Remodeling and self-healing of individual amyloid tactoids via multiphoton absorption

Domain: physics
Published: 2025-12-02
Editorial outcome: minor_revision

Abstract
Colloidal self-assembly can typically be controlled only globally, at ≥101 μm scale, such as in liquid-liquid crystalline phase separation (LLCPS) of anisotropic tactoidal droplets. Here, we introduce a stimuli-responsive approach allowing a fully reversible structural control of LLCPS morphologies at the individual droplet level. Using amyloid-based tactoids and multiphoton absorption with localized photothermal effect, we can cut, ablate, and self-heal individual tactoids, and - when desired - print, erase, and store structural information within at sub-micron resolution. We exploit the nematic-isotropic-nematic transition within single tactoids to locally melt the liquid crystalline (LC) order into the isotropic phase, leaving unchanged the bulk dispersion structure. The locally melted nematic field recovers its ground-state LC order within minutes after the exposure, showing both self-healing and short-term memory-storage features. Furthermore, hybrid cholesteric tactoids functionalized by guest nanoparticles can be re-modeled into new tactoids with different symmetry and functionalities, for example, featuring laser-induced fluorescent encoding. These results introduce a general strategy to direct phase separation-within-phase separation and to store, control, and engineer information at the sub-micron level in heterogeneous complex fluids.

Introduction
Liquid crystalline (LC) droplets, also referred to as tactoids, spontaneously nucleate and grow within an isotropic solution, forming a heterogeneous water-in-water system in which the dispersed phase emerges primarily (but not exclusively) as a trade-off between orientational and free volume packing entropies1–5. These droplets hold various spherical, ellipsoidal, and spindle-shaped morphologies with intricate internal symmetries and physical properties6–8. Tactoids have been observed in a variety of LC systems, including polypeptides9,10, supramolecules6, molecular aggregates11, biological colloids of viruses12,13, nanocrystals14, and fibrils8,15,16. Significant efforts have been devoted to understanding the tactoids’ origin, shape, and structure, which may not only enable novel applications5,17,18, but also enhance our understanding of a number of biological phenomena19, including the formation of collagen byssal threads20, amyloid protein condensates and plaques21,22, and P. aeruginosa resistance to antibiotics13,23. Numerous studies have explored controlling the pathway leading to the formation of tactoids emerging from liquid–liquid crystalline phase separation (LLCPS)5,24,25, using intensive variables, such as temperature26,27, pressure28, ion type29, concentration25,30, pH31, and other additives6,9,16, which therefore affect the entire system. At best, tuning the morphology of a restricted number of tactoids has been achieved through microfluidics approaches15,32,33. However, manipulation of the internal structure, shape, and physical properties at the single tactoid level is yet to be reported. Because this can only be attempted by departing from the thermodynamic ground state, non-invasive localized stimuli have to be considered to pursue this task.

Unlike the linear absorption of light by matter, multiphoton absorption (MPA) is a second and third-order nonlinear effect, in which the absorption is greatly strengthened as energy density increases34–36. Previous research confirmed that highly ordered amyloid fibrils and silk fibroin exhibit strong nonlinear optical absorption with large multiphoton cross-section, which is absent in their native protein precursors37,38. Infrared pulses with low average energy exhibit high penetration in biological colloidal media and are absorbed by amyloids in multiphoton mode39,40. This property allows minimal surface absorption (in accordance with the Lambert–Beer law) and enables the formation of a focal spot, causing MPA deep within the materials. Multiphoton-induced structural modification and patterning have been investigated in various biocompatible materials, including silk fibroin36, collagen41, poly(vinyl-alcohol)42, poly(methylmethacrylate)43, and gelatin hydrogels44, but to the best of our knowledge, this technique has not yet been considered as a tool to control LLCPS.

In this article, we report a method to manipulate amyloid LC droplets via nonlinear MPA in three dimensions (3D) with sub-micron resolution (named 3D-Heating). Amyloid tactoids show appealing capability of being locally heated through nonlinear absorption, in situ, with a high degree of spatial selectivity. The tactoids could be easily erased, encoded with structural information, and modulated into new tactoids by 3D-Heating. We demonstrate that the amyloid tactoids exhibit a strong memory effect, pitch-tunable function, and fast self-healing ability, thereby underscoring their potential as smart biomaterials and bio-devices through LLCPS. The resultant 3D-Heating on amyloid tactoids offers a promising strategy in the manipulation of complex fluids, with potential applications in engineering, printing, memory-storage, and self-healing in nanotechnology, life sciences, and biomaterials design.

Results
We realized LLCPS and the formation of various tactoids symmetries using amyloid fibrils made of β-lactoglobulin (BLG) (see Fig. 1a), a readily available protein associated with dairy byproducts. Regarding pathological, functional, and artificial amyloid fibrils, their highly ordered and tightly packed core with β-sheet structures endows BLG amyloid fibrils with high mechanical rigidity and thermodynamic stability. To form amyloid tactoids, the long semi-flexible BLG amyloid fibrils with a persistence length Lp of 1.8 μm were broken down by stirring into shorter fibrils (Fig. 1b and Supplementary Fig. 1). The average fibril length, measured via atomic force microscopy (AFM) images using FiberApp contour tracking software45, is L = 435 nm and the average height ~4 nm, corresponding to an aspect ratio of ~109. Next, an aqueous suspension of shortened amyloid fibrils was prepared at a concentration such that it falls within the isotropic-nematic two-phase region (Supplementary Fig. 2). At such concentration, when sealed in a capillary tube, the amyloid fibril suspension undergoes LLCPS, forming, over the course of the following weeks, tactoids with a rich palette of symmetries, sizes, and morphologies (Fig. 1b and Supplementary Fig. 3).

Multiphoton absorption of tactoids
We discovered that a pulsed infrared laser could destruct the tactoids, whereas a laser in the visible light region did not, while performing fluorescence imaging of amyloid tactoids. Inspired by this, we set out to systematically study the laser-induced manipulation of amyloid tactoids, using the layer-by-layer exposure concepts from 3D printing technology46–48. We exposed individual tactoids to a scanning focused pulsed laser (810 nm, ~100 fs, 80 MHz ± 1 MHz) following spatially predefined paths (Fig. 1c). During laser pulse exposure, the virtual-state lifetime is determined by the pulse duration. Intuitively, the transition of an electron from the ground state to the virtual state, and finally to the excited state, is induced by two or more sequential photons. Using z-scan techniques, we found that BLG fibrils exhibited three-photon absorption (3PA) capabilities (Fig. 1d and the theory in Supplementary Note 1). The linear absorption spectrum showed a single absorption peak at ~280 nm from the amyloid fibrillar suspension (Fig. 1e), providing further evidence of the nonlinear absorption process under pulsed laser. Previously, the 3PA process was observed in silk-fibroin36, amyloid fibrils37, and spider silk49. The enhanced 3PA in amyloid fibrils is attributed to through-space dipolar coupling between the excited states of aromatic amino acids densely packed within the fibrous structures37. This highly ordered β-sheet structure was confirmed by Thioflavin T fluorescence and Fourier transform infrared spectroscopy (Supplementary Fig. 4). The heating accumulation from direct 3PA allowed us to raise the temperature of amyloid mesogens precisely at the focused point. We found that the local transition from the anisotropic to isotropic phase in tactoids required only a weak laser power of ~0.25 nJ per pulse, which was one order of magnitude lower than the energy needed for patterning in soft silk protein hydrogels (2 nJ per pulse to break hydrogels)36.

