Compression of colloidal monolayers at liquid interfaces: in situ vs. ex situ investigation

The assembly of colloidal particles at liquid/liquid or air/liquid interfaces is a versatile procedure to create microstructured monolayers and study their behavior under compression. When combined with soft and deformable particles such as microgels, compression is used to tune not only the interparticle distance but also the underlying microstructure of the monolayer. So far, the great majority of studies on microgel-laden interfaces is conducted ex situ after transfer to solid substrates, for example, via Langmuir-Blodgett deposition. This type of analysis relies on the stringent assumption that the microstructure is conserved during transfer and subsequent drying. In this work, we couple a Langmuir trough to a custom-built small-angle light scattering setup to monitor colloidal monolayers in situ during compression. By comparing the results with ex situ and in situ microscopy measurements, we conclude that Langmuir-Blodgett deposition can alter the structural properties of the colloidal monolayers significantly.


Abstract:
The assembly of colloidal particles at liquid/liquid or air/liquid interfaces is a versatile procedure to create microstructured monolayers and study their behavior under compression.
When combined with soft and deformable particles such as microgels, compression is used to tune not only the interparticle distance but also the underlying microstructure of the monolayer. So far, the great majority of studies on microgel-laden interfaces is conducted ex situ after transfer to solid substrates, for example, via Langmuir-Blodgett deposition. This type of analysis relies on the stringent assumption that the microstructure is conserved during transfer and subsequent drying.
In this work, we couple a Langmuir trough to a custom-built small-angle light scattering setup to monitor colloidal monolayers in situ during compression. By comparing the results with ex situ and in situ microscopy measurements, we conclude that Langmuir-Blodgett deposition can alter the structural properties of the colloidal monolayers significantly.

INRODUCTION
Colloidal monolayers at liquid interfaces, namely, micro-and nanoparticle-laden liquid interfaces, are widely used in fundamental and applied studies. Colloidal particles can self-assemble, in fact, in two-dimensional materials with properties (e.g. photonic or electronic) similar to those of atomic structures. However, unlike atomic counterparts, the colloidal building blocks can be engineered in terms of the chemical composition, 1-4 shape, [4][5][6][7] and morphology, 8,9 in order to tailor the assembly behavior and spatial arrangement. One of the methods for preparing colloidal monolayers is via confining particles at the flat interfacial plane between two immiscible fluids (e.g. an air/water or oil/water interface). 10 This approach offers great advantages not only for studies of gas-liquid-solid phase transitions as the particle concentration (i.e., the number of particles per unit area) can be tuned in situ by means of lateral barriers, 11,12 but also for scalable fabrications for both planar and curved surfaces with areas ranging from cm 2 up to m 2 scales. [13][14][15] In the latter approach, the microstructures at the liquid interface are transferred and deposited on solid surfaces (Langmuir-Blodgett deposition) to obtain dried colloidal films, e.g. for coating or photonic applications. [16][17][18][19][20] In contrast to assemblies of rigid spheres, soft colloidal objects like microgels and nanogels 21 can be deformed, for example, under external compression giving access to richer phase diagrams and complex superstructures. 22,23 The structural properties of colloid-laden interfaces are typically extracted from microscopy images by detecting the centers of mass of the colloidal units. [24][25][26][27][28] This "particle-tracking" method quickly becomes time-consuming and computationally demanding in the presence of many particles. Even more importantly, it can be only applied above the Abbe diffraction limit. For example, due to the mostly small sizes synthesized to date, assemblies of coreless and core-shell microgels have been mainly characterized ex situ -by looking at dried samples with atomic force or electron microscopes -under the assumption that the structure is unaltered during Langmuir-Blodgett deposition. 8,11,12,[29][30][31][32][33] Only recently, in situ observation of local regions of the particleladen interface was achieved via atomic force microscopy. 34 In this study, we propose an in situ method -a Langmuir trough combined with small-angle light scattering (LT-SALS) -to characterize colloidal monolayers at the air/water interface. To demonstrate its versatility, colloids of different morphologies were monitored during compression: silica particles (rigid spheres), poly-N-isopropylacrylamide (PNIPAM) microgels (soft spheres) and silica-PNIPAM core-shell microgels (hard core-soft shell spheres). The focus of our study, however, lies on the assembly of the core-shell (CS) microgel system. We first present the results from an ex situ structural analysis using Langmuir-Blodgett deposition. Then, we compare these results to an in situ analysis performed using LT-SALS as well as fluorescence microscopy. Our results indicate that there are severe structural differences between the microstructures of CS microgels at air/water interfaces and after transfer to a solid substrate. These drying effects are in stark contrast with the widely accepted assumption that the interfacial structure is replicated during Langmuir-Blodgett deposition for microgel type building blocks. We discuss analogies and differences with existing works as well as possible reasons for the observed structural changes during drying.

