Department of Physics, Sapienza University of Rome, Piazzale Aldo Moro 2,
00185 Roma, Italy, European Union Department of Physics, Sapienza University
of Rome, Piazzale Aldo Moro 2, 00185 Roma, Italy, European Union Centro de
Fısica de Materiales (CSIC, UPV/EHU) - Materials Physics Center MPC, Paseo
Manuel de Lardizabal 5, 20018 San Sebastián, Spain, European Union.
IKERBASQUE—Basque Foundation for Science, Plaza Euskadi 5, 48009 Bilbao,
Spain, European Union.

# In-situ study of the impact of temperature and architecture on the
interfacial structure of microgels

Steffen Bochenek  Institute of Physical Chemistry, RWTH Aachen University,
Landoltweg 2, 52056 Aachen, Germany, European Union  Fabrizio Camerin  CNR-
ISC, Sapienza University of Rome, Piazzale Aldo Moro 2, 00185 Roma, Italy,
European Union  Emanuela Zaccarelli  CNR-ISC, Sapienza University of Rome,
Piazzale Aldo Moro 2, 00185 Roma, Italy, European Union  Armando Maestro
Institut Laue-Langevin ILL DS/LSS, 71 Avenue des Martyrs, 38000 Grenoble,
France, European Union  Maximilian M. Schmidt  Institute of Physical
Chemistry, RWTH Aachen University, Landoltweg 2, 52056 Aachen, Germany,
European Union  Walter Richtering  Institute of Physical Chemistry, RWTH
Aachen University, Landoltweg 2, 52056 Aachen, Germany, European Union  Andrea
Scotti  Institute of Physical Chemistry, RWTH Aachen University, Landoltweg 2,
52056 Aachen, Germany, European Union<EMAIL_ADDRESS>
###### Abstract

The structural characterization of microgels at interfaces is fundamental to
understand both their 2D phase behavior and their role as stabilizers that
enable emulsions to be broken on demand. However, this characterization is
usually limited by available experimental techniques, which do not allow a
direct investigation at interfaces. To overcome this difficulty, here we
employ neutron reflectometry, which allows us to probe the structure and
responsiveness of the microgels in-situ at the air-water interface. We
investigate two types of microgels with different cross-link density, thus
having different softness and deformability, both below and above their volume
phase transition temperature, combining experiments with computer simulations
of realistic in silico synthesized microgels. We find that temperature only
affects the portion of microgels in water, while the strongest effect of the
microgels softness is observed in their ability to protrude into the air. In
particular, standard microgels have an apparent contact angle of few degrees,
while ultra-low cross-linked microgels form a flat polymeric layer with zero
contact angle. Altogether, this study provides an in-depth microscopic
description of how different microgel architectures affect their arrangements
at interfaces, and will be the foundation for a better understanding of their
phase behavior and assembly. This manuscript has been accepted for publication
in Nature Communications (open access). The final version of the manuscript
including the Supplementary Information will be available in the future.

##  1 Introduction

Soft nano- and microgels - cross-linked polymer networks swollen in a good
solvent - reveal peculiar properties that are different from those of other
colloidal systems such as hard nanoparticles, polymers and surfactants.1, 2,
3, 4, 5 The impact of softness, for instance, emerges when micro- and nanogels
adsorb at interfaces: they stretch and deform to maximize the coverage of the
interface and minimize the interfacial energy. 6, 7, 8, 9, 10, 11 At the same
time, they do not completely disassemble but remain individual particles, in
contrast to other macromolecules such as block copolymer micelles, which
irreversibly change their internal conformation upon adsorption at an
interface 12, 13.

Nano- and microgels based on poly-_N_ -isopropylacrylamide (pNIPAM) have a
high interfacial activity14 and at the same time maintain their thermo-
responsiveness once adsorbed to air-15, 16, 17, liquid-18, 19, 20, 21, or
solid interfaces 22, 23, 24, 25. They can be used to prepare smart emulsions
18, 19, 26, 27, 28 that can be broken on demand as a function of external
stimuli such as temperature and pH 18, 29, 19, 30, 31, 32.

A detailed knowledge of the 3D structure of microgels at an interface is
essential to understand fundamental aspects such as their 2D-phase behavior
33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43 or their functionality in emulsion
stabilization. While there has been significant progress in studying microgels
at solid substrates, in-situ experiments at fluid interfaces are still scarce.
A powerful technique to obtain experimental insight into the structure and
composition of surfaces and/or thin films with sub-nanometric resolution is
specular neutron reflectometry (SNR), which has been employed to study NIPAM-
based systems, such as linear polymers and nanogels. 44, 45

Recently, Zielińska et al. probed the structure of pNIPAM nanogels (with
diameter smaller than 40 nm) below and at the lower critical solution
temperature of pNIPAM of 32$\,{}^{\circ}$C.44, 46 They found that nanogels
protrude for $\approx 2$ nanometers in the air phase and form a thick
polymeric layer at the interface. After this, two layers of highly solvated
pNIPAM were observed. As highlighted in these studies, a key aspect which
determines the monolayer structure is represented by the nanogel
deformability. More generally, the extent of the microgels’ deformation, their
final shape, and their phase behaviour strongly depend on their softness and
internal architecture.

It can be expected that size and cross-linker density of the microgels
strongly influence the structure of the microgel-covered interface and indeed
a transition from particle-to-polymer-like behavior has been observed for
ultra-soft microgels adsorbed to solid interfaces.39 Atomic force microscopy
(AFM), cryo-scanning electron (cryoSEM) microscopy, and computer simulations
show that adsorbed standard microgels, i.e. microgels with a cross-linker
content of few mol %, have a core-corona or fried-egg-like shape when dried,
where the fuzzy shell of the microgels forms a thin layer at the interface
with the more cross-linked core in the center. 8, 47, 6, 33, 48 The core-
corona structure gives rise to a rich 2D-phase behavior of the microgel
monolayer characterized by a solid-to-solid phase transition 33. In contrast,
AFM measurements demonstrate that ultra-soft microgels have a flat and
homogeneous pancake-like structure 25. Furthermore, depending on the monolayer
concentration, they can form both flat films and behave as polymers or as a
disordered arrangement of particles 39.

In this contribution, we address the following questions: Do microgels
protrude into the air and if so how far? Is it possible to determine a contact
angle for microgels? How are these quantities affected by the cross-linking
density and by the collapse of the microgels in the water phase? In
particular, we employ SNR to determine in-situ the structure of microgels
along the normal to the interface and compare the resulting polymer fraction
profiles with those obtained by computer simulations.