Systematically, the relationship between the laser-induced nematic-isotropic transition rate and laser power was investigated. As shown in Fig. 1f, we found that a power of 13.6 mW (total pixel dwell time of 16 μs) could have distinct breaking effect on cholesteric structures. Here, to show the relation between the irradiation area and the actual area affected within the sample, we designed three different irradiation areas (48.6, 176.4 and 360.6 μm2) as shown in Supplementary Fig. 5 and irradiated them under the same conditions. Following the irradiation, we immediately measured the disordered regions within the sample, marked with red dash circles in Supplementary Fig. 5b. Our measurements show that the area set by the program and the disordered region measured within the samples are of comparable size (Supplementary Fig. 5c), indicating the same irradiation effects on different areas. We note that the slightly lower values for disordered areas compared to the set region are due to a measurement delay (>10 s) between the switch of NIR irradiation and POM imaging. Additionally, the kinetic irradiation was also measured in Supplementary Fig. 5d. The result shows that the isotropic region (121.9 μm2) obtained by ON-OFF laser period (pixel dwell time of 0.4 μs with three repetitions) was smaller than the single continuous exposure (167.1 μm2, pixel dwell time of 1.2 μs). Overall, this method provided us with a powerful and versatile method for generating complex 3D disordered phase patterns within the otherwise LC-ordered state of the tactoid.

Erasing and cutting amyloid tactoids
First, a bipolar tactoid, in which the director field follows the tactoid interface, was selected and irradiated entirely by exposing a cuboid volume at its center (Fig. 2a). Monitoring the morphology over time under polarized optical microscopy (POM), we observed that the tactoid with an initial volume of 17,236 μm3 disappeared within ~155 s, following an initial lag phase, indicating an accumulation of light energy before the order was erased (for volume calculation see Supplementary Fig. 6 and Supplementary Note 2). The aspect ratio (H/2R) of the exposed tactoid exhibited an initial decrease followed by an increase after reaching a critical point (80 s, 4,836 μm3). To explore this phenomenon, we compared the dynamics of H and 2R during this exposure (see POM intensity maps in Supplementary Fig. 7). The length of the H dimension was reduced on both poles of the bipolar, while 2R decreased slowly until ~80 s. We attribute this phenomenon to the different fibrillar alignments along H and 2R, leading to different ablating and expanding features. In contrast, when only half of the bipolar tactoid was exposed (Fig. 2c), the aspect ratio showed a linear increase. Remarkably, the bipolar volume decreased with time while the tactoid always maintained a spindle shape, and finally reached zero. These findings strongly suggest a rapid dynamic reconstruction of the amyloid tactoids under localized irradiation, similar to proteins in coacervates, which remain very dynamic on sub-microsecond timescales50. The same protocol was applied to cholesteric tactoid (with an oblate shape, and thus with an initial aspect ratio <1) with chiral alignment of the mesogens (Fig. 2e). The 3D-Heating technique ablated the entire cholesteric tactoid in 1,718 s, despite its volume being 42 times larger than the bipolar tactoid in Fig. 2a. In this case, the system revealed a clear lag phase in the initial ~350 s (Fig. 2f). When only half of the cholesteric tactoid was irradiated, the unexposed part retained its original chiral symmetry, but underwent internal dynamic changes into a new cholesteric tactoid (Fig. 2g). By examining the unexposed part in more detail, it was found that the half pitch increased gradually from 6.6 ± 0.2 μm to 7.7 ± 0.2 μm, consistent with our general observation that smaller tactoids show larger half pitch values (Fig. 2h and Supplementary Table 1)8. Furthermore, the aspect ratio of the new tactoid increased from ~0.8 to ~1.2, evolving from an oblate to a prolate ellipsoidal morphology (Supplementary Fig. 7c)16.

We further explored the potential of our 3D-Heating technique for manipulating tactoids. As shown in Fig. 2i, a “laser knife” was created to cut the bipolar tactoid into two separated bipolar tactoids. The laser was scanned along the long axis (H) of the bipolar tactoid, with the length > H, width > 2R, and the thickness of the laser box was set to 5 μm to ensure complete separation. This operation produced two stable bipolar tactoids with a 16 μm separation gap in solution at 197 s (Supplementary Fig. 7d). When tracking the dynamics of the internal structure of the new bipolar tactoid (B1 in Fig. 2i), an increase in the aspect ratio with respect to exposure time was observed (Fig. 2j). Roughly, we could draw the conclusion that the manipulation of bipolar tactoids triggers the growing of aspect ratio, eventually even leading to the formation of homogeneous tactoids (Supplementary Fig. 3). Similarly, the cholesteric tactoid (O1) was cut into two smaller cholesteric tactoids using the laser knife along its band (2R) axis (Fig. 2k). After the cutting, the half-pitch value increased from 6.6 ± 0.2 μm to 9.8 ± 0.1 μm, providing direct evidence again that the half pitch increased in small cholesteric droplets (Fig. 2l and Supplementary Table 2). Notably, the cholesteric pitch values of neighboring tactoids, measured as the control experiment (see O2 in Fig. 2k) remained unchanged, confirming the resolution of our method in manipulating the tactoids. To further test the versatility of the “laser knife”, we performed cuts at different operating directions where there was a 45° angle between the long axis (H) and the laser knife, as shown in Supplementary Fig. 8. The post-dynamic rearrangement of the separated bipolar tactoids was also recorded (Supplementary Movie 1). The beveled half tactoid reformed into a standard spindle-shaped bipolar within 400 s, providing insight into the phase recovery timescale of amyloid tactoids. In the case of cholesteric tactoids, cutting along the short axis (H) also yielded two separated tactoids (Supplementary Fig. 9), both of which showed increased half-pitch values. These results demonstrate the flexibility and precision of the 3D-Heating technique, offering a powerful tool for manipulating amyloid tactoids into a wide range of morphologies. Conversely, tactoids formed by cellulose nanocrystals (CNCs)14,51, and exposed to the same laser dosage and conditions maintained their cholesteric structure without noticeable changes (Supplementary Fig. 10), as expected by the corresponding UV-Vis spectrum, which shows nearly no absorption at about 281 nm. This provides direct evidence that our 3D-Heating is highly specific to MPA materials, such as β-sheet-rich amyloid fibrils.

Self-healing of amyloid tactoids
To investigate the LC phase recovery properties, a localized isotropic region was induced within a single tactoid using the 3D-Heating method. Owing to its high-resolution, as shown in Fig. 3a, a thin disk exposure region (red color) with a depth of 5 μm and a radius of 30 μm was created in the center of a cholesteric tactoid. Here, we found that the exposure area appeared as a shadow under a cross polarizer, indicating that a local isotropic phase was induced, but recovered quickly (Fig. 3b and Supplementary Movie 2). The shrinking speed of the disordered region revealed that the recovery along the POM bands (Sl, the length of the disordered region parallel to the cholesteric bands) was initially faster than the recovery perpendicular to the bands (Ll), as shown in Fig. 3c, indicating that mesogens realigned more easily along the bands where the mesogens were parallel to each other. Unexpectedly, from 20 to 40 s, 50 to 90 s, 100 to 130 s, and beyond 140 s, Ll kept the same value and showed steps. These pinning effects strongly suggested that the process involved several transient energy minima, i.e., metastable states. It can be interpreted that the discrete behavior of Ll relaxation is consistent with the number of bands (one band represents half a period of rotation of the mesogens). In contrast, the short axis (Sl) shrank rapidly within 40 s to a constant value, showing the largest ratio (Ll/Sl) of the isotropic region at 1.9 in Fig. 3d. Eventually, at around 183 s, the region transformed into a negative tactoid, maintaining an isotropic internal phase within the anisotropic medium, with the aspect ratio different than 1 and the H aligned with the cholesteric bands. Furthermore, the recovery rate of the disordered area followed an exponential decay (Supplementary Fig. 11).