Core-Shell Microgels
We prepared monolayers of CS microgels at an air/water interface in a Langmuir trough and studied their structure under compression using ex situ light microscopy (Method 1), in situ fluorescence microscopy (Method 2) and in situ small-angle light scattering (Method 3).
CS microgels possess two relevant length scales: the diameter of the incompressible core (here, silica) and the thickness of the soft, deformable shell (here, PNIPAM). In bulk, these length scales simply define the boundaries of the interparticle interactions. When the microgels are confined and spread at the air/water (or oil/water) interface, the situation becomes more complex because the shells laterally deform at the interface leading to changes in shell morphology and shape, and consequently the total diameter, Di (interfacial diameter), which is larger than the bulk hydrodynamic diameter, Dh. 12,23,35,36 Generally, there are three different scenarios for the spatial arrangement of CS microgels at air/water or oil/water interfaces: At very low number of particles per unit area (nP/A), i.e., for near-zero surface pressures, the CS microgels mostly stay apart in an unordered, fluid-like state. In the second regime, as the nP/A increases, the microgel shells start to touch (shell-shell contact) more frequently. Finally, in the third regime, the shells are squeezed and/or interpenetrated (core-core contact) until the critical point, where the monolayer buckles, breaks and/or is pushed into the subphase (water). Theoretically, if the energy difference between the partially and fully overlapped shells is small enough, energy minimization is achieved by the overlap of shells in some directions at the cost of other neighboring shells, 37-39 leading to a change in the symmetry of the monolayer. In experimental studies, however, such a symmetry change of the microgel monolayer with increasing nP/A has only been partially observed. 40 In most cases, core-shell structured microgels 12,29,30 and coreless microgels [31][32][33][41][42][43] seem to undergo an "isostructural solid-solid phase transition" characterized by microgels in hexagonal arrangement with "shell-shell" contact versus a denser phase, also of hexagonal order, where the microgels are in "core-core" contact. 11,44 Note that the interparticle distance in "core-core" contact includes the diameter of the core, Dc, as well as the compressed microgel shell. The discrepancies between numerical and experimental studies concerning the phase behavior at interfaces have been ascribed to capillary forces and a highly nonlinear mechanical response of the polymer chains (i.e. the PNIPAM corona) under compression 11,30,33,40 In the following, we report the same "isostructural solid-solid phase transition" only during ex situ characterization (Method 1) of monolayers of micron-sized CS microgels. Remarkably, this phenomenon is not observed during in situ experiments (Methods 2 and 3).