We investigate two different types of microgels. The first one is a standard
microgel synthesized with a cross-linker content of 5 mol %. This has an
architecture characterized by a more cross-linked core and a gradual decrease
of the cross-linking density and the polymer segment density towards the
periphery. Finally, dangling chains decorate the outer shells. 49. This
architecture is a consequence of the fact that the cross-linker agent reacts
faster than the monomer during the precipitation polymerization. 50 We
prepared two separate batches, where in one case the isopropyl group of the
monomer was deuterated to improve the contrast for neutron reflectivity (NR).

pNIPAM microgels can also be synthesized via precipitation polymerization
without addition of a cross-linker agent 51. The network is formed by self-
cross-linking of NIPAM due to transfer reactions 52. As with the standard
microgels, we use a partially deuterated monomer in which the vinyl group is
deuterated 52 to increase and vary the contrast in neutron reflectometry.
Given the absence of cross-linker agent, these ultra-low cross-linked (ULC)
microgels are ultra-soft 54, 53 and have an almost uniform, albeit very low,
internal density of polymer segments. 39. Nonetheless, such particles remain
fundamentally different from linear polymers. For instance, in bulk solution,
ULC microgels were found to form colloidal crystals in clear contrast to
linear or branched chains 55, 54. Furthermore, their behavior can be tuned
between that of polymer and the one of colloidal particle depending on the
compression of the monolayer 39. These microgels also differ from linear
polymers once adsorbed at a solid interface where their architecture is the
one of ultra-soft disks 25.

The differences in internal architecture between standard and ULC microgel
affect their compressibility and deformability. For instance, the presence of
a more cross-linked and denser core inhibits large compression in bulk, 56
whereas the poorly cross-linked network of the ULC microgels is easy to
compress in crowded solutions 57, 53. While compressibility is the key aspect
for the three-dimensional response of microgels, their deformability is
pivotal once they are confined in two dimensions, i.e. onto liquid or solid
interfaces.

The analysis of our data shows the effects of the microgel internal
architecture on their structure orthogonal to the interface. For both systems,
the protrusion in the air and the polymeric layer sitting at the interface are
independent of the temperature, T. Furthermore, simple geometrical
considerations on the density profiles combined with the in-plane microgel
radius determined by AFM, allow us to determine the apparent contact angle of
the adsorbed microgels. We show that the morphology of ULC microgels is more
similar to linear polymers and macromolecules, while standard microgels
resemble more closely hard colloids.

##  2 Results

###  2.1 Microgel structure in bulk solution

The ratio between the hydrodynamic radius in the swollen and collapsed state -
swelling ratio - is a good measurement of the softness of the microgel
network: The larger this ratio, the softer the microgel 58, 59, 60. All
microgels studied here have a comparable hydrodynamic radius at
20$\,{}^{\circ}$C, see Table 1 and Supplementary Fig. 1a. They do however
exhibit different swelling ratios, see Supplementary Fig. 1b.

Table 1: Characteristic lengths of the individual pNIPAM based microgels below
and above their VPTT.

Name | T | $R_{h}$ | $R_{\text{SANS}}$ | $R_{\text{SANS,c}}$ | 2$\sigma_{\text{SANS}}$ | 2$R_{\text{2D}}$ | 2$R_{\text{2D,c}}$ | h${}_{\text{2D}}$
---|---|---|---|---|---|---|---|---
| (∘C) | (nm) | (nm) | (nm) | (nm) | (nm) | (nm) | (nm)
5 mol% D0 | 20 | 150 | 151 | 32 | 119 | 688 | 360 | 21
5 mol% D0 | 40 | 85 | 72 | 59 | 13 | 651 | 289 | 26
5 mol% D7 | 20 | 153 | 120 | 33 | 87 | - | - | -
5 mol% D7 | 40 | 72 | 62 | 57 | 5 | - | - | -
ULC D3 | 20 | 138 | 134 | 53 | 81 | 733 | - | 3
ULC D3 | 40 | 54 | 56 | 41 | 15 | 689 | - | 4

  * •

Hydrodynamic radius in water, $R_{\text{h}}$, radius from SANS in D2O,
$R_{\text{SANS}}~{}=~{}R_{\text{SANS,c}}~{}+~{}$2$\sigma_{\text{SANS}}$ where
$R_{\text{SANS,c}}$ is the core radius in bulk and 2$\sigma_{\text{SANS}}$ is
the fuzziness of the shell in bulk determined by SANS. 2$R_{\text{2D}}$ is the
interfacial (dry) diameter and 2$R_{\text{2D,c}}$ is the interfacial (dry)
diameter of the core. h${}_{\text{2D}}$ is the maximum height once adsorbed
(dry). The last three quantities are determined by AFM, see Supplementary
Figs. 3 and 4. The values including the errors are given in Supplementary
Table 1.

For the hydrogenated 5 mol% cross-linked standard pNIPAM microgels, 5 mol% D0,
the swelling ratio is $1.76\pm 0.03$. For the deuterated pNIPAM microgels
synthesized with the same amount of cross-linker - 5 mol% D7 - the swelling
ratio is $2.12\pm 0.04$. Finally, the swelling ratio of the deuterated pNIPAM
ULC microgels, ULC D3, is $2.56\pm 0.05$. This confirms that the ULC microgels
are the softest, according to this parameter.

Small-angle neutron scattering (SANS) is used to determine the characteristic
lengths of the microgels, such as total radius, $R_{\text{SANS}}$, radius of
the more cross-linked core, $R_{\text{SANS,c}}$, and extension of the fuzzy
shell, 2$\sigma_{\text{SANS}}$. The values of these quantities are determined
fitting the form factors with the fuzzy-sphere model 49 and are reported in
Table. 1. The data and the fits in Supplementary Figs. 2a-d confirm the
different internal architecture between standard and ULC microgels.

We note that the main effects of selective deuteration and of using deuterated
solvents is to shift the VPTT of pNIPAM to a higher temperature 61, 62, 63,
64. However, at the lowest and highest temperatures measured, the microgels
are in the fully swollen and collapsed state (see Supplementary Figs. 1c and
2a-d), respectively, allowing for an appropriate comparison of the different
architectures.

###  2.2 Standard Microgels at the interface

For each monolayer of hydrogenated and deuterated microgels studied here, the
intensities of the reflected neutrons, R(Q), were recorded as a function of
momentum transfer normal to the interface, $Q$, in two isotopic contrasts: D2O
and air contrast matched water (ACMW). The latter consists in a mixture of D2O
and H2O (8.92% v/v), which matches the scattering length density (SLD) of air
($b_{\text{air}}$ = 0 $\cdot~{}10^{-4}$ nm-2), and therefore only the polymer
contributes to the reflected signal of the curves in Figures 1a and b. R(Q)
for the same microgels, measured in D2O as sub-phase, is plotted in the insets
of the Figures 1a and b. In this case, when a neutron beam is reflected from
air at D2O, which has a higher SLD ($b_{\text{D${}_{2}$O}}=6.36\cdot 10^{-4}$
nm-2) or a lower refractive index $n=1-\lambda^{2}/2\pi b$ (with $\lambda$ the
neutron wavelength), respectively, total reflection occurs below a critical
value of the momentum transfer $Q_{\text{c}}=0.16$ nm-1. Above this value the
reflectivity decays as a function of $Q^{4}$.

