/cm²; (4) 1.2 W/cm2; (5) 0.9 W/cm². (g) Comparison between FL spectra of Alexa Fluo 405 dye, FFF nanodots, and GFP.

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3.2 FL spectra of PVA/FL-dots films

Optical measurements of FL spectra were conducted with PVA/peptide FL dots film samples deposited on a glass substrate (Experimental section). The FL spectrum is acquired during ∼1.5 seconds of excitation to avoid a photo-bleaching effect. Figure 1(d) demonstrates a series of FL spectra for five different levels of excitation intensity in the range (0.9-3.3) W/cm² measured from PVA/FL-dots films (excitation wavelength is 405 nm). The higher the excitation intensity, the stronger the fluorescence is. The maximum FL intensity is observed at 465 nm wavelength. The fluorescence spectra spread across the range between 450 nm to 650 nm (the short FL wavelength spectra less than 450 nm are cut off due to the long-pass filter that blocks the excitation laser radiation of 405 nm). The observed FL spectral range (Fig. 1(d)) is similar to those studied in the bioinspired and biological peptides/proteins folded into beta-sheet structures regardless of their origin and chemical compositions. These FL spectra can be regarded as an optical signature of the beta-sheet secondary structure. [20]

3.3 Photobleaching effect in PVA/peptide FL nanodots

In this work, we revealed the photobleaching effect in thin polymer PVA films with embedded FL peptide nanodots and applied it for direct micro-patterning, encoding hidden images. While studying the spectral properties of the peptide thin films, we observed that the FL intensity of the evaluated samples gradually fades under continuous illumination by the excitation laser beam. The experimental studies of the photobleaching in FL peptide nanodots were performed using a high-grade optical spectrometer equipped with a low-noise cooled linear array sensor. FL excitation in the applied PVA polymer film incorporating FL peptide nanodots was achieved by focusing a beam of a laser diode with a wavelength of 405 nm. (Experimental section).

Figure 1(e) demonstrates the primary results of the photobleaching effect observed under continuous laser illumination (excitation intensity 4.5 W/cm²) for two FL spectra measured in 5- minutes intervals. Both FL curves cover a similar spectral visible range and exhibit spectral maxima at unchanged wavelength positions. The detailed bleaching process (Fig. 1(f)) is presented by five graphs acquired for five different levels of the laser excitation intensities (0.94-3.32) W/cm². Integrated over the entire spectral range, the FL emitted power gradually decreases and exhibits FL bleaching in ∼300 sec. Conducting FL studies in the mode “switching off/on” of the excitation laser showed that photo-bleaching in the studied FL peptide dots is irreversible.

Photobleaching is a phenomenon that occurs when a fluorophore loses its ability to emit fluorescence due to prolonged exposure to excitation light. [9,10,22] It is a well-known and commonly considered problem in high-resolution fluorescence microscopy [23]. This undesirable outcome in fluorescence imaging was studied and applied for various applications, particularly in the field of hidden imaging. One such application is in fluorescence microscopy for super-resolution imaging. [24] Another application of the photo-bleaching effect is related to the deep modification of functional surface properties for creating diverse high-resolution spatially selective patterns. [2,3,8,25] It involves spatially selectively photo-bleached certain areas using light irradiation to alter the photo-physical and photochemical properties through the photo-bleaching of fluorophores originating from different sources.

Photobleaching characteristic time is the most critical parameter for technological applications in hidden imaging and photobleached-patterned structures. Its fundamental mechanism considers diverse photo-physical and photochemical reactions such as photooxidation, photodegradation, and photobleaching processes initiating molecular triplet-state formation and oxidative processes, which indicate the breaking and restructuring of fluorophores’ chemical bonds. The characteristic time of the photo-bleaching process can vary depending on the specific fluorophore (quantum dots, organic dyes, fluorescent proteins) being used. Understanding these mechanisms is crucial for harnessing photobleaching for hidden imaging applications effectively. [9,10,23,24]

Quantum dots (QDs): Quantum dots are nanoscale inorganic semiconductor particles that exhibit unique optical properties associated with the quantum confinement effect. They are known for their high photostability, which means they have long photo-bleaching times that could reach for extended periods, from minutes to hours. Organic dyes: Organic dyes are commonly used as fluorophores in various imaging applications. Some dyes may exhibit relatively rapid photobleaching, while others may be more photostable and exhibit slower photobleaching rates. The photobleaching times for organic dyes vary significantly, ranging from milliseconds to minutes. Fluorescent Proteins: fluorescent proteins, such as green fluorescent protein (GFP), are genetically encoded fluorophores commonly used in biological imaging. The fluorescent protein photobleaching times depend on the specific variant and environmental conditions. Some fluorescent proteins can exhibit relatively fast photobleaching, while others are more photostable and have longer photobleaching times. GFP’s photobleaching time ranges from minutes to hours.