Method 1 (ex situ microscopy)
The ex situ microstructural analysis relies on the microscopic investigation of the colloidal monolayer upon transfer from the liquid interface to a solid substrate followed by drying. This leads to dry, substrate-supported colloidal monolayers. When the transfer to the solid substrate is done continuously while the monolayer is compressed in the Langmuir trough, the monolayer position on the substrate can be linked to the corresponding surface pressure at the liquid interface. 11,44 In this study, the CS microgel system used for the in situ and ex situ comparison has a core diameter Dc = 340 ± 20 nm and a total hydrodynamic diameter Dh = 920 ± 18 nm (see Synthesis Section for more details). Figure 1A shows the measured compression isotherm during the Langmuir-Blodgett deposition along with the corresponding microscopy images. Note that we use a linear color coding from light blue to black linked with low to high surface pressure (Π) throughout this article. From the mid Π regime (16.3 mN/m and higher), the images were taken in dark field mode to facilitate image analysis. The monolayer images at lower Π were recorded in bright field mode. With increasing compression, i.e., decreasing available area, A, the surface pressure increases continuously. In the low Π regime, the CS microgels are not homogeneously distributed over the accessible area (see microscopy images) but rather show hexagonal arrangements with shell-shell contacts and some voids among numerous crystalline domains. This indicates the presence of attractive interparticle interactions despite the large interparticle distances, in agreement with previously reported results from in situ and ex situ analysis of CS microgel monolayers. 8,12,44 As Π increases, the crystalline domains grow while the voids close.
In the high Π regime, we observe the formation of CS microgel clusters in "core-core" contact.
The critical surface pressure for the start of this "isostructural solid-solid phase transition" is around 16 -18 mN/m for the presented CS microgel, which can be also identified both in the splitting of the first peak of the radial distribution functions (RDFs, Figure 1B) and in the diffraction patterns of the dried monolayers (Figure 2). Although the ex situ "core-core" distance should lie within the detection limit of our current SALS setup, the microstructures produce diffuse scattering patterns (Figure 2C and 2D), instead of revealing two distinctive length scales. This is due to the fact that the "isostructural phase transitions" is only locally isostructural, i.e. the monolayers, on mm 2 scale, do not show a defined symmetry. The transition is more pronounced for higher values of Π, i.e. the diffuse contribution to the scattering patterns increases with increasing Π. For low to medium values of Π, the RDFs are characterized by the first peak corresponding to the interparticle distance, i.e. center-to-center distance from ex situ image ( − , ), whereas for high Π above the critical value (e.g. 23.2 mN/m), the peak splits near Dh. Figure 1C reflects the appearance of these two distinct interparticle distances, as the value of − , approaches Dh.
In summary, the ex situ analysis reveals that the CS microgel monolayers undergo an

Method 2 (in situ microscopy)
In situ analysis of the monolayers of CS microgels at the air/water interface under compression was done by combining fluorescence microscopy with a microscopy trough, i.e., a trough equipped with an optical window. Figure 3A shows representative microscopy images taken at various values of Π during compression. At near zero Π, we observe clusters of CS microgels due to attractive (capillary) interparticle interactions (see Figure S1 in Supporting Information (SI)), as also reported for other large (Dh > 700 nm) coreless and CS microgels. 8,[44][45][46] In the regime of low Π, we observe similar microstructures as for the ex situ analysis after transfer to a substrate.
However, the degree of order appears to be lower at the air/water interface. For medium-to-high values of Π, the comparison with ex situ results reveals striking differences: unlike − , , the in situ interparticle distances, − , , continuously decrease and the degrees of order increase with increasing Π. An "isostructural solid-solid phase transition" is not observed, in contrast to the assembly behavior reported for other similarly-sized coreless and CS microgels. 29,44 This becomes even more evident when looking at selected RDFs as presented in Figure 3B. The first double peak in the RDFs for higher Π is not present. Furthermore, the higher degree of order is reflected by the large number of distinct peaks in the RDF computed for the highest Π. In contrast to the high Π regime studied in the ex situ analysis, the monolayer possesses pronounced long-range order when studied at the air/water interface. Figure 3C shows are slightly larger than half the initial − , and lie -until the monolayer buckles -in between the two distinct distances (shell-shell and "core-core") determined by the ex situ analysis.
To summarize, the in situ measurements using fluorescence microscopy revealed significant differences not only in the microstructure of the monolayer but also in terms of the evolution of the interparticle distance and a noticeable shift in np/A for corresponding Π (see Figure S2A and B in SI for more details). For the studied CS microgels, these findings point towards a pronounced drying and/or substrate effect upon transfer by Langmuir-Blodgett deposition, as typically performed for such ex situ microstructure analysis. We address this further when discussing the LT-SALS experiments in the next section.