Figure 1: Reflectivity curves of 5 mol% cross-linked microgels at different
temperatures. a Reflectivity curves, reflectivity, R(Q), versus momentum
transfer, $Q$, of pNIPAM microgels at the air-ACMW interface and corresponding
fits. b Reflectivity curves of D7-NIPAM microgels at the air-ACMW interface
with fits. Insets: Reflectivity curves at air-D2O interfaces. The curves are
shifted in y-direction for clarity. The unshifted curves are shown in
Supplementary Figs. 6a and b. The error bars represent the statistical errors
on R(Q).

The samples studied here yielded laterally homogeneous interfaces on the
length scale of the in-plane neutron coherence length, on the order of several
microns 65. This implies that the measured SNR can be correlated with the
averaged SLD depth profile across the interface delimited by this coherence
length and, therefore, the in-situ structure of the microgels as a function of
the distance from the interface $z$ can be determined. This is done by fitting
the reflectivity curves with a model consisting in different layers
characterized by a thickness, $d$, a roughness, $\sigma$, and a SLD, $b$. The
latter contains information on the atomic density of the NIPAM molecules and,
therefore, is linked to the polymer concentration and solvation of the
different layers (see Methods section for further details). Here, we find that
a model composed by 4 layers is the most suitable to describe the density
profile of the standard pNIPAM microgels perpendicular to the plane of the
interface where the layers 1-to-4 are sandwiched between the bulk air (layer
0) and the bulk solvent (background layer).

The length and width of the slab are delimited by the illuminated area that is
roughly 109 times the interfacial diameter of the measured microgel.
Therefore, in contrast to microscopy-based techniques, our measurements probe
a statistically significant ensemble of microgels. We fit the R(Q)-curves of
the same sample at the same temperature for both contrasts simultaneously to
reduce the number of free parameter. The best fits are shown by the black full
lines in Figures 1a and 1b. The parameters of the fits are reported in Table
2. The use of models with a smaller number of layers cannot reproduce the
experimental data or it leads to density profile inconsistent with previous
studies 66, 6, 67, 10, 35, 68, 69, see Supplementary Figs. 7a-c.

Additionally, to verify the validity of the four slab-models, the data for the
deuterated microgels at 20$\,{}^{\circ}$C have been fitted using a continuous
variation of the SLD profile sliced into many ($>$ 1000) thin layers of 1.5 Å
thickness. As shown in the Supporting Information (Supplementary Fig. 10a and
b), the fit leads to identical results and, therefore, validates the findings
from the four slab-models used. From this discussion, it is clear that the
model employed here can reproduce the data with the due accuracy and the
lowest number of free fitting parameters.

Table 2: Parameters of the 4-layers fit for the 5% cross-linked microgels in
Figure 1.

T | Layer 1 | Layer 2 | Layer 3 | Layer 4 | Background
---|---|---|---|---|---
| $d_{\text{1}}$ | $\sigma_{\text{1}}$ | $b_{1}$ | $d_{\text{2}}$ | $\sigma_{\text{2}}$ | $b_{2}$ | $d_{\text{3}}$ | $\sigma_{\text{3}}$ | $b_{3}$ | $d_{\text{4}}$ | $\sigma_{\text{4}}$ | $b_{4}$ | $\sigma_{\text{bkg}}$ | $d_{\text{total}}$
(∘C) | (nm) | (nm) | ($10^{-6}$ Å-2) | (nm) | (nm) | ($10^{-6}$ Å-2) | (nm) | (nm) | ($10^{-6}$ Å-2) | (nm) | (nm) | ($10^{-6}$ Å-2) | (nm) | (nm)
5 mol% D0 Microgels, $b_{\text{theo}}$ = 0.93 $\cdot$ $10^{-6}$ Å-2
10 | 14 | 8 | 0.06 | 2.1 | 0.7 | 0.32 | 4.4 | 0.4 | 0.14 | 122 | 3.5 | 0.06 | 31 | 220
20 | 14 | 8 | 0.06 | 2.1 | 0.7 | 0.31 | 4.3 | 0.8 | 0.19 | 117 | 3.5 | 0.07 | 28 | 210
30 | 14 | 8 | 0.08 | 2.2 | 1.0 | 0.35 | 4.7 | 0.6 | 0.20 | 99 | 4.0 | 0.08 | 29 | 194
40 | 14 | 7 | 0.10 | 2.7 | 0.5 | 0.35 | 6.8 | 1.0 | 0.23 | 48 | 3.2 | 0.10 | 26 | 140
5 mol% D7 Microgels, $b_{\text{theo}}$ = 4.78 $\cdot$ $10^{-6}$ Å-2
20 | 16 | 11 | 0.1 | 2.3 | 0.5 | 1.58 | 3.0 | 0.2 | 0.49 | 136 | 3.4 | 0.21 | 33 | 245
40 | 16 | 8 | 0.2 | 2.6 | 0.2 | 1.73 | 4.7 | 0.3 | 0.62 | 66 | 2.6 | 0.26 | 27 | 160

  * •

$d_{\text{i}}$ is the thickness of a layer with the scattering length density
$b_{i}$. $\sigma_{i}$ is the roughness between a layer and the layer above it.
$d_{\text{total}}$ is the total film thickness and $\sigma_{\text{bkg}}$ is
the roughness between the last layer and the background. The Uncertainties
from the fits are given as errors in Supplementary Table 2.

Figures 2a and b show the polymer fraction normal to the interface
(z-distance) of the hydrogenated (5 mol% D0) and deuterated (5 mol% D7)
microgels, respectively. These curves are calculated from the SLD profiles
obtained from the fits and shown in Supplementary Figs. 8a and b.

We note that the extension of the dangling, highly hydrated polymeric chains
at the end of the swollen microgels is accounted considering the roughness
between the last layer and the background, i.e. equals $2\sigma_{\text{bkg}}$.
The profiles of the polymer fraction normal to the interface show that the
microgels deswell in the vertical direction with increasing temperature. The
total film thickness
$d_{\text{total}}=d_{\text{1}}+...+d_{\text{N}}+2\sigma_{\text{1}}+2\sigma_{\text{bkg}}$
is reported in the last column of Table 2.

Below the VPTT, the 5 mol% D0 microgels are fully swollen and have a
$d_{\text{total}}$ in between $210\pm 6$ and $220\pm 5$ nm. Once the microgels
are collapsed at 40$\,{}^{\circ}$C, they are deswollen and have a thickness of
$d_{\text{total}}$ = (140 $\pm$ 5) nm. In the literature, a very similar value
of the thickness was measured for the same microgels in the swollen and
collapsed state with ellipsometry 34. Also the deuterated microgels show the
deswelling with temperature. The thickness of the monolayer in the swollen and
the deswollen state is $d_{\text{total}}=245\pm 14$ nm and
$d_{\text{total}}=160\pm 2$ nm, respectively; see Table 2.