A further LC phase recovery study was carried out by the exposure of narrow rectangular areas with different orientations, as shown in Fig. 3e. Three narrow rectangular regions confining the isotropic phase were induced by 3D-Heating within a single cholesteric tactoid and designed with angles of 0°, 60°, and 90° relative to the cholesteric bands. Their morphological evolution (laser off) showed that isotropic regions with 0° maintained their orientation during recovery, while the ones with 60° and 90° demonstrated rotational behavior and aligned with the same directions as the cholesteric bands after 409 s (Fig. 3f). A detailed analysis of the changes in the relative angles of the isotropic region (60°), similar to the results in Fig. 3c, revealed that the angle changes occur in steps at ~ 40 s, ~ 65 s, and 90 s (Fig. 3g). In other words, the process of healing passed through metastable states associated with cholesteric bands during the path towards the full recovery of the ground-state LC configuration. This stepwise behavior was confirmed in the analysis of the time evolution of the length of the narrow rectangular regions in Fig. 3h. In addition, the recovery speeds for isotropic regions demonstrate a clear behavior that the region parallel (0°) to the bands shows the fastest shrinking speed, while the orthogonal cut (90°) has the slowest speed. Finally, the length of three isotropic regions stabilized at ~20 μm after 120 s. Further study with a cross pattern and spots array pattern in Supplementary Fig. 12 additionally confirmed that the recovery of the isotropic region parallel to the bands was quicker than in the vertical direction, resulting in a negative tactoid with the long axis parallel to the bands.

We further monitored the evolution of the laser-induced negative tactoid. Supplementary Fig. 13 shows a negative tactoid crossing three bands in the center of a cholesteric tactoid, induced by the 3D-Heating method. The negative tactoid faded after 308 s, with full vanishing (LC reconstruction) completed in 1,000 s, a process nonetheless accompanied by a slight reduction in the total volume of the cholesteric tactoid. Additionally, as the negative tactoid shrank, the recovery rate increased. The half pitch, which broadened during laser irradiation, exhibited a shrinking behavior when rebuilding the cholesteric structures in the absence of the pulsed laser. Interestingly, from the monitoring of laser-induced negative tactoid arrays (Supplementary Fig. 14), the isotropic regions tended to fuse with their neighboring regions along the 2R direction (the fibrils were parallel to each other), but never combined in the H direction (the fibrils were rotated to each other). When the laser-induced isotropic region was large (50 μm in diameter) and its depth reached ~10 μm (near the pitch value), we captured the entire localized nucleation process (Fig. 3i). The cylindrical isotropic region evolved into a negative spindle-like shape after 36 s. A cholesteric nucleus formed at 142 s in the center of the isotropic region (Supplementary Movie 3). Although the cholesteric nucleus had an initially different director compared to the surrounding cholesteric boundary, it merged and connected with the surrounding structure in subsequent steps, completing the rebuilding process in 1,052 s. These results directly confirmed that amyloid fibril tactoids possess a strong self-healing ability to restore the LC ground state via a localized process of nucleation and growth.

In order to understand the self-healing ability of the tactoids under conditions of extreme, disruptive deformation, experiments were designed in which half of the cholesteric tactoids were ablated using a higher laser power (∼0.3 nJ per pulse). Two cholesteric tactoids with different orientations (H1 and H2) were selected according to the model illustrated in Fig. 3j. Half of both tactoids were exposed to the laser at the same laser exposure. POM images exhibited two half-shaped cholesteric tactoids in the beginning (marked as 0 s in Fig. 3j) after laser irradiation. Importantly, we captured the rapid formation of cholesteric structures on the cutting face (cross section) within the next 222 s. The two half tactoids recovered into new prolate tactoids, with an aspect ratio (H/2R) larger than 1 (Supplementary Fig. 15 and Supplementary Table 3), which demonstrate the ultrafast and accurate manipulation by the 3D-Heating method and remarkable self-healing features of the tactoids even under extreme deformation conditions.

Operation in nanoparticle anchored tactoids
The 3D-Heating method provides us with the opportunity to build complex colloidal assembly structures and patterns. As a proof of concept, amyloid tactoids were functionalized with fluorescent nanoparticles (d = 30 nm) to create fluorescent-LC hybrid droplets. As shown in Fig. 4a, the nanoparticles became self-enriched in the cholesteric tactoid, whereby LC properties were fully preserved (as evidenced by the bands) under confocal fluorescence microscopy. In contrast to previous studies on fluorescence-labeled tactoids, in which fluorescent signals were uniformly distributed within the cholesteric tactoid6,9, the bright and dark band phenomenon suggests that the nanoparticles aligned with the amyloid mesogens in an orderly manner. Additionally, the fluorescent nanoparticles enabled 3D-Imaging of the hybrid droplets (Supplementary Fig. 16). For example, we could quickly monitor the tactoid along the xy and xz axes (which cannot be measured in POM) in situ and perform 3D modeling of the tactoid, enabling precise monitoring of the internal structures of tactoids (Fig. 4b and Supplementary Movie 4).

Combining the 3D-Heating and 3D-Imaging methods, as shown in Supplementary Fig. 17, six ordered isotropic points were printed inside the hybrid tactoids spatially, which gradually recovered to the cholesteric phase one by one after 956 s. Here, with the help of xz axes scanning, we show that six isotropic points recovered to the cholesteric phase from ~284 s to ~956 s (Supplementary Fig. 17b). We further show that patterns could be printed on a single layer (exhibiting a homogeneous fluorescence signal) in 3D space. As a demonstration, the ETH logo was printed on one of the bright bands in the hybrid tactoid (Fig. 4c). The pattern evolved first into a negative tactoid within 356 s and then fully recovered to the ground LC state after 1,051 s. Upon examining the fluorescence intensity in the bright layer (Fig. 4d), the logo region revealed an increasing average intensity as it returned to the cholesteric phase. However, the intensity in the surrounding area (above and below the logo) decreased slightly in approximately 400 s, but recovered after 1,051 s. At the same time, the area of the operated layer recovered with a slight decrease in the area (Fig. 4e), thus providing full evidence that the large-scale reconstruction of the cholesteric structure is governed by fast dynamic and cooperative nature.

To showcase the strong manipulation capabilities of 3D-Heating and the tactoid’s self-healing features, the tactoid could be separated into three (Fig. 4f and Supplementary Movie 5) or multiple pieces (Supplementary Fig. 18 and Supplementary Movie 6), and then quickly recovered within 353 s and 411 s. The half pitches were dynamically monitored, shrinking quickly in the first 75 s and expanding in a stepwise manner during the subsequent process (Fig. 4g). Notably, two key steps occurred when the separated tactoids began reconnecting. Additionally, the length of 2R increased to 93 μm and then returned to its original value after 340 s (Supplementary Fig. 19). To better understand the dynamics of connection, one cholesteric tactoid was selected and cut along both horizontal and vertical directions consecutively (Fig. 4h). First, cutting along the bands (2R) created an open zipper, which closed rapidly and completed in 116 s (see Supplementary Movie 7). The zoomed image of separated tactoids is shown in Supplementary Fig. 20a. Similar to the decrease of area in the logo printing (Fig. 4e), the cutting of the tactoids along the bands also shows the reduction of 2R (Supplementary Fig. 20b). In contrast, the H has a temporary increase under equilibrium. When the cutting direction was rotated 90° and operated on the same tactoid that recovered to its original shape, the zipper took much longer to close - starting at 165 s and finishing at 387 s (Fig. 4i and Supplementary Movie 8). This finding strongly suggests that the recovery of the original cholesteric structure is more difficult when mesogen rotation is involved across several bands. Unlike the cutting along the bands, 2R demonstrated a temporary increase under recovery, while the H decreased accordingly (Supplementary Fig. 21). As a result, the cutting along the different directions results in markedly different recovery dynamics. Additional laser manipulations were also performed. From the random cutting of tactoids in Supplementary Fig. 22, the tactoids can be constructed into flexible patterns. However, all of the patterns exhibited strong self-healing abilities and recovered to their original shapes. The sub-micron resolution of our laser operation is best showcased in Supplementary Fig. 23, where a 1.4 μm length gap was created to separate the cholesteric tactoid into two parts.