Method 3 (in situ LT-SALS)
We realized a custom-built setup that combines a Langmuir trough featuring a transparent glass window in the trough bottom (microscopy trough) with a custom-built SALS setup that allows to measure diffraction patterns at high frame rates (up to 30 frames per second, in our case). The details of this setup are provided in the Experimental Section and in the SI. Furthermore, the SI addresses the achievable q-range for various laser wavelength highlighting the versatility of the presented LT-SALS method. Figure     In conclusion, the results obtained using the three Methods (ex situ microscopy, in situ microscopy and in situ LT-SALS) are compared in Figure 6A. The grey and blue shadowed areas illustrate the calculated interparticle distances from LT-SALS and in situ fluorescence microscopy, respectively, whereas the filled squares correspond to the ex situ measurements. The graph highlights the conflict between the ex situ and in situ analysis of our CS microgel monolayers. The continuous evolution of interparticle distance in monolayers at the air/water interface during the continuous compression was also observed for other CS microgels, as illustrated in Figure 6B and 6C, where the interparticle distance (normalized by the core diameter) is plotted as a function of Π for CS microgels with different shell thickness ( Figure 6B) and overall hydrodynamic diameters ranging from 770 to 1170 nm ( Figure 6C).

Application of LT-SALS to Other Colloidal Systems
In this section, we would like to briefly emphasize the versatility of LT-SALS by showing data for two additional representative colloidal systems, i.e., silica particles (as an example of rigid spheres) and PNIPAM microgels without rigid cores (as an example of soft spheres). In i.e. in situ interparticle spacing, of the silica particle monolayer is also present in the ex situ microscopic images and the diffraction patterns of the dried silica monolayer as shown in Figure   S3 (SI).
The diffraction pattern obtained from the monolayers of the PNIPAM microgels (Dh = 858 ± 41 nm) goes through a transition from a diffraction ring (unordered state) to six distinct Bragg peaks (hexagonally ordered state) near the maximum Π. The monolayers showed rather small changes in Π per area reduced over the course of compression. The experiment was conducted in a highly compressed state not only because the high Π regime is where the structural change is most visible but also because the interparticle distance in the low Π regime is far too large to be resolved by diffraction analysis with our current setup. Figure 7B illustrates  Figure S4 (SI) confirms that the interparticle distance at the air/water interface evolves in a continuous manner throughout the compression also for these coreless microgels. Additionally, the contrast in the spatial arrangements between the in situ monolayer and the dried microgels ( Figure S5), which resembles reported dried monolayer of similarly sized CS microgels, 40 further supports our conclusion that the drying process accompanies structural changes. which would otherwise prevent "core-core" contact. Both contributions depend on various parameters including the degree of deformability of the shells (which is mainly determined by the crosslinker density 50 ), the size of the core and the shell, the materials, and the overall synthesis protocol. Ex situ measurements suggest that microgels with low crosslinker density show a more continuous evolution of the interparticle distance, whereas higher crosslinker densities give rise to "isostructural phase transitions". 29, 31-33 However, we want to note that small microgels with low crosslinker density tend to self-assemble into less ordered structures. 51 Phase transitions seem also to be more likely when large microgels (e.g., Dh = 1450 nm 44 ) are used or when the polymer shells are thicker in the case of CS microgels. 29 Nonetheless, there exist still several controversial results; for example, Vogel et al. 52 and Rauh et al. 12 studied CS microgels of similar size, but "isostructural phase transitions" were only observed in reference. 12 Importantly, all these results are based on ex situ measurements and only recent works started to provide in situ data. 34,53 In particular, acquiring structural information on statistically relevant areas remains challenging.
In this manuscript, we used in situ methods (Method 2 and Method 3) to investigate monolayers of CS microgels of size and crosslinker density similar to references 29, 33, 40, 46 and, to a smaller extent, monolayers made of coreless microgels similar to references. 44,45 In all cases, our results strongly point towards a continuous evolution of the interparticle distance, i.e. no "isostructural phase transition". The direct comparison with ex situ measurements (Method 1) suggests that the "structural transitions" are an artifact of the transfer and/or drying process. As such, the conflicting literature can be partly explained by taking into account the further complexity introduced by the ex situ measurement protocol. For example, in contrast to the often applied synchronized Langmuir-Blodgett deposition during compression also depositions at fixed surface pressures were performed. 31 What happens when a microgel-laden monolayer is transferred onto a solid substrate? Figure 8A illustrates a sketch of a typical CS microgel, while Figures 8B1 and 8C1 depict CS microgels with shells of different deformability at the air/water interface. As water evaporates, the microgels approach the substrate and the bottom part of the microgels will start to touch the substrate, most likely causing further deformations as illustrated in Figures 8B2 and 8C2. The contact area between the microgels and the substrate and the resulting adhesion depends on (1) the properties of the microgels (e.g., their morphology), the ones of the underlying surface (e.g., its wettability) and the transfer protocol (e.g., deposition speed). 54,55 As the level of subphase lowers further, the microgels protrude more and more from the liquid film as shown in Figures 8B3 and   8C3, leading to a deformation of the meniscus and attractive immersion capillary forces. [56][57][58] Although these forces have not been measured experimentally for CS microgels, they qualitatively explain the formation of clusters as monolayers are transferred to the substrate.
We briefly verified that the transfer protocol affects the ex situ assemblies by drying CS microgel monolayers with overall hydrodynamic diameters ranging from approximately 500 to 1000 nm 59 (5 mol.% crosslinker density) with two different drying conditions; 'slow' drying at ambient conditions against open air, and 'fast' drying using a heat gun. Figure 9 shows that structural changes ̶ consistent with an "isostructural phase transition" at the interface ̶ appear only after slow evaporation (blue panels). This observation implies that the microgels have enough time to rearrange when the monolayer is dried slowly under ambient conditions. This is in line with the experimental and theoretical findings of Volk et al. 60 It is noteworthy, however, that the "freezing" of monolayers by fast drying has its limits and will depend on the core dimension, the shell-to-core size ratio, and the crosslinker density. A similar conclusion was drawn by Vasudevan et al. 46 Furthermore, as the temperature influences the microgel fraction in the water subphase and mostly along the vertical direction, 53   and shell-to-core size ratios. The scale bars correspond to 5 μm. The inset in C2 is an AFM image of the corresponding monolayer. The scale bar corresponds to 2 μm. The microgels are labeled with core diameter along with their shell-to-core size ratio in parentheses.