Figure 2: Structure of 5 mol% cross-linked microgels at liquid interfaces.
Polymer fractions of the adsorbed 5 mol% D0 a and 5 mol% D7 b microgels at
different temperatures. c Density profiles of simulated microgels at different
effective temperatures, corresponding to $\alpha=0,0.5$. Horizontal and
vertical dashed lines are guidelines for the eyes and represent zero polymer
fraction/density and zero z-distance from the interface, respectively.
Negative values of z represent the air phase and positive values represent the
water phase. d Simulation snapshots showing the side perspective of an
adsorbed standard microgel for $\alpha=0,0.5$. Solvent particles are not shown
for visual clarity.

In our model, the protrusion of the microgel into the air is
$d_{p}=d_{\text{1}}+2\sigma_{\text{1}}$ and is calculated using the values
given in Table 2. For clarity, we have shifted the position of the polymer
profiles along the z-distance to have this protrusion layer at negative
distances from the interface, Figures 2a and b. The unshifted polymer fraction
profiles are shown in the Supporting Information, Supplementary Fig. 9a and b.

At 20$\,{}^{\circ}$C, the 5 mol% D0 and 5 mol% D7 microgels protrude
approximately $30\pm 2$ and $37\pm 2$ nm into the air, respectively. This
corresponds to about 10$\%$ of the diameter of the swollen microgels in
solution or 15$\%$ of their $d_{\text{total}}$. The protrusion into the air
phase does not change significantly with increasing temperature. Geisel et al.
determined a protrusion height below 70 nm for microgels of similar size. They
noted that this value is the maximum protrusion height according to
geometrical calculations from the cryoSEM images and has to be interpreted as
an upper limit 6.

The estimated values of $d_{\text{p}}$ allow us to calculate the apparent
contact angles of the microgels assuming a simple orthogonal triangle. To this
aim, we make use of the total interfacial diameter 2$R_{\text{2D}}$ of the
individual microgels determined by AFM measurements, see Table 1. The apparent
contact angle, $\theta_{\text{C,app}}=\arctan(d_{p}/R_{2D})$ is found to be
approximately 5$\,{}^{\circ}$ at 20 and 40$\,{}^{\circ}$C. Since the corona of
the microgels is expected to form a flat layer within the interfacial plane,
the interfacial diameter of the core, 2$R_{\text{2D,c}}$, can be used instead.
This results in $\theta_{\text{C,app}}$ of 9$\,{}^{\circ}$ and
11$\,{}^{\circ}$ at 20 and 40$\,{}^{\circ}$C, respectively.

The second region is a thin, polymer-rich layer lying at $z=0$ ( Figures 2a
and b). In our model, this region is described by layer 2 in Table 2. We
assume slabs parallel to the interface and, therefore, we only determine an
average SLD which is proportional to the average polymer fraction at the
interface. Similarly to the protrusion of the microgels in air, also this
polymer-rich layer is temperature independent and has a constant volume
fraction of $\approx 0.33$, as indicated by the constant values of SLD
reported in Table 2. The high polymer content in these regions implies that
the network expelled a significant amount of solvent compared to the solvated
part in water. Therefore, we can compare the thickness of these two layers
($\approx 40$ nm) to the length of the collapsed shell at high temperatures in
bulk, see Table 1, which is found to be much smaller than the layers
thickness. From this, we can infer that also a part of the more cross-linked
core protrudes into the air, as shown in Figure 2 and in the sketch in Figure
3a-c.

Our model also reproduces the portion of a microgel in the aqueous phase, i.e.
the third region, as shown by the polymer fractions at $z>0$ in Figures 2a and
b. This portion of the microgel is described by the third and fourth layers,
and the corresponding parameters are reported in Table 2. Its extension is
calculated as $d_{\text{water}}=d_{3}+d_{4}+2\sigma_{\text{bkg}}$ and shows
the strongest reaction to a change of temperature. For the hydrogenated
microgels, $d_{\text{water}}$ goes from $178\pm 5$ to $106\pm 5$ nm when
temperature increases from 20 to 40$\,{}^{\circ}$C. A change in
$d_{\text{water}}$ from $205\pm 6$ to $125\pm 2$ nm for the same temperature
increase is determined for the 5 mol% D7 microgels. This collapse is
accompanied by an increase of the polymer fraction in the layers 3 and 4 for
both microgels as indicated by the increases in the values of $b_{i}$. We note
that both below and above the VPTT, the values of $d_{\text{water}}$ are
smaller than the hydrodynamic diameters of the swollen and collapsed microgels
in bulk, $2R_{h}$ in Table 1. This observation, combined with the large values
of the interfacial diameters, indicates a strong deformation of the adsorbed
microgels, see Figure 3a-c. On the other hand, the swelling ratio in 2D,
defined as the ratio between $d_{\text{water}}$ at 20 and 40$\,{}^{\circ}$C,
is found to be $1.68\pm 0.09$ and $1.65\pm 0.05$ for the hydrogenated and
deuterated 5 mol% cross-linked microgels, respectively. These values are
smaller than the corresponding ratios in 3D, implying that the adsorption
leads to a stiffening of the polymeric networks swollen in water, as also
found in computer simulations 37. Furthermore, provided both microgels have
the same 2D swelling ratio, the 5 mol% cross-linked standard microgels have
similar softness at the interface, whereas in bulk the deuterated ones appear
to be slightly softer.

We also note that the slight difference in polymer fraction in the water phase
between deuterated and hydrogenated nanogels depends on the fact that they
have slightly different masses and molecular weights $M_{\text{w}}$. Combining
viscosimetry measurements and dynamic light scattering measurements 70, we
found that the 5 mol% D7 microgels have a mass of $6.3\pm 0.6\cdot 10^{-19}$
kg ($M_{\text{w}}=3.8\pm 0.4\cdot 10^{8}$ gmol-1), while the 5 mol% D0
microgels have a mass of $7.7\pm 0.7\cdot 10^{-19}$ kg ($M_{\text{w}}=4.6\pm
0.4\cdot 10^{8}$ gmol-1).

The conformation of the regular microgel at the interface is in excellent
agreement with numerical simulations. In this case, microgels are synthesized
in silico through the self-assembly of patchy particles 71, 37. The resulting
polymer network is disordered and accounts for a higher concentration of
cross-linkers in the core of the particle, with a bulk density profile that
progressively rarefies in the outer corona. The microgel is embedded within
two different types of immiscible solvents, mimicking air and water, which
gives rise to a surface tension similar to experiments. In this way, the
simulated microgel spontaneously acquires the typical fried-egg-like shape.
More details on the assembly process and on the simulations at the interface
can be found in the Methods section.