The characteristic time is large in the studied beta-sheets peptide nanodots embedded into PVA polymer films. It reaches hundreds of seconds, τ ∼300 sec in the range of the excitation power (0.9-3.3) W/cm² (Fig. 1(f)). The mechanism of visible FL in the beta-sheet peptide/protein amyloid and amyloidogenic structures of different origins, compositions, and scales is similar and related to intermolecular hydrogen bonds binding peptide/protein molecules into beta-sheet elongated structures. [21]

The general concept of the FL-beta-sheet supramolecular arrangements considers peptide/protein structures as two subsystems within biomolecular organizations. One subsystem comprises non-fluorescent peptide/protein biomolecules, while the other involves a network of intermolecular noncovalent hydrogen bonds that bind these biomolecules together, forming a stable beta-sheet structure. The basic alpha-helical-to-beta-sheet refolding phenomenon preserves the composition of biomolecules but restructures the hydrogen bonds. This refolding converts the intramolecular hydrogen bonds of the native state into intermolecular hydrogen bonds in the beta-sheet state. Molecular dynamics simulations [23,24] have demonstrated that the reconstructed H-bonds within the beta-sheet peptide/protein structures enable proton transfer between the N- or C-terminus, forming a double-well ground state potential. This precise modification of the electronic structure of the intermolecular hydrogen bonds is responsible for the visible FL in the beta-sheet state [26].

In this study, it was discovered that the light-induced effect of the FL-bleaching (Fig. 1(e),(f)) results in the complete decay of visible FL-centers within the beta-sheet peptide structures. This decay signifies the destruction of their intermolecular noncovalent hydrogen bonds. It is worth noting that the beta-sheet peptide structure can be light-switched by ultrashort light pulses, forming unfolded clusters. This observation indicates the disruption or weakening of hydrogen bonds and the consequent generation of peptide structures with broken hydrogen bonds. [27] The same effect of disassembled amyloid Ab-protein aggregates associated with Alzheimer's disease was observed by destabilizing the beta-sheet secondary structure under illumination by white light-emitting diode light. [28]

In Fig. 1 g, the normalized FL spectrum of FFF peptide nanodots is shown in comparison with emission spectra of established fluorophores Alexa Fluor 405 and GFP. [29]

3.4 Recording hidden images

In order to pattern the photo-bleached areas, the fragments of 2D masks were illuminated by the excitation diode laser beam and projected on the surfaces of the fluorescent peptide dots PVA films by the 40X microscope objective. The resolution test targets are typically used to estimate the resolution of an imaging system (Fig. 2). They consist of reference line patterns with well-defined thicknesses and spacings and are designed to be placed in the same plane as the object being imaged. The exposition time for the photo-bleaching process and writing the bleached patterned images was about 145 seconds. The excitation light spot spatially modulated by the mask selectively irradiated the film area and created spatially photobleached patterns according to the mask structure. When removing the mask, the CCD camera read out the fluorescent photobleached-patterned image under homogenous excitation. The image can be totally erased by homogenous illumination for ∼200 seconds.

 

Fig. 2. Photo-bleaching patterning of PVA thin polymer films incorporating fluorescent peptide dots using different 2D masks structures. Scale bars in (a) – (c) are 25 µm. (a) Resolution test target on a glass substrate; (b) 8 × 8 array of opaque squares on a glass substrate; (c) transparency film with halftone photographic image. The test masks (first column), irradiation patterns (the second column), and photo-beached patterns (the third column).

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Figure 2 exhibits photo-bleached patterns obtained with 2D masks of different structures: (a) resolution test target on a glass substrate; (b) 8 × 8 array of opaque squares on a glass substrate; (c) transparency film with halftone photographic image. The presented data demonstrate full consistency of the photo-bleached patterns-images with the test masks structure.

Figure 3(a) presents the periodic Ronchi gating test with a 1.6 lines/mm spatial frequency. Figures 3(b) and 3(c) show the microscopic FL images of this test object at two different stages: during photobleaching patterning (Fig. 3(b)) and the visualization of the resulting bleached pattern (Fig. 3(c)). The specific regions of interest are indicated by red and blue semicircles in Figs. 3(b) and 3(c). Figure 3(d) shows that the irradiation and the photobleached patterns are shifted by half of the grating's period. This shift is also illustrated through line profiles of fluorescence intensity, as shown in Fig. 3(e). These profiles are recorded along the red and blue lines in Fig. 3(d). Additionally, Fig. 3(f) demonstrates the hiddenness of the photobleached pattern without excitation (left panel) and its visualization when subjected to excitation (right panel).