CONCLUSION
In this work, we have investigated the isothermal compression of different colloidal monolayers assembled at air/water interfaces using a Langmuir trough in combination with a self-built setup for small-angle light scattering measurements (LT-SALS). This setup allowed us to measure the interparticle distances and characterize the structural order of the monolayers in situ, while the total available surface area was continuously reduced by the barriers of the Langmuir trough. When using core-shell microgels with rigid cores and soft and deformable shells, we found stark differences between microstructures analyzed ex situ (i.e., monolayers that were transferred to solid substrates) in comparison with the in situ structural analysis based on optical diffraction. The ex situ analysis revealed an "isostructural phase transition" from core-shell microgels in shell-shell contact to "core-core" contact during compression. In contrast, the in situ analysis revealed a continuous decrease of interparticle distance as the monolayer is compressed. No phase transition was observed. This key result was also confirmed by in situ real space analysis of the monolayer using fluorescence microscopy. As a proof of concept, we also demonstrated that the in situ investigation using small-angle light scattering can be also applied to monolayers of rigid particles as well as low optical contrast PNIPAM microgels.
LT-SALS is fast, non-destructive and relatively easy to set up from low cost components.
Compared to in situ optical microscopy, it has several important advantages: 1) Very large monolayer areas > 1 mm 2 can be probed. Such large areas correspond to, for example, > 40 × 10 4 core-shell microgels that are simultaneously probed. 2) It is not necessary to have markers or strong refractive index contrast in colloid systems under investigation.
3) The measurement is less sensitive to external interferences such as vibrations. 4) Microstructural phase transitions become evident immediately due to changes of the diffraction pattern, i.e., a transition from a diffraction ring to Bragg peaks revealing the transition from a disordered to an ordered state. 5) The processing and analysis (e.g., radial averaging, peak position and width) of the diffraction patterns is much less prone to errors and less time-consuming when compared to real space analysis of microscopy images for which the centers of mass of all imaged particles have to be identified.
We believe that the presented methodology will stimulate further research on colloidal monolayers at liquid/liquid or air/liquid interfaces, in particular when softness and deformability of objects are studied. 50,61 Furthermore, the fact that phase transitions can be directly monitored in situ at the respective interface will allow systematic studies required to achieve a more comprehensive understanding of colloidal assembly at interfaces 22