In order to compare with the experimental profiles of the microgel parallel to
the plane of the interface, we calculate the numerical number density profiles
by dividing the simulation box into three-dimensional slabs along the
z-direction, i.e. orthogonally to the interfacial plane. In this way, we have
direct access to the polymer network, without any interference given by the
presence of the solvent. The resulting profiles are reported in Fig. 2c for
two different effective temperatures.

The three regions described experimentally are also present in the numerical
profiles. At all temperatures, we detect the presence of a protrusion into the
air phase and a polymer layer lying on the interface. As shown by the
snapshots reported in Fig. 2d, the protrusion is given by the fact that the
more cross-linked core cannot fully expand, as it happens for the corona, on
the interfacial plane. In fact, the corona creates the second part of the
density profile that is characterized by a pronounced peak at the interface.
The polymer network accumulates onto the interface to reduce the surface
tension between the two fluids as much as possible. The third region of the
profile is inside the aqueous phase. As in the experiments, this region is
most affected by temperature. While at low temperatures a large portion of the
microgel protrudes significantly into the aqueous phase, at high temperatures
the microgel tends to assume a more spherical and compact shape, contracting
the polymer chains toward the interfacial plane. The consistency between
simulations and experiments also allows us to confirm the robustness of the
four layers fitting model used in experiments.

Figure 3: Sketch of the adsorbed microgels. Panel a shows the vertical
profiles of standard microgels and f the vertical profiles of ULC microgels
below and above the VPTT. Their corresponding shapes are outlined in b-e. The
shapes are based on the combination of our polymer fraction profiles,
simulations and AFM measurements at the liquid-solid interface from the
literature25.

###  2.3 ULC microgels at the interface

The reflectivity curves of deuterated ULC microgels at the air-ACMW interface
are shown in Figure 4. In the inset, the measurements with pure D2O as sub-
phase are shown. In contrast to standard microgels, a three-layer model can
successfully fit the data (solid lines in Figure 4). The fit parameters are
obtained by fitting neutron reflectivity (NR) curves of the same sample at the
same temperature simultaneously for both contrasts. Their values are reported
in Table 3.

Once more, we checked the validity of the three-layer model by comparing the
results from a fit with a model consisting of a continuous variation of the
SLD with many thin layers. In the Supplementary Information, it is shown that
the results from the two models are identical (Supplementary Figs. 10c and d).
This further demonstrates that a slab model including a Gaussian error
function can successfully reproduce the experimental NR data of ULC microgels
with the smallest number of free parameters.

Figure 4: Reflectivity curves of ULC D3 microgels at different temperatures.
Reflectivity, R(Q), versus momentum transfer, $Q$, at the air-ACMW interface.
The fits are shown by continuous lines. Inset: Reflectivity curves at air-D2O
interfaces. The curves are shifted in y-direction for clarity. The unshifted
curves are shown in Supplementary Figs. 6c. The error bars represent the
statistical errors on R(Q).

The structure of the deuterated ULC microgels as a function of the distance to
the interface is described by the shifted and unshifted polymer fraction
profiles in Figure 5a and Supplementary Fig. 9c, respectively. At
$20~{}^{\circ}$C, the length of the protrusion of ULC microgels into air is
$d_{p}=~{}8~{}\pm~{}3$ nm. This is less than 3% of the ULC swollen diameter in
solution and approximately 5% of the total thickness of the ULC,
$d_{\text{total}}=157\pm 7$ nm, see Table 3. Similarly to standard microgels,
the ULC protrusion into air does not change once temperature rises above the
VPTT. Another similarity with the standard microgels is the presence of a
dense layer of polymer sitting on the interface of $\approx 3$ nm. Adding the
length of the protrusion in air, $d_{\text{p}}$, to this extension, we obtain
$\approx 11-15$ nm which is consistent with the extension of the collapsed
fuzzy shell measured by SANS for the D3-ULC microgels, see Table 1. This
indicates that, in contrast to standard microgels, only the collapsed external
shell protrudes into air and lies on the interface, as shown in Figure 5 and
sketched in Figure 3d-f.

Table 3: Summary of the model fits of the reflectivity curves of the ULC D3
microgels in Figure 4.

T | Layer 1 | Layer 2 | Layer 3 | Background
---|---|---|---|---
| $d_{\text{1}}$ | $\sigma_{\text{1}}$ | $b_{1}$ | $d_{\text{2}}$ | $\sigma_{\text{2}}$ | $b_{2}$ | $d_{\text{3}}$ | $\sigma_{\text{3}}$ | $b_{3}$ | $\sigma_{\text{bkg}}$ | $d_{\text{total}}$
(∘C) | (nm) | (nm) | ($10^{-6}$ Å-2) | (nm) | (nm) | ($10^{-6}$ Å-2) | (nm) | (nm) | ($10^{-6}$ Å-2) | (nm) | (nm)
ULC D3 Microgels, $b_{\text{theo}}$ = 2.57 $\cdot$ $10^{-6}$ Å-2
20 | 3 | 2 | 0.04 | 2.2 | 0.4 | 1.01 | 86 | 0.4 | 0.09 | 30 | 157
30 | 3 | 2 | 0.070 | 2.4 | 0.4 | 1.08 | 64 | 0.2 | 0.09 | 26 | 125
36 | 3 | 2 | 0.110 | 2.6 | 0.2 | 1.08 | 61 | 0.2 | 0.05 | 25 | 120
40 | 3 | 1 | 0.120 | 2.7 | 0.4 | 1.08 | 52 | 0.4 | 0.008 | 15 | 89

  * •

$d_{\text{i}}$ is the thickness of a layer with the scattering length density
$b_{i}$. $\sigma_{i}$ denotes the roughness between a layer and the layer
above it. $d_{\text{total}}$ the approximated total film thickness and
$\sigma_{\text{bkg}}$ the roughness between the last layer and the background.
The uncertainties from the fits are given as errors in Supplementary Table 3.

The third region of the ULC microgels has a lower polymer fraction (below
0.04, Fig. 5a) compared to the standard microgels (above 0.05 Figs. 2a and b)
below the VPTT. Unfortunately, due to the resolution of NR and the fact that
we average the SLD over the entire monolayer, it is not possible to finely
resolve the structure of the collapsed ULC. Above the VPTT, the polymer
fraction in the third region of the collapsed ULC is estimated from the value
of $b_{3}$ to be $\approx 0.003$. This small value might result from the
average between regions with no polymer and denser globules of collapsed
microgels around the few cross-linking points. Such globules have been
observed by AFM on re-hydrated ULC microgels adsorbed onto solid interfaces
after transferring from a Langmuir-Blodgett trough 38.