Pitch-tunable amyloid tactoids
Our 3D-Heating method can be applied not only for the cutting and self-healing of individual tactoids, but also for controlling their internal structure. For example, tunable pitch control is crucial for optical applications, such as producing structural color and circularly polarized luminescence52,53. When the laser was set to a moderate power (∼0.2 nJ per pulse), we found that their radiation could first trigger the melting of the local order, and then induce a new band in the exposed region. As illustrated in Fig. 5a, the half pitch length expanded under exposure, and new periods were induced, i.e., a new light band appeared in each laser-exposed region. Our experimental results in Fig. 5b demonstrated that a 10-band cholesteric tactoid was rebuilt into a 12-band tactoid by triggering the onset of the new bands. The length (H) of the tactoid increased dramatically from the original length of 65.7 μm to 98 μm. As illustrated by the fluorescence intensity evolution map (Fig. 5c and Supplementary Fig. 24a), the exposed tactoid exhibited a relaxation period of 150 s, during which the tactoid grew from 94.3 μm to 98 μm. As the tactoid stabilized (shrank) into a steady state, the formation of new bands was observed in the relaxation regions between 110 and 150 s (Fig. 5b). At this point, as marked with white arrows in Fig. 5c, new bands appeared and equilibrated with the surrounding cholesteric structures. The final length (H) of the tactoid shrank to 76 μm after 500 s, and the half-pitch sizes remained heterogeneous; the newly formed bands revealed half-pitch values between 5.6 and 6.1 μm (Supplementary Fig. 24b), smaller than the original one (~7.5 μm) (see Supplementary Movie 9). Notably, not only did the number of bands change, but the half-pitch length was also reduced.

The 3D-Heating method may also allow for erasing the bands. As shown in Fig. 5d, the newly built 12-band tactoid was engineered into tactoids with 11, 10, and 9 bands through step-by-step laser irradiation. In this case, a relatively high laser power (∼0.3 nJ per pulse) was applied to dissolve the mesogens into the surrounding isotropic environment, achieving the new stable structure with the desired number of bands after a 10-min equilibrium. Moreover, this method can also fix structural dislocation in tactoids, as shown in Supplementary Fig. 24c. A two-phase (heterogeneous) tactoid was restored to a single-phase tactoid after laser exposure in the marked region. To further explore this function, we exposed the tactoid to low-power irradiation (∼0.14 nJ per pulse) in dark bands. The bands expanded without the formation of new bands and returned to their original state after ~300 s, as illustrated by the fluorescence intensity map in Fig. 5e over five cycles. The half pitch was tuned between ~5.6 μm and ~7.5 μm (Fig. 5f). In aggregate, and similarly to a micron-blade scalpel, we were able to precisely manipulate the local half-pitch of the chiral nematic bands, which may be of paramount importance for optical applications exploiting cholesteric phases.

Finally, due to the strong self-healing features and the high degree of control over the 3D-Heating process, the fluorescent amyloid hybrid tactoid was utilized as a smart optical switch, equipped with self-healing and light converting functionalities (Fig. 5g). The rotation of the tactoid (0-360°) exhibited high optical response, switching between ON and OFF states based on light intensity (Fig. 5h). As an advantage compared with the POM image, the fluorescence imaging exhibits a stable background intensity (Supplementary Fig. 25). In this case, the rotation of the tactoid produced a fluorescence signal that clearly followed an ON-OFF-ON-OFF-ON sequence, while the emitting light color was dependent on the dyes. When the focused layer of the tactoid was selectively erased by the laser (Supplementary Fig. 26), the switching capability vanished, resulting in a flat-line response (Fig. 5i). After 10 min, however, the switch regained functionality as the cholesteric structures recovered. This simple example demonstrates how fluorescent signals can also be precisely engineered by combining LLCPS, fluorescent probes, and the 3D-Heating process.

Methods
Preparation of amyloid tactoids
The BLG amyloid fibrils were prepared using the β-lactoglobulin protein (whey protein). β-lactoglobulin protein powder was dissolved in Milli-Q water, and the pH of the suspension was adjusted to 2. Then, the suspension (2 wt.%) was filtered with a nylon syringe filter (0.45 μm, Huberlab) to remove protein aggregates. In order to obtain the amyloid fibrils, the solution was heated at 90 °C for 8 h. Amyloid fibrils were then cut into short fibrils by mechanical shear forces. Next, a dialysis was performed for 7 d using a 100 kDa (MWCO) Spectra/Por dialysis membrane (Biotech CE Tubing) with daily bath replacement. The final concentration of short BLG fibrils with an average length of ~435 nm was achieved by up-concentrating the solution with the reverse osmosis method, in which a dialysis membrane (standard RC tubing) was used against a 10 wt.% polyethylene glycol (Sigma Aldrich) solution under pH = 2. Phase separation of the amyloid fibril suspension was found after 3–4 weeks when stored in bottles.

All glass capillaries and containers were cleaned by piranha solution, ethanol, and Milli-Q water. The carboxylate-modified latex bead solutions (λex ~520 nm; λem ~540 nm, Sigma Aldrich) were centrifuged twice at 16500 × g and replaced with pH = 2 Milli-Q water. The BLG fibril suspension was used at a concentration in the coexistence region of the isotropic-nematic phases and incubated in a capillary tube with the size of 50 mm × 4 mm × 200 μm. For fluorescent tactoids, a designed volume (2~5 μl, 500 nm, 1%) of latex bead solution mixed with the BLG suspension (34~37 μl) was injected into the capillary tube gently by pipette. Finally, both ends of the capillary tube were sealed with UV-curable glue. The equilibrium was performed at room temperature.

Multiphoton heating
Experiments were performed using a multiphoton microscope with Mai Tai XF (Spectra-Physics) femtosecond laser, tunable from 720–950 nm. Ultrashort (100 fs) laser pulses at a pulse repetition frequency of 80 MHz were focused into the bulk of the amyloid tactoids using a 25× 0.95 NA L Water HCX IR APO microscope objective. The scheduled process is shown in Fig. 1. A capillary tube with well grown tactoids was put on the scanning platform of an inverted confocal fluorescence microscope (Leica SP8). The microscope equipped with a polarizing filter was used to check the tactoids without fluorescence dyes. The switch between femtosecond laser exposure and fluorescence imaging had a 7–10 s delay in the full frame scanning.

The microscope can sequentially scan a single point or a small region of interest (ROI) within a sample. The full frame resolution (laser points) was set as 1024×1024 or 512×512. As a result, ROI scanning provides us with the possibility to scan designed patterns. Both the laser beam can be moved orthogonally to the xy plane by high-speed oscillating mirrors and the sample along the z axis by a piezo stage. The single scan speed is 5–7 full frames per second.