Silica particles and silica-PNIPAM CS microgels
The detailed synthesis protocol for both silica nanoparticles and micron-sized silica-PNIPAM microgels can be found in. 59 In short, silica particles were synthesized via the well-known Stöber procedure. RITC dye was incorporated in the particles that were used for fluorescence microscopy experiments. The PNIPAM shell encapsulation was done via seeded precipitation polymerization.
The silica particles used to create monolayers at the air/water interface were measured to be 695 ± 22 nm (126 particles counted) in diameter by TEM. Its ethanolic dispersion was mixed with chloroform with 1:4 volume ratio to assist the spreading of the silica particles at the air/water interface. Surface charges were screened by adding 100 mM NaCl in the aqueous subphase of the Langmuir trough in order to achieve rigid sphere-like interactions.
The main CS microgels used for the in situ and ex situ comparison had a core with a diameter of 340 ± 20 nm. The Dh of the total CS microgel was measured to be 920 ± 18 nm at 20 °C using dynamic light scattering (DLS). The purified dispersion was freeze-dried, re-dispersed in ethanol with 5 w/v% and stored on a 3D shaker overnight prior to the monolayer deposition at the air/water interface. Ethanol was used as spreading agent.

PNIPAM microgel synthesis
The synthesis protocol for the PNIPAM microgels was adopted from a previously published work. 62

Ex situ investigation after Langmuir-Blodgett deposition (Method 1)
For the ex situ analysis, we followed the well-established protocol to study the phase behavior of CS microgel monolayers at the air/water interface. 12,29,30 According to the protocol, the microgel monolayer at the air/water interface is simultaneously transferred to a substrate during the compression, dried as the substrate is pulled out and examined under a microscope, hence referred to as an ex situ approach. The total duration of the substrate pulled out is often matched with the total duration of the compression, consequently enabling the position of the substrate to the corresponding Π tracing. This link between the substrate position to Π was established under the assumption that the number of particles transferred from the air/water interface per time is negligible thus does not influence the measured value of Π.
The transfer of the monolayer was carried out using a Langmuir-Blodgett deposition trough (Microtrough G2, Kibron Inc.) equipped with a film balance, two Delrin barriers, a dip coater and an acrylic cover box. A standard microscope glass slide was treated in an ultrasonic bath sequentially with Hellmanex aqueous solution (2 vol%), water (2 ×) and in ethanol (2 ×) for 15 minutes each. The cleaned glass slide was then cut in half along its length and the position markings were carved on its back to trace the corresponding Π at the moment of monolayer transfer (see Figure 10A for a schematic illustration of the procedure). Before the deposition of the particle monolayer, the trough and the barriers were thoroughly cleaned with water, ethanol and again rinsed with water. The trough was then filled with water with the barriers closed. The glass slide was thoroughly rinsed with water before being mounted to the dip coater (parallel to the barriers and perpendicular to the air/water interface), positioned at the center of the trough and lowered 55 mm below the interface. An aspirator with a narrow tip was used to remove any residual floating substances at the interface between the two barriers as well as to flatten the interface by lowering its level to the height of the trough wall. A Wilhelmy plate was rinsed with water and ethanol and held over a flame to remove any impurities and cooled before it was mounted to the film balance.

In situ investigation by LT-SALS (Method 3)
The

Transmission electron microscopy (TEM)
TEM measurements were performed using a JEOL JEM-2100 Plus microscope operated in brightfield mode at 80 kV acceleration voltage. The sample preparation was done by applying a drop of the respective particle dispersion on a carbon-coated copper grid (200 mesh, Science Services) and drying at room temperature. The captured images were then processed with ImageJ for the size analysis.