As for the regular microgels, we can use the estimated $d_{\text{p}}$ and the
2D radius of the ULC microgels to compute their apparent contact angles. The
resulting angles are negligible, $\approx$ 1$\,{}^{\circ}$, at both
temperatures. This behavior is close to what one can expect for macromolecules
adsorbed at interfaces in contrast to colloidal particles. This is consistent
with recent literature on these ultra-soft microgels. Indeed, it has been
shown that, due to their high compressibility and deformability 25, 54, these
microgels show the typical behavior of polymers. For instance, their bulk
viscosity does not diverge in proximity of the glass transition but at much
higher concentrations, indicating a high degree of deformability 72. Also at
the interface it has been shown that, depending on their concentration, they
can cover the interface uniformly as a linear polymer or create a disordered
array of individual particles as hard colloids 39.

Figure 5: Structure of ULC microgels at liquid interfaces. a Results of the
fits of the experimental data for the ULC D3 microgels. Inset: Zoom of polymer
fraction profiles. b Density profiles of simulated ultra-low cross-linked
microgels at different effective temperatures, corresponding to
$\alpha=0,0.5$. Horizontal and vertical dashed lines are guidelines for the
eyes and represent zero polymer fraction and zero z-distance from the
interface, respectively. Negative values of z represent the air phase and
positive values represent the water phase. c Simulation snapshots showing the
side perspective of an adsorbed ULC microgel for $\alpha=0,0.5$. Solvent
particles are not shown for visual clarity.

To gain more information on the adsorbed ULC microgels, we also performed
computer simulations of such system. The corresponding density profiles and
simulation snapshots are reported in Figure 5b and 5c, respectively. At both
effective temperatures, the ULC microgels show a flat profile. The polymer
network appears to be equally distributed across the interface, with only a
slight preference for the water phase. Consistently with experiments, no
effect of temperature change is observed for the fraction of polymer in the
air side and on the plane of the interface. Furthermore, as in the experiment,
the contact angle for the ULC microgels is virtually zero.

For standard microgels, the presence of the well-defined core generates a
noticeable dense protrusion into the aqueous phase (Fig. 2a); for the ULC
microgels, the amount of polymer in water is considerably lower (Fig. 5a). The
ULC microgels extend into the aqueous phase for
$d_{\text{water}}=d_{4}+2\sigma_{\text{bkg}}=144\pm 8$ nm at T =
20$\,{}^{\circ}$C. Furthermore, they remain thermo-responsive and their
extension in water decreases to $d_{\text{water}}=83\pm 3$ nm when temperature
changes from 20 to 40$\,{}^{\circ}$C. The 2D swelling ratio equals $1.7\pm
0.1$, a value much smaller than the corresponding 3D ratio and comparable to
the swelling ratio of the standard microgels in 2D. This implies that also ULC
microgels experience a significant stiffening of the polymeric network in
water due their the large deformation. This takes place both in the lateral
and in the vertical directions, as indicated by their large in-plane diameter
and by the fact that $d_{\text{water}}\ll 2R_{h}$, see Table 1. Furthermore,
$d_{\text{water}}$ at 40$\,{}^{\circ}$C is slightly larger than the region
with more homogeneous polymer distribution of the collapsed ULC as measured by
SANS, see Table 1. Therefore, we can assume that this region does not protrude
into the air as shown in the sketch in Figure 3d-e.

While the experimental and numerical descriptions of ULC microgels agree
regarding the microgel portion which protrudes in air and sits onto the
interface, there is a difference in what we observe in the water phase. This
is most likely generated by the presence of few dangling chains that do not
absorb on the plane of the interface and, therefore, protrude into the aqueous
phase. The reason why this protrusion is not observed in the numerical
profiles is most likely due to the small size of the simulated microgel. In
fact, the number of monomers and the minimal percentage of cross-linkers
employed for the in silico synthesis cause the microgel to be highly extended
allowing for all simulated monomers to absorb at the interface. On the
contrary, we expect that a significantly larger microgel would have enough
monomers to form a plain layer at the interface so that some chains would be
desorbed into the aqueous phase, as is the case in experiments. Nevertheless,
at present, this is computationally unfeasible due to the huge number of
particles that would be involved in an explicit solvent simulation with such a
large-sized microgel. For the same reason, an accurate quantitative comparison
between numerical and experimental density profiles is, at the moment, out of
reach.

##  3 Discussion

In this article, we used neutron reflectometry and computer simulations to
probe the structure of microgels orthogonal to the air-water interface, below
and above the VPTT. The advantage of neutron reflectometry is that it allows
to probe the structure of a statistically significant ensemble of microgels
in-situ at the interface. Using NR, we can directly measure the protrusion of
the microgels in the air and estimate how it changes with temperature.
Microscopy-based techniques such as transmission X-ray microscopy (TXM) or
cryoSEM are usually limited by the small number of observed particles, the
size of the particles, an observation direction perpendicular to the
interface, and complicated sample preparation6, 66, 10, 8. The latter makes it
particularly difficult, for example, to observe the effect of temperature on
the swelling of microgels.

In the future, super-resolved fluorescence microscopy techniques, which in
principle can resolve sizes below 30 nm 5, could also be used at the air-water
interface to obtain complementary data. To date, however, even these
techniques are limited by the spatial resolution in the $z$-direction that is
$\approx 60$ nm 73 and by the difficulties in the analysis of the point clouds
generated by the blinking of the dyes 74, 75.

For both 5 mol% cross-linked and ultra-low cross-linked microgels, we find
that the portion of microgels protruding in air is insensitive to changes in
temperature (Figs. 3a and f). Concerning standard microgels, the more cross-
linked core is found to partially protrude in the air, leading to an estimate
of the apparent contact angle of a few degrees (Figs. 3b and c). This value is
significantly smaller than the angle estimated using cryoSEM and TXM of
microgels protruding into different n-alkanes.6, 66 The reason for this
discrepancy is probably that the cryoSEM estimates were limited either by the
smallest angle employed, which was about 30$\,{}^{\circ}$,6 or by the size of
the employed microgels.66

In contrast, ULC microgels form a flat polymer layer that protrudes only a few
nanometers into the air, resulting in a nearly null apparent contact angle
(Figs. 3d and e). We also note that the length of such a layer is
approximately equal to the extent of its collapsed fuzzy shell (Table 1),
supporting the idea that only this part protrudes into the air. Again, since
these microgels are ultra-soft and extremely deformable, they stretch as much
as possible after adsorption at the interface to minimize the interfacial
energy. This behavior is consistent with the experiments of Richardson and co-
workers that used neutron reflectivity to probe linear pNIPAM solutions and
nanogels with a mesh-size comparable to their dimensions and, therefore,
highly stretchable at the interfaces 45, 44. Above the pNIPAM LCST, the
collapsed film protrudes about 4 nm into air 45, which is practically the same
as the protrusion height estimated here for the ULC microgels. These
observations are consistent with the fact that the adsorbed ULC microgels
behave more like linear polymers rather than rigid particles 39.

The present study can also contribute to the current debate on the role and
importance of capillary interactions for microgels adsorbed at the interface,
which seem to be significant only for large particles 76, 77. Indeed, the
strength of capillary interactions depends on the size of the particles, the
density difference between the particles and the liquid, and the contact angle
78. Therefore, our measurements reinforce the idea that for small microgels
with low contact angle, such as the one investigated here, capillary forces
are negligible.