 

Fig. 3. Comparison between the spatial structure of periodic grating images at the stages of photobleaching patterning and visualization of the bleached pattern. (a) Ronchi test grating; (b) irradiation pattern; (c) photo-bleached pattern; (d) spatial alignment of irradiation (top) and photobleached (bottom) patterns; (e) line profiles of fluorescence intensity, recorded along the red and blue lines shown in Fig.3d; (f) microscopic images of a small part of the photobleached area without excitation (left panel) and under excitation (right panel); (g) line profiles of normalized FL intensities of periodic photobleached patterns with various spatial frequencies; (h) estimated modulation transfer function of whole writing/reading system.

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Closer examination of Fig. 2 and Fig. 3(b),(c) reveals two types of spatial inhomogeneities of fluorescence signals: the wide, monotonically distributed variations of FL intensity across the field of view and the well-localized defects. The first type can be attributed to the inherently non-uniform (gaussian-elliptical) intensity distribution of the writing-reading beam of the laser diode used in the optical setup. The second type of irregularities arises from micro-defects on the surfaces of the optical elements in writing-reading channels and PVA films. Both types of inhomogeneities can be significantly reduced by optimizing the optical setup design and improving the thin film deposition process.

We estimated the modulation transfer function (MTF) of the whole writing/reading system using the normalized line profiles of periodic photobleached patterns with various spatial frequencies obtained from Fig. 2(a) and 3(c). The results of MTF estimation are presented in Fig. 3(g),(h). It can be seen from Fig. 3(h), that full width at half maximum (FWHM) of the MTF is 0.18 µm^-1 which corresponds to 2.8 lines pairs per micrometer.

This newly introduced approach enables us to find a long-term optical memory by writing photo-bleached hidden images and patterns within thin PVA polymer films containing a densely arranged array of FL peptide nanodots.

Our research demonstrates a significant photobleaching effect in the developed PVA/FFF-peptide nanodots thin film material. The unlimited area of the films and their homogenous flat surface combined with irreversible photo-bleaching allow their use for a wide range of potential applications, [2,30,31] such as product labeling, biocell tagging and tracking, selective attachment of proteins with sub-diffraction resolution, anti-counterfeiting of drugs, security printing, and photobleached patterned structures and bar codes.

4. Experimental section

Materials and methods: An investigation was conducted on ultrashort aromatic peptide nanodots, with a specific focus on the self-assembly of aromatic triphenylalanine (FFF) peptide biomolecules into nanodots, followed by their thermally-induced refolding into beta-sheet peptide secondary structure.

FL peptide nanodots: The fabrication process of FL peptide nanodots involves two steps. In the first step, monomer peptide tri-phenylalanine (FFF) biomolecules were self-assembled in organic solvents using a bottom-up approach to form peptide nanodots. This process can be viewed as the nucleation of peptide/protein monomers into seeds with a critical size, which serves as the fundamental building blocks based on the supramolecular concept. [11,13,14] At this stage, a solution of native tri-phenylalanine (FFF), was prepared by the following procedure: the lyophilized powder of FFF (Bachem, Switzerland) was dissolved in HFIP (Sigma-Aldrich) to an initial concentration of 100 mg/mL, mixed in a vortex mixer (VELP Scientifica) and then further diluted to a final concentration of 1 mg/mL in ethylene glycol (EG) (Sigma-Aldrich). This self-assembly process of native tri-peptide FFF-biomolecules was inhibited at the first initial stage of the nucleation to the nanodots by using polar solvents. At the second step, the FFF-nanodots were subjected to a thermal treatment at 160 °C (at this final temperature, the heating was fixed for three hours) in a high boiling temperature solvent of EG (boiling point is 195°C) and then cooled down back to room temperature. This thermal treatment procedure allowed the transformation of the non-fluorescent peptide nanodots into visible FL bio-nanodots with a refolded beta-sheet secondary structure. [11,12]

Fabrication of polymer polyvinyl alcohol (PVA) films with embedded FL peptide nanodots: In the current investigation, we have developed and examined a novel category of fluorescent materials: fluorescent peptide films. These films consist of colloidal solutions of fluorescent nanodots made from tri-phenylalanine (FFF) dispersed in polyvinyl alcohol (PVA). To achieve uniformity, the PVA colloidal solution was spin-coated onto a glass substrate, resulting in homogeneous films composed of fluorescent peptide dots embedded in PVA. This technique enables the creation of thin films ranging in thickness from nanometers to micrometers. To prepare the colloidal solution of peptide nanodots, a 1 ml volume containing 1 mg/ml of fluorescent FFF- dots (at a 1:1 volume ratio) was combined with 1 ml of a 10% polyvinyl alcohol (PVA) solution. The mixture was vigorously shaken for approximately 1 hour, after which 200 µl of the resulting solution was carefully applied to the cleaned glass substrate. The desired film thickness can be controlled by adjusting the angular speed, solution viscosity, and spinning time. Subsequently, the deposited colloidal solution was air-dried for 24 hours, resulting in a uniform, solid polymer film incorporating FL peptide nanodots.