SUPPORTING INFORMATION
Additional comparison of ex and in situ measurement, interparticle distance calculation from diffraction images, LT-SALS measurement with different wavelength lasers, LT-SALS setup and monolayer preparation for LT-SALS, image processing steps and other details (PDF), a video of LT-SALS measurement (AVI).

NOTES
The authors declare no competing financial interest.   Additional ex situ vs in situ differences for the main CS microgels Figure S2A shows Dc-c plotted as a function of the particle number per unit area (nP/A). Theoretical values of Dc-c for close-packed, perfectly hexagonally ordered monolayers were calculated according to our already published work. 59 These values are compared with ex and in situ microscopy data. Figure S2B illustrates the systematic shift of nP/A in ex situ measurement compared (filled squares) to the in situ method (open squares) at the same measured Π.

Monolayers of silica particles
The monolayer of silica particles shown in Figure 7A of the main manuscript was transferred and dried onto a solid substrate using Langmuir-Blodgett deposition. Figure S3 shows microscopic images and SALS patterns of these monolayers for different values of Π (see caption). The diffraction patterns vary from ring-like to peak-like depending on the position on the substrate.

Monolayers of (coreless) PNIPAM microgels
The system of coreless microgels shown in Figure 7B of the main manuscript was also investigated in situ using fluorescence microscopy (Method 2), also in a range of Πs where the interparticle distance is too large to be resolved in our current LT-SALS setup. Figure S4 shows microscopic images of the microgel monolayer at different Π. We do not observe an "isostructural phase transition". The same monolayer of microgels was then studied ex situ after Langmuir-Blodgett deposition. Figure S5A and C show optical light microscopy images of the dried microgel monolayers prepared at two different Π of 30.2 (black) and 31.2 mN/m (red). Figure S5B and D show AFM images of the corresponding microgel monolayers. Figure S5E shows calculated radial distribution functions (RDFs) as well as nearest neighbor center-to-center distances, Dc-c. Figure   S5F displays the diffraction pattern recorded by SALS, representative for the microgel monolayers taken from Π between 30.2 and 31.2 mN/m. The microscopic image of CS microgel (Dc: 105 nm, shell-to-core size ratio: 4.8) in Figure 9C2 (dried in open air) of the main manuscript could not be resolved due to the relatively homogeneous refractive index of the microgel, in comparison with Figure 9C1 (dried with heat gun). Figure   S6A and C show AFM measurements on these monolayers at lower Π (10 mN/m), and Figure   S6C and D are the corresponding phase images.

Interparticle distance from LT-SALS
The interparticle distance measured by LT-SALS ( − ) was calculated as below: where q is the magnitude of the scattering vector in µm -1 , n the refractive index (refractive index of air, n = 1), λ the wavelength of the light in µm, x the distance from primary beam in pixel, y the conversion factor in mm per pixel and DS-D the sample-to-detector distance in mm. The scattering vector yields the lattice spacing ℎ = 2 . For a two-dimensional, hexagonally ordered system, the interparticle distance is − = 2 √3 ℎ . With a blue diode laser (λ = 405 nm), the available q-

LT-SALS setup
The level of accuracy was checked with all the involved components in the laser path using a circular level. The laser was aligned with the camera center with two mirrors and through the microscopy window of the trough by using a pinhole on a rail, which consisted of two parallel rods screwed into the optical plate. After the alignment, the rail and the pinhole were removed. The Langmuir trough and the camera were placed back in the laser path. The paper screen (width: 90 mm) was rolled around two metal rods, fastened parallel to the trough and fixed on the customized frame, see Figure S8. The laser beam center was marked on the screen for various size of beam stops to be glued on when required. The sample-to-detector distance (DS-D) was measured with a ruler ensuring all four corners of the screen have the same distance to the trough wall. The pixel/mm value was determined using millimeter paper after all the involved components were fixed on their positions. The screen was rolled back and put aside on one metal rod for the cleaning of the trough. The trough was filled again with water before the screen was rolled out and fixed back to its position. Then the monolayer was deposited at the air/water interface. The DS-D of our current setup could be varied from 25 to 200 mm (scattering angle ranges from 2 -74°). Figure S8. Photograph of the LT-SALS setup.