Finally, our work is important to shed light on the collective behavior of
microgels at interfaces. The differences we highlighted in the structure may
be relevant for a more comprehensive understanding of microgels’ effective
interactions, paving the way for a better description of their 2D assembly and
for a clever design of their applications such as emulsion stabilizers.

Recent literature has also shown that the substitution between air and
alkanes, such as decane, only slightly changes the stretching of the microgels
at the interface 36. This is due to high interfacial tension of the two
systems and the insolubility of the microgels in the alkane/oil. However, at
lower interfacial tensions, a greater reduction in the spreading of the
microgels is observed 79. Therefore, we expect that our results on the
protrusion of the microgels into the hydrophobic phase and the observed
difference between ULC and standard microgels at an alkane/(oil)-water
interface will not change qualitatively.

##  4 Methods

###  4.1 Synthesis

Standard 5 mol% D0 (SFB985_B8_SB_M000325), 5 mol% D7 (SFB985_A3_MB_M000238),
and ULC D3 (SFB985_A3_MB_M000301) Microgels were synthesized by precipitation
polymerization. 34, 56, 57, 52 The main monomers for all microgels were NIPAM
(D0) or deuterated NIPAM, in which three (D3) or seven (D7) hydrogen atoms
have been exchanged by deuterium. The deuterated monomers were obtained from
Polymer Source, Canada, hydrogenated monomers were obtained from Acros
Organics, Belgium. Surfactants, sodium dodecyl sulfate (SDS) or
cetyltrimethylammonium bromide (CTAB), were added during the synthesis to
control the size polydispersity and final microgel size. Briefly, for the
three different synthesis, 5.4546 g of D0-NIPAM (5 mol% D0 microgels), or
1.5072 g of D7-NIPAM (5 mol% D7 microgels), or 1.0093 g of D3-NIPAM (ULC D3
microgels) were dissolved in 330 mL, 83 mL, and 70 mL double-distilled water,
respectively. For the 5 mol% microgels 0.3398 g (5 mol% D0) or 0.1021 g (5
mol% D7) of the cross-linker $N$,$N$’-methylenebisacrylamide (BIS) were added.
No additional cross-linker was included during the synthesis of the ULC D3
microgels. The reaction flask of the 5 mol% D0 microgels contained
additionally 0.1474 g of $N$-(3-aminopropyl) methacrylamide hydrochloride
(APMH) as co-monomer. The monomer solutions were purged with nitrogen under
stirring and heated to 65$\,{}^{\circ}$C (5 mol% D0), 70$\,{}^{\circ}$C (5
mol% D7) and 70$\,{}^{\circ}$C (ULC D3). The initiators and the surfactants
were dissolved in a few milliliters of double-distilled water in separated
vessels and degassed for at least one hour. For the deuterated 5 mol% D7 and
ULC D3 microgels 0.372 g and 0.0506 mg of potassium peroxydisulfate (KPS) and
0.202 g and 0.0277 g of SDS were used, respectively. For the 5 mol% D0
microgels, 0.2253 g $2$,$2$’-Azobis-(2-methyl-propionamidin) dihydrochlorid
(V50) and 0.0334 g of CTAB were used. After adding the surfactant to the
reaction flask, the polymerization was initiated by injecting the dissolved
initiators. The reactions were carried out for 4 h at the given temperatures
and under constant nitrogen flow and stirring. The obtained microgels were
purified by threefold ultra-centrifugation and re-dispersion in fresh double-
distilled water. Lyophilization was applied for storage for all microgels.

###  4.2 Dynamic light scattering

A laser with vacuum wavelength $\lambda_{0}=633$ nm was used to probe diluted
suspensions of the different microgels in water and heavy water. The
temperature was change from 20$\,{}^{\circ}$C to 50$\,{}^{\circ}$C in steps of
2$\,{}^{\circ}$C using a thermal bath filled with toluene to match the
refractive index of the glass. The momentum transfer
$Q=4\pi/\lambda\sin{\theta}$, was changed by varying the scattering angle,
$\theta$, between 30 and 130 degrees, in steps of 5 degrees.

###  4.3 Small-angle neutron scattering

SANS experiments were performed at the KWS-2 instrument operated by the JCNS
at the MLZ, Garching, Germany, and at the D11 instrument at the Institut Laue-
Langevin (ILL, Grenoble, France). For the KWS-2 the $q$-range of interest was
covered by using a wavelength for the neutron beam of $\lambda=0.5$ and $1$ nm
and three sample-detector distances: 20, 8 and 2 m. The detector is a 2D-3He
tubes array with a pixel-size of 0.75 cm and a $\Delta\lambda/\lambda=10\%$.
For the D11 three configurations were used: sample detector distance,
$d_{\text{SD}}=34\,$m with $\lambda=0.6\,$nm; $d_{\text{SD}}=8\,$m with
$\lambda=0.6\,$nm; and $d_{\text{SD}}=2\,$m with $\lambda=0.6\,$nm. Due to the
velocity selector, the resolution in $\lambda$ was 9 %. The D11 is equipped
with a 3He detector with a pixel size of $7.5$ mm.

###  4.4 Compression isotherms and depositions

Gradient Langmuir-Blodgett type deposition 33, 34, 36 from air-water
interfaces were performed to study the mechanical properties of the microgels
and microgel monolayers and visualize them ex-situ. The Langmuir-Blodgett
trough was made from polyoxymethylene (POM) and was equipped with two movable
POM barriers. For each deposition, the trough was carefully cleaned, heated to
the appropriated temperature (20 or 40$\,{}^{\circ}$C) with an external water
bath, and a fresh air-water interface was created. The surface pressure was
monitored during the depositions with an electric balance fitted with a
platinum Wilhelmy plate. The substrates were rectangular pieces of ultra-flat
silicon wafer ($\approx$ 1.1 x 6 cm, P100). The substrates were carefully
cleaned with distilled water, isopropyl alcohol and ultrasonication. They were
mounted to the dipper arm of the Langmuir-Blodgett trough with an inclination
with respect to the liquid interface of about 25 $\,{}^{\circ}$. After moving
the substrate to the starting position, the microgels were spread at the air-
water interface. For this purpose, microgels were suspended either in 50/50
vol$\%$ mixtures of water-propan-2-ol or in pure chloroform. This was done to
maximize the adsorption of the microgels to air-water interfaces and minimize
partial loss of microgels into the sub-phase. This loss is unavoidable if the
surface-active component is soluble in either phase. After equilibration for
at least 30 minutes, the substrates were lifted through the interface while
the barriers of the Langmuir-Blodgett trough compressed the interface. The
speed of the barriers ($v_{\rm barrier}=6.48$ cm2 min-1) was matched to the
speed of the dipper arm ($v_{\rm dipper}=0.15$ mm min-1). This, together with
the tilt of the substrate, allowed the microgels to be deposited on the
substrate with increasing concentration 33.