Polarized optical microscopy and fluorescence microscopy
Cross-polarized optical microscopies (Zeiss Axio Imager M1m and Leica SP8) were used to record the images of the tactoids. The microscope (Zeiss Axio Imager M1m) equipped with LC (liquid crystal)-PolScope was used to capture the colorful PolScope images of the tactoids. To obtain the multiphoton image and three-dimensional fluorescent image, the multiphoton excitation confocal microscopy (Leica TCS SP8) with a water-immersed 25× objective was used. The excitation source used in our experiments was a continuous laser at 561 nm and a femtosecond laser at 780–950 nm. The confocal microscopy was also used to record the polarized image by an additional polarized filter. All analyses were performed using ImageJ, Zen software, and Laica X software.

Atomic force microscopy
The amyloid fibril samples were diluted to a final concentration of 0.02 wt.% to prepare for scanning by AFM. An aliquot (5 μL) of the diluted solution was deposited on a freshly cleaved mica, followed by rinsing with Milli-Q water and drying with a gentle compressed airflow after 3 min. A multimode VIII scanning probe microscope (Bruker) with a commercial silicon nitride cantilever (Bruker) at a vibration frequency of 150 kHz was used to obtain the morphologies. Finally, AFM images were simply flattened using the Nanoscope software (Bruker). The histogram of the fibril length was fitted with the Gaussian distribution.

Absorption spectrum and z-scan spectrum
The fabricated samples were diluted to a final concentration of 0.2 mg/mL in a 10 mm glass cuvette for the absorption spectrum measurements (JASCO spectrophotometer). Z-scan experiments were carried out using a laser system consisting of a femtosecond pulse generated by an amplified femtosecond system at an 80 MHz repetition rate, ~100 fs pulses operating as an 810 nm pump. Amyloid solutions were placed in a 1 mm deep glass capillary tube sealed with UV glue prior to scanning. The laser was focused onto the sample by a lens to a minimum beam waist radius of 30 μm measured by the knife edge method. The narrow path length cuvette ensured that the length was shorter than the Rayleigh length of the beam (≈3.5 mm). A silicon photodetector (ThorLabs, Newton, NJ, U.S.A.) was used to collect signals. Samples were driven along the beam path at a constant speed of 2 mm/s by an electronic control platform. Z-scans were collected from three independent positions in a capillary tube.