Fluorescence spectra measurements in PVA polymer films with embedded FL peptide nanodots: A custom-designed, reconfigurable microscope spectrometer setup (Fig. 4) was used to conduct an experimental study on the optical spectra of thin PVA films containing FL peptide dots. This setup facilitated precise sample positioning, FL excitation, and FL spectral characterization. The experimental arrangement involved placing a glass substrate with a deposited thin PVA/peptide FL dots film sample in front of the microscope objective. FL excitation in the film was achieved by focusing a beam of a purple laser diode with a wavelength of 405 nm. Spectral separation between excitation and FL channels was accomplished using dichroic and broadband beam splitters. Color images of the analyzed structures were captured using a CCD camera and transferred to a computer for post-processing. FL spectra were acquired using a high-grade scientific spectrometer (Ocean QEPro model) with a low-noise cooled linear array sensor. The investigated sample was placed in the focal plane of the microscope objective and precisely aligned using a piezo-motor translation stage.

 

Fig. 4. Experimental setup for spectral characterization of thin PVA films with embedded peptide nanodots.

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Experimental study of photobleaching effect: While studying the spectral properties of the peptide thin films, we observed that the FL intensity of the evaluated samples gradually fades under continuous illumination by the excitation laser beam. This phenomenon is well-known in fluorescence microscopy as a photobleaching effect. The photobleaching effect involves a photochemical modification of the fluorescent material, resulting in the irreversible loss of its ability to fluoresce. In some cases, a fluorescent sample can be switched on again after an apparent loss of emission ability or from a natural initial dark state or is able to switch on and off by itself within a short timescale. This study refers to photobleaching as an irreversible loss of fluorescence properties. The photobleaching effect was studied using the experimental setup shown in Fig. 4.

Recording of hidden images: Photobleaching-based patterning and reading hidden images in polymer PVA thin films with embedded fluorescent peptide nanodots were recorded in the modified FL setup, shown in Fig. 5. The modification involves a mechanism for inserting/removing 2D patterning masks in the excitation laser beam path.

 

Fig. 5. Experimental setup modified for photobleaching patterning/reading.

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The patterning/reading technique involves two steps. In the first step, the patterned illumination of the PVA/FL peptide dots film was performed by the laser diode (405 nm) via 2D patterned mask. The latter was inserted between the laser diode and dichroic filter and projected by the 40X microscope objective on the surfaces of the tested PVA/FL peptide dots film. The excitation laser light spot, spatially modulated by the patterned mask, irradiated the film's surface for about 145 sec. During this exposition time, the corresponding fluorescent film's area was spatially bleached in accordance with the mask structure, forming two regions: illuminated and non-illuminated. The dynamics of the photobleaching process in the illuminated regions were visually observed and recorded by the digital CCD camera. At the next step, the mask was removed, and the uniform laser excitation of the spatially patterned PVA/FL peptide dots sample allowed read out by the camera the bright FL spatially modulated image, which was patterned under the preliminary photobleaching procedure. As soon as the mask is removed, the fluorescence from the regions non-illuminated at the first step gradually fades due to the photobleaching effect. The patterned image of the PVA/FL peptide dots film sample is totally erased under continuous laser illumination for ∼300 seconds.

4. Conclusion

The photobleaching phenomenon is recognized as a detrimental factor affecting optical memory that relies on fluorescent nanostructures. Our research reveals that beta-sheet peptide FL bio-nanodots also display a notable and irreversible photobleaching effect. This effect arises from the light-induced disruption of intermolecular hydrogen bonds that stabilize the beta-sheet secondary structure of the peptide dots. However, exploiting this photobleaching effect in beta-sheet bioinspired peptide nanodots, we have developed a novel method for high-resolution hidden imaging. Our findings introduce a new approach to achieving long-term optical memory with exceptional clarity by customizing diverse concealed images within thin polyvinyl alcohol (PVA) polymer films, incorporating a densely arranged array of FL peptide nanodots. This innovative technology facilitates recording photo-bleached patterns, barcodes, and high-resolution hidden images.