Data availability
All data that support the findings of this study are available within the article and the article and the Supplementary Information. Source data are provided with this paper.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript reports laser-induced manipulation of amyloid tactoids formed by isotropic-nematic transition in colloidal systems of anisotropic particles. Various types of manipulations such as local metlting, cutting, and self-healing are examined. Although the results are predictable and very reasonable, direct observations and experimental analysis with optical microscopy are new and valuable. For these reasons, I recommend publication after some revisions suggested below.\n- Various types of manipulations shown in the manuscript should require different conditions of laser-beam irradiation. Laser power should be the most important but other factors such as irradiation area can also contribute. Kinetic reasons such as appropriate irradiation period are also possible. Thus, summary of the relationship between the manipulation type and laser conditions is very valuable for readers.\n- Recover of the namatic structure after the perturbation by the laser irradiation is reasonable because thermodynamic stability of the nematic state. However, the tactoid reformation would not be explained by thermodynamics. Non-recoverable damages might happen under some irradiation conditions.\n- In the Discussion section, the authors concluded the contribution of enthalpic process in the tactoid formation and manipulation against the Onsager theorem assuming entropic process. For my understanding, however, the Onsager theorem does not predict the tactoid formation but only explains the isotropic-nematic transition. Also, many experimental studies have demonstrated that real systems do not rigidly obey the theorem. Contribution of non-entropic, i.e. enthalpic process shoud not be a new finding. In that sense, the results should be discussed in relation to the formation mechanism of tactoids.\n- Molecular mechanism of the tactoid collapse, reformation, and structural modification should be given.", "reviewer_2": "This work describes manipulation of amyloid colloidal liquid crystalline structures, tactoids, by micron scale targeted heating with a laser. The efforts done to visualize these phenomena as images, in short time scale, are very impressive and results highly interesting to learn from.\nEffects due to manipulation have been demonstrated from multiple angles making it interesting from application point of view. The different responses between bipolar and cholesteric tactoids to manipulation are very well documented and explained for further utilization.\nIt is curious to think that the 3PA technic would work on cellulose nanocrystal (CNC) tactoids as well, but since theres no aromatic functionalities, should the CNC material be somehow functionalized (chemically or with additives) to probe the technic on CNCs?\nI recommend publishing as it is. Beautiful work!", "reviewer_3": "Lin et al. show that amyloid tactoids can be manipulated using a pulsed laser. Heating droplets with high spatial accuracy, they control individual droplet morphology. The authors demonstrated this principle using two types of tactoids: bipolar and cholesteric. The morphological changes, particularly in the cholesteric tactoids, were interesting, as they could manipulate the number of bands. The authors described their process in detail and characterized the internal morphological changes. A particularly notable aspect of the work is the tactoids' ability to self-heal, which opens possibilities for many technological applications as well as fundamental physical studies. The authors also presented an application of their technique using a cholesteric tactoid as an optical switch, demonstrating both fundamental insights and practical applications. The reviewer would like to congratulate the authors on this work and finds it to be of high quality and suitable for the standards of Nature Communications. The reviewer particularly appreciates that this is curiosity-driven research, which led to interesting fundamental discoveries. The high-quality images and videos will certainly engage readers and demonstrate the possibilities of these findings. The work is ready for publication, but the reviewer would like to address minor corrections and clarifications that will increase the reproducibility and clarity of the work.\n\nThe reviewer recommends acceptance of this manuscript following these minor revisions."}, "author_response": "Responses to Reviewers\nNCOMMS-25-63118-T (Writing, reading, erasing, cutting and self-healing individual\namyloid tactoids by multiphoton absorption)\nReviewer #1\nThis manuscript reports laser-induced manipulation of amyloid tactoids formed by\nisotropic-nematic transition in colloidal systems of anisotropic particles. Various types\nof manipulations such as local metlting, cutting, and self-healing are examined.\nAlthough the results are predictable and very reasonable, direct observations and\nexperimental analysis with optical microscopy are new and valuable. For these reasons,\nI recommend publication after some revisions suggested below.\nResponse: We thank the Reviewer for their positive comments on our work. We\naddressed all the suggested revisions in the revised manuscript.\nComment 1: Various types of manipulations shown in the manuscript should require\ndifferent conditions of laser-beam irradiation. Laser power should be the most\nimportant but other factors such as irradiation area can also contribute. Kinetic\nreasons such as appropriate irradiation period are also possible. Thus, summary of the\nrelationship between the manipulation type and laser conditions is very valuable for\nreaders.\nResponse: We agree with the Reviewer, and in the revised manuscript, we added the\nfollowing section to the SI to clearly explain the relationship between the manipulation\ntype and laser conditions.\nIn our experiments, we applied irradiation using Point Scanning Confocal\nMicroscopy, which employs a focused laser beam to sequentially scan a single point or\nsmall region of interest (ROI) within a sample. The full frame resolution (laser points)\nwas set to 1024 × 1024 or 512 × 512, where the ROI feature allows for scanning\ndesigned patterns. To show the relation between the irradiated area and the actual area\naffected within the sample, we performed the following experiments. We designed three\ndifferent irradiation areas (48.6, 176.4 and 360.6 μm2) in ROI as shown in\nSupplementary Fig. 5 and irradiated them under the same conditions. Following the\nirradiation, we immediately measured the disordered regions within the sample, marked\nwith red dash circles in Supplementary Fig. 5b. Our measurements show that the area\nset by the ROI and the disordered region measured within the samples are of\ncomparable size, indicating the same irradiation effects on different areas. We note that\nthe slightly lower values for disordered areas compared to the ROI region are due to a\nmeasurement delay (> 10 s) between the NIR irradiation and POM imaging, which is\nunavoidable for technical reasons.\nAdditionally, to show how the kinetics of the irradiation plays a role in disordering\nthe ordered amyloid fibril samples, we performed two sets of experiments with different\nexposure time intervals, but the same total irradiation time (Supplementary Fig. 5d). In\nthe first set of experiments, a sample was irradiated with a pixel dwell time of 1.2 μs to\nobtain an isotropic region (167.1 μm2). In the second set, the pixel dwell time was\nchanged to 0.4 μs with three repetitions, and the system was given the same amount of\ntime to recover after each exposure. The result shows that, despite the same total\nexposure time (and thus energy), the isotropic region obtained by laser with recovery\nintervals was smaller (121.9 μm2) than that from a single continuous exposure.\nThe discussion was added in revised manuscript and highlighted with yellow color\nas follows:\n“Systematically, the relationship between the laser-induced nematic-isotropic\ntransition rate and laser power was investigated. As shown in Fig.1f, we found that a\npower of 13.6 mW (total pixel dwell time of 16 μs) could have distinct breaking effect\non cholesteric structures. Here, to show the relation between the irradiation area and the\nactual area affected within the sample, we designed three different irradiation areas\n(48.6, 176.4 and 360.6 μm2) as shown in Supplementary Fig. 5 and irradiated them\nunder the same conditions. Following the irradiation, we immediately measured the\ndisordered regions within the sample, marked with red dash circles in Supplementary\nFig. 5b. Our measurements show that the area set by program and the disordered region\nmeasured within the samples are of comparable size (Supplementary Fig. 5c),\nindicating the same irradiation effects on different areas. We note that the slightly lower\nvalues for disordered areas compared to the set region are due to a measurement delay\n(> 10 s) between the NIR irradiation and POM imaging. Additionally, the kinetic\nirradiation was also measured in Supplementary Fig. 5d. The result shows that the\nisotropic region (121.9 μm2) obtained by ON-OFF laser period (pixel dwell time of 0.4\nμs with three repetitions) was smaller than the single continuous exposure (167.1 μm2,\npixel dwell time of 1.2 μs). Over all, this method provided us with a powerful and\nversatile method for generating complex 3D disordered phase patterns within the\notherwise LC-ordered state of the tactoid.”\nSupplementary Fig. 5 | The effects of irradiation area and time on samples of\nordered amyloid fibrils. a. Three different irradiation areas were designed. b. The\nensued disordered regions within the sample as a consequence of irradiation are marked\nwith red dashed circles. Scale bar is 30 μm. c. The irradiation area and the resulting\ndisordered region (isotropic area) measured within the samples are of comparable size.\nd. The comparison between a sample irradiated with a pixel dwell time of 1.2 μs and a\nsample irradiated with the same total exposure time, but divided into three times with\nrecovery intervals. Scale bar is 10 μm.\nComment 2: Recover of the namatic structure after the perturbation by the laser\nirradiation is reasonable because thermodynamic stability of the nematic state.\nHowever, the tactoid reformation would not be explained by thermodynamics. Non-\nrecoverable damages might happen under some irradiation conditions.\nResponse: We first note that, in our experiments, we set the laser irradiation power to\nbe less than 20 mW, which does not result in non-recoverable damage in the amyloid\nfibrils (Fig.1). Previously, with a series of femtosecond laser irradiation experiments\n(ACS Nano 2023, 17, 10, 9429–9441; ACS Chem. Neurosci. 2016, 7, 12, 1728–1736),\nwe and others have shown that amyloid fibrils are not damaged with the laser power as\nhigh as 200 mW. Thus, we conclude that the laser irradiation at the intensities used only\ninduces reversible order-disorder transition in the exposed area. Additionally, in other\nearlier works from our group on amyloid fibrils and cellulose nanocrystals, we have\nshown experimentally and numerically the formation of tactoids with timescales of the\norder of a few minutes (Nat Commun 2022, 13, 2778, Nat Commun 2023, 14, 607).\nThis behavior of tactoid formation, the symmetry of the nematic field and the structural\nrelaxation of mesogens were successfully explained following thermodynamics\nprinciples in these works.\nComment 3: In the Discussion section, the authors concluded the contribution of\nenthalpic process in the tactoid formation and manipulation against the Onsager\ntheorem assuming entropic process. For my understanding, however, the Onsager\ntheorem does not predict the tactoid formation but only explains the isotropic-nematic\ntransition. Also, many experimental studies have demonstrated that real systems do not\nrigidly obey the theorem. Contribution of non-entropic, i.e. enthalpic process shoud not\nbe a new finding. In that sense, the results should be discussed in relation to the\nformation mechanism of tactoids.\nResponse: The referee is entirely correct by saying that Onsager theory determines only\nthe boundaries of isotropic and nematic phase, and that tactoids formation is more\nproperly described by other theories involving elastic energy distortion, first in line\nbeing the Frank-Oseen energy functional (see for example our works starting with\nNature Nanotechnology, 13, 330–336 (2018)). However, we note that in our work the\ninput of energy in the form of NIR irradiation (i.e. heat dissipation, enthalpy) leads of\na nematic-to-isotropic transition in the entire irradiated volume irrespectively of the\ntactoid volume or symmetry. Therefore, the order-disorder transition discussed in the\nmanuscript remains of “bulk” nature, where by “bulk” we mean the entire irradiation\nvolume. Therefore, under the conditions used, the Onsager theory remains the reference\ntheory to understand the energetic driving force restoring the liquid crystalline ground\nstate after irradiation. From there, the observation that non-entropic (enthalpic)\ncontributions lead to liquid crystalline melting, as opposed to the Onsager theory\nfindings, is relevant and highlight a non-trivial finding. The observation made by the\nreferee becomes important if the irradiation volume is larger than the single tactoid\nvolume. These experimental conditions are not used in the manuscript, but we take\ninspiration from this point to design corresponding experiments in our future work.\nComment 4: Molecular mechanism of the tactoid collapse, reformation, and structural\nmodification should be given.\nResponse: We are thankful to the reviewer for this constructive suggestion. Again, we\nmust note to start that order-disorder-order transitions occur within the full irradiation\nvolume, which makes the problem under the lens a “bulk” study. This being said, if by\n“molecular” mechanisms, the reviewer refers to “mechanistic”, the melting process\nupon NIR irradiation is (in the ideal case) an order-disorder process associated with a\nchange in the relative contributions of free volume and rotational entropies, as predicted\nby the original Onsager theory, with the main difference that the trigger is here the\ninjection of enthalpy via NIR irradiation. As noted on the comment above, this is a\nsurprising finding that we have already commented in the manuscript. The reformation\nand structural evolution back to the ground LC state from the isotropic phase is, on the\ncontrary, a disorder-order transition which is very well described by the Frank-Oseen\ntheory and already discussed in several earlier publications from us and other groups.\nFor a review on this process, we address the Reviewer to our recent review: Reports on\nProgress in Physics 88 (3), 036601 (2025).\nIf, on the other hand, by “molecular” mechanisms, the reviewer refers to “molecular\nscale”, the amyloid mesogen length scale of ~ 435 nm in length and 4 nm in diameter\nmakes the complexity of the problem intractable at the molecular level. Any study on\nmolecular mechanisms of first order transitions, which we agree is of high importance,\ndeserves a full separate story possibly via coarse-grained MD simulations, as fully\natomistic simulations would be extremely time consuming. This is a topic which\ndeserves a fully study on its own.\nReviewer #2\nThis work describes manipulation of amyloid colloidal liquid crystalline structures,\ntactoids, by micron scale targeted heating with a laser. The efforts done to visualize\nthese phenomena as images, in short time scale, are very impressive and results highly\ninteresting to learn from. Effects due to manipulation have been demonstrated from\nmultiple angles making it interesting from application point of view. The different\nresponses between bipolar and cholesteric tactoids to manipulation are very well\ndocumented and explained for further utilization. It is curious to think that the 3PA\ntechnic would work on cellulose nanocrystal (CNC) tactoids as well, but since theres\nno aromatic functionalities, should the CNC material be somehow functionalized\n(chemically or with additives) to probe the technic on CNCs? I recommend publishing\nas it is. Beautiful work!\nResponse: We are happy that the Reviewer finds our work impressive and beautiful.\nWe thank the Reviewer for his/her encouraging comments. We tried the effect of the\n3PA on CNCs tactoids (no functionalities, Supplementary Fig. 10) and observed no\nvisible changes in the tactoids' structure. CNCs tactoids were actually used as control,\nprecisely because free of aromatic groups capable of absorbing radiation. As the\nReviewer precisely suggested, functionalizing the CNC would be a next promising step\nin an attempt to manipulate CNC tactoids with MPA, and we may take inspiration from\nthis comment for our future work.\nSupplementary Fig. 10 | Exposure of CNCs tactoids. a. The molecular structure of\nCNCs and the UV-VIS spectrum of CNCs suspension. The 4% CNCs suspension shows\nthe formation of birefringent domains. b. AFM images of CNCs (diluted ×500). The\ninset image is the CNCs film on Mica. The scale bar is 500 nm. c. POM image of CNCs\ncholesteric tactoids after an incubation of 4 days. The red region is prepared for the\nlaser exposure. POM image was captured under a crossed polarizer. The scale bar is 60\nμm. d. POM image checked immediately after the exposure. No visible changes occur\nin the cholesteric structure exposed in the marked region. The scale bar is 60 μm.\nReviewer #3\nLin et al. show that amyloid tactoids can be manipulated using a pulsed laser. Heating\ndroplets with high spatial accuracy, they control individual droplet morphology. The\nauthors demonstrated this principle using two types of tactoids: bipolar and cholesteric.\nThe morphological changes, particularly in the cholesteric tactoids, were interesting,\nas they could manipulate the number of bands. The authors described their process in\ndetail and characterized the internal morphological changes. A particularly notable\naspect of the work is the tactoids' ability to self-heal, which opens possibilities for many\ntechnological applications as well as fundamental physical studies. The authors also\npresented an application of their technique using a cholesteric tactoid as an optical\nswitch, demonstrating both fundamental insights and practical applications. The\nreviewer would like to congratulate the authors on this work and finds it to be of high\nquality and suitable for the standards of Nature Communications. The reviewer\nparticularly appreciates that this is curiosity-driven research, which led to interesting\nfundamental discoveries. The high-quality images and videos will certainly engage\nreaders and demonstrate the possibilities of these findings. The work is ready for\npublication, but the reviewer would like to address minor corrections and clarifications\nthat will increase the reproducibility and clarity of the work.\n\nThe reviewer recommends acceptance of this manuscript following these minor\nrevisions.\nResponse: We are happy that the Reviewer finds our work high quality study and would\nlike to thank them for their positive comments. We addressed all the suggested revisions\nin the revised manuscript.\nComment 1: The reviewer is particularly interested in the experimental details of the\nheating protocol. As far as the reviewer understood, the authors are using a scanning\nlaser microscope, but it is not clear from the methods section whether the laser is\nmoving, or the sample is moving. Scanning inherently involves a delay between different\nlocations. The reviewer would like to know how fast this scanning is, which raises the\nquestion of whether any delay can be ignored. The authors should comment on whether\nthis delay in heating times could affect the dynamics of tactoid self-healing. For\nexample, in supplementary movie 5, the zipping and recovery of the tactoid begins at\none end and then zips back. It is unclear whether the laser cuts one part first and then\nthe second part, or if it scans back and forth between the two locations. This also\napplies to the more complex patterns described in Figures 3e and 3j. Additionally,\nFigure 1f is not clear regarding power density units. The x-axis should be labeled as\npower per cm², which for a pulsed laser should be on the order of TW/cm² or GW/cm²\ngiven the description of the optical setup. The y-axis units are also difficult to\nunderstand— is this related to the scanning rate? From the insets, the reviewer cannot\ndetermine at what power the laser induced the isotropic transition.\nResponse: We are thankful for this comment of the reviewer which allows us to clarify\nimportant experimental details. The irradiation we applied is from a point scanning\nconfocal microscopy (Leica SP8 equipped with Mai Tai XF (Spectra-Physics), tunable\nfrom 720–950 nm). The microscopy can sequentially scan a single point or small region\nof interest (ROI) within a sample. The full frame resolution (laser points) was set as\n1024×1024 or 512×512. As a result, ROI scanning provides us possibility to scanning\ndesigned patterns. Both the laser beam can be moved orthogonally to the xy plane by\nhigh-speed oscillating mirrors and the sample along the z axis by a z piezo stage. The\nsingle scan speed is 5-7 full frames per second. Thus, once the designs (for example,\nthe three striped-shaped regions as in Movie 5) are set, the laser irradiation takes less\nthan one second, indicating that delay between patterns is on the order of microseconds.\nConsidering that in all our discussions about zipping and recovery (for example, see\nFig. 3c,d,g,h and Fig. 4d-g,i) involve time scales on the order of hundreds of seconds,\nthe delay can be ignored.\nThe relevant descriptions have been added to the methods section and tracked in\nyellow color:\n“The microscopy can sequentially scan a single point or small region of interest (ROI)\nwithin a sample. The full frame resolution (laser points) was set as 1024×1024 or\n512×512. As a result, ROI scanning provides us possibility to scanning designed\npatterns. Both the laser beam can be moved orthogonally to the xy plane by high-speed\noscillating mirrors and the sample along the z axis by a z piezo stage. The\nsingle scan speed is 5-7 full frames per second.”\nWe thank the Reviewer for pointing out the issues regarding the axes in Fig. 1f. It\nshould be laser power (mW). Power meter was used here to measure the power of laser\nfrom objective (25× 0.95NA L Water HCX IRAPO). In addition, we now changed the\ny axis to the scanning time of each pixel (laser point) from nematic to isotropic. The\nscanning time was calculated following single pixel dwell time multiplied by the\nnumber of repetitions. The result shows that the irradiation of 4.6 and 9.2 mW would\nnot trigger the structure transition with a total pixel dwell time of 40 μs. The obvious\ntransition was induced when the laser power increased to 13.6 mW (total pixel dwell\ntime of 16 μs).\nFig. 1. | The LLCPS of β-lactoglobulin (BLG) amyloid fibrils and the multiphoton\nadsorption of amyloid tactoids. a. Amyloid fibrils with β-sheet-rich structures were\nobtained from the incubation of β-lactoglobulin (BLG) protein at pH=2 and 90 ℃\nconditions. Protein structure is available from the Protein Data Bank (PDB ID: 3BLG).\nb. The AFM images of amyloid fibrils before (left panel: top-left) and after (left panel:\ntop-right) being shortened into uniform length, and TEM image (left panel: bottom) of\noriginal amyloid fibrils. The scale bar is 500 nm. When prepared at a concentration\nwithin the Onsager binodal lines, the suspension of short amyloid fibrils shows the\nformation of a rich variety of tactoids through LLCPS (right panel, image is taken\nbetween crossed polarizers). The schematic shows the director field orientation for\nthree different classes of homogenous, bipolar and cholesteric tactoids. The scale bar is\n200 μm. c. Schematic showing the exposure of LC cholesteric structure to the pulsed\nlaser. Compared to the ordinary laser focus, the multiphoton absorption can trigger the\nheating precisely on a small focused spot. This spot can be scanned spatially in xyz-\naxes. d. Open aperture z-scan transmission curve from BLG fibrils solution with\ntheoretical fitting for three-photon absorption (3PA). The sample was exposed with 100\nfs pulses at 810 nm wavelength with a pulse repetition rate of 80 MHz. e. Linear\nabsorption spectrum of BLG fibrils with background subtracted at one-photon\nabsorption (1PA) mode. The absorption was measured in the range between 200 and\n800 nm. f. The relationship between the scanning time (pixel dwell time) of laser-\ninduced nematic-isotropic transition and laser power. The scanning time was calculated\nfollowing single pixel dwell time multiplied by the number of repetitions. Here, a two-\ndimensional single scanning layer inside of a cholesteric tactoid was exposed under\ndifferent laser power. The dark region represents the localized disordered state of\namyloid fibrils induced by the laser exposure.\nComment 2: Following the previous question, the reviewer would like to know how the\nauthors controlled the thickness of the exposure region depicted in Figure 3a. Is the\nobjective or sample moving up and down? Given that the laser spot should be smaller\nthan 5 μm, the reviewer wonders what the protocol is for controlling these regions. The\nreviewer thinks these questions could be easily answered with a short description of the\nprotocol in the methods section, which would give readers a clearer picture of how the\nexperimental setup works.\nResponse: The scanning of the thickness is based on the z-piezo stage (not the\nobjective). To control these small regions, we use ROI + z-stack protocol to realize the\nirradiation of required volumes. In our experiments, we fixed distance between each\nlayer in z stack irradiation at 1 μm. As a result, a 5 μm deep region would cover 5 layers\nof scanning.\nWe agree with the Reviewer, and, in the revised manuscript, we added following\nnotes to the Methods section to clearly explain our protocol:\n“The scanning of the thickness is based on the z-piezo stage. We used ROI + z-stack\nprotocol to realize the irradiation of required volumes where the distance between each\nlayer in z stack irradiation was fixed at 1 μm.”\nComment 3: Can the authors explain how they concluded that the Rayleigh length of\nthe beam is around 3.5 mm given the numerical aperture of the objective and the\nwavelength used? This Rayleigh length should be in the submicron realm.\nResponse: Thank you for the comments. We did not use the objective here. The\nwavelength is 810 nm and the beam waist radius is 30 μm (measured by the knife edge\nmethod). In air,\n𝜋𝜔2\n𝑍 = 0\n𝑅\n𝜆0\nthis result in Z =3.5 mm. We added following notes to the Methods section to clearly\nR\nexplain our protocol:\n“The laser was focused onto the sample by a lens to a minimum beam waist radius\nof 30 μm measured by the knife edge method.”\nComment 4: Could the authors comment on why cutting along the tactoid horizontally,\nas depicted in figure 4h, did not change the number of bands, given that figures 5b and\n5d show cuts in a similar fashion? Is the pitch control a function of the laser power?\nResponse: The Reviewer is correct. Cutting the tactoid or manipulating the bands is\nachieved by controlling the laser power. Cutting or manipulating the pitch depends\ndirectly on the laser power. A higher laser power (∼0.2 nJ per pulse) results in breaking\nthe LC order of amyloid fibrils and consequently results in the separation of tactoids,\nwhile a lower laser power (∼0.14 nJ per pulse) induces a relaxation gap that grow a\nnew band. In the manuscript, we mentioned the laser power in each manipulation.\nComment 5: When describing the optical switch, how did the authors induce or control\nthe tactoid rotation? Do the authors know why the fluorescence is angle-dependent?\nResponse: The rotation is realized by rotating the laser. In terms of the angle-dependent\nfluorescence, we believe that fluorescence, as other optical and physical properties of\nthe tactoids has a tensorial anisotropic distribution with respect to the polar coordinates\ntaken from the helical axis of the chiral nematic field.\nComment 6: It is difficult to discern B1 and B2 in Figure 2i showing the cutting of a\nbipolar tactoid. Supplementary Figure 6 helped considerably, but during the initial\nreading it was difficult to understand what B1 and B2 represented. The authors could\nconsider using the B1 and B2 images from the supplementary information as insets.\nResponse: We drew a new cartoon to help the understanding of B1 and B2 in Fig.2i.\nFig. 2 | Manipulation of amyloid tactoids by 3D-Heating. a. Time-lapse images of\nirradiation of a single bipolar tactoid with marked exposed region (fully covered). The\nscale bar is 50 μm. b. Aspect ratio and volume of fully exposed bipolar tactoids in (a)\nplotted vs. exposure time. The gray curves represent the fittings. c. Irradiation of half a\nbipolar tactoid. The scale bar is 50 μm. d. Aspect ratio and volume of half exposed\nbipolar tactoids in (c) vs. exposure time. e. Time-lapse images of a cholesteric tactoid\nbeing fully exposed to irradiation. The scale bar is 60 μm. f. Aspect ratio and volume\nof the cholesteric tactoid in (e). g. Exposure of half cholesteric tactoid. The scale bar is\n60 μm. h. Dynamics of half pitch measured in the non-exposed region of the tactoid in\n(g). The color bar represents the value of half pitch. i. Break-up of a bipolar tactoid by\n3D-Heating. The images show the cutting process of a large bipolar tactoid into two\nsmaller bipolar tactoids, labeled B1 (inside) and B2 (outside). The thickness of the\nexposure region Δz = 5 μm. The scale bar is 30 μm. j. The aspect ratio and volume of\nthe tactoid (B1) during the bipolar cutting. The gray curve represents the fittings. k. A\ncholesteric tactoid (O1) was cut into two smaller cholesteric tactoids along the band\n(2R) with Δz = 15 μm. The resulting tactoids are labeled C1 and C2. The tactoid O2 is\nmarked as the control experiment. The scale bar is 30 μm. l. Measurement of half pitch\nwhile O1 transforms to C1 and C2 tactoid. All of the images were obtained under a\ncrossed polarizer.\nComment 7: The reviewer found Supplementary Figure 3 very useful, as it provides a\nclear picture of the different types of tactoids and how the authors are studying them.\nThe reviewer recommends that the authors consider incorporating this figure into\nthe main text.\nResponse: Thank you for the suggestion. We added schematics of tactoids from\nSupplementary Figure 3 into Fig.1b now (see Fig. 1b in Comment 1). In addition, the\nLC-PolScope images of the three classes of tactoids are kept in Supplementary Fig. 3.\nComment 8: Really minor comments: Figure 1e would benefit from arrows pointing to\nthe peak absorption wavelength and the laser excitation wavelength. Figure 2l\nregarding the pitch of the bands was much more useful than the heat maps.\nResponse: We added an arrow pointing to the peak absorption wavelengths in the\nrevised Fig. 1e (shown in comment 1). For the UV-Vis absorption spectrum, we added\nthe description of “The absorption was measured in the range between 200 and 800 nm”.\nThe modified figure is exhibited in comment 1.\nIn Fig. 2h, the heat map was made to show the half pitch distribution of each band.\nIn order to show the half pitch value directly and clearly, we followed the suggestion\nfrom the Reviewer and changed the heat map into cross section profiles like Fig. 2l.\nPlease find the Fig.2h in Comment 6."}, {"round": 2, "reviewer_comments": {"reviewer_1": "I satisfied the revision of the manuscript. I recommend publication as is.", "reviewer_2": "Nice work, I recommend publishing.", "reviewer_3": "The authors have adequately addressed all the reviewer's comments and concerns. The reviewer notes that the other reviewers' assessments were similarly positive, which reinforces that this manuscript is suitable for Nature Communications. The manuscript is now ready for publication."}, "author_response": ""}]

Decision letter