###  4.5 Atomic force microscopy

Deposited, dried microgels were imaged using a Dimension Icon atomic force
microscope with closed loop (Veeco Instruments Inc., USA, Software: Nanoscope
9.4, Bruker Co., USA) in tapping mode. The probes were OTESPA tips with a
resonance frequency of 300 kHz, a nominal spring constant of 26 N m-1 of the
cantilever and a nominal tip radius of $<$ 7 nm (Opus by Micromasch, Germany).

###  4.6 Image analysis

The open-source analysis software Gwyddion 2.54 was used to process the AFM
images. All images were leveled to remove the tilt and zero height was fixed
as the minimum z-value of the image.

Height profiles of single dried microgels were extracted through their apices
and at different angles with respect to the fast scan direction. Multiple
height profiles of one image were summarized and aligned to the apices (zero
coordinate of the x-axis) to obtain averaged microgel profiles and not to bias
the results. The profiles are presented with standard deviations as the error.
The apices and heights of microgels were computed using the Matlab function
findpeaks.

The AFM phase images were used to determine the interfacial (dry) diameter,
2$R_{\text{2D}}$, of the all microgels and the interfacial (dry) diameter of
the core, 2$R_{\text{2D,c}}$, of the standard microgels. For this, the
interfacial areas, $A_{\text{2D}}$ and $A_{\text{2D,core}}$, of at least 200
well separated, isolated, and uncompressed microgels were measured.
2$R_{\text{2D}}$ and 2$R_{\text{2D,c}}$ were calculated by 2$R_{\text{2D}}$ =
$\sqrt{{(4\cdot A_{2D})/\pi}}$

###  4.7 Specular neutron reflectometry

Specular neutron reflectometry measurements were conducted on FIGARO, a time-
of-flight reflectometer at the Institute Laue-Langevin, Grenoble, France. Two
angles of incidence ($\theta_{\rm in}=$ 0.615 and 3.766$\,{}^{\circ}$) and a
wavelength resolution of 7% $\Delta\lambda/\lambda$ were used yielding a
momentum transfer of 0.089 $<Q<$ 3.5 nm-1, normal to the interface. The
wavelength of the neutron beam, $\lambda$, was 0.2 to 3 nm.

An area of $\approx$ 10 $\times$ 40 mm2 was illuminated with the neutron beam.
The reflected neutrons were detected by a two-dimensional 3He detector. The
raw time-of-flight experimental data at these two angles of incidence were
calibrated with respect to the incident wavelength distribution and the
efficiency of the detector. Using COSMOS80, in the framework of LAMPS 81, this
yielded the resulting reflectivity profiles R(Q), where $R$ is defined as the
ratio of the intensity of the neutrons scattered at the air-water interface
over the incident intensity of the neutron beam.

SNR experiments were performed using D2O and 8.92% v/v D2O:H2O mixtures as
sub-phase. The latter is generally known as air contrast matched water (ACMW)
since its scattering length density is equal to the one of air. A
polytetrafluoroethylene (PTFE) Langmuir trough with an area of 100 cm2 and a
volume of $\approx$ 60 mL equipped with two parallel moving PTFE barriers was
used. The trough was placed inside an gas-tight box with heated sapphire or
quartz glass windows to prevent condensation. The box is placed on an active
anti-vibration stage which can be moved vertically and horizontally. Prior to
a measurement series (measurements at different temperatures), the trough was
carefully cleaned and a fresh air-water (D2O or ACMW) interface was created.
For temperature control, the trough was connected to an external water bath.
The trough was cooled down to the lowest temperature and left to equilibrate
for 30 mins. The microgels were added to the interface from solution with a
concentration of 1 mg mL-1 in deuterated chloroform or 50/50 vol$\%$ mixtures
of water-propan-2-ol. Subsequently, the interface was compressed to $\approx$
13 mN m-1 and the first measurement was conducted. At this surface pressure
the average nearest neighbour distance between the microgels is $\approx 500$
nm as determined from AFM, see Supplementary Fig. 5. Afterwards the trough was
tempered to the next temperature, left to equilibrate for 30 mins, and
subsequently a measurement conducted. This was repeated until
40$\,{}^{\circ}$C was reached. A feedback loop controlled and adjusted the
surface pressure during the experiments. Surface pressures were measured with
electric balances equipped with paper Wilhelmy plates.

In the literature it is shown that the polymer fraction within a ULC microgels
in bulk is much lower than for cross-linked microgels.39, 72, 54 As a
consequence, their contrast is very low both in the bulk and at the interface,
and long measurement times would be required to collect statistically reliable
data. For this reason, only deuterated ULC microgel were measured at the
interface. The substitution of 3 atoms of hydrogen with 3 atoms of deuterium
improves the contrast of the ULC microgels when both ACMW and pure D2O are
used for the water-phase.

###  4.8 Analysis and model for neutron reflectometry data

As mentioned above, SNR allowed us to determine the density profile of the
microgel monolayer in-situ along the z-direction, normal to the interface. The
measured R(Q) profile can be linked to an in-plane averaged scattering length
density (SLD) profile of the monolayer along the $z$-direction, $b(z)$, thus
giving information of a statistically significant number of microgels.

Here, SNR data modeling was performed by minimizing the difference between the
experimental and the calculated reflectivity profile using the Parratt’s
recursive formalism 82. The calculated profiles were obtained under the
assumption that the $z$-profile of the SLD can be decomposed in $N$-layers,
with an error function connecting adjacent layers. Every layer was
characterized by a constant scattering length density $b_{\rm i}$, which
depends on the volume fraction of polymer and solvent in this layer. Data
analysis was performed using constraints between layer parameters (thickness,
roughness, and degree of hydration or SLD) and simultaneous co-refinement of
data sets at two contrasts (D2O and ACMW) to reduce ambiguity in modeling with
Motofit83 in IGOR Pro (Wavemetrics). Thus, all parameters in Table 2 and 3,
except $b_{i}$, were co-refined for the two contrasts. The model was fitted to
the data using global minimization of a least squares function $\chi^{\rm 2}$.
In each $i$-layer, the SLD and the polymer fraction $x$ follows $b_{\rm
i}=xb_{\rm pNIPAM}+(1-x)b_{\rm solvent}$, where $b_{\rm pNIPAM}$ and $b_{\rm
solvent}$ are the theoretically calculated values. The polymer fraction
distribution x(z) normal to the plane of the interface for each i-layer was
calculated as the sum of two error functions as follows

$x(z)=\frac{1}{2}x_{i}\left[\operatorname{erf}\left(\frac{z-d_{i}/2}{\sqrt{2}\sigma_{i}}\right)-\operatorname{erf}\left(\frac{z+d_{i}/2}{\sqrt{2}\sigma_{i+1}}\right)\right],d_{i}<z<d_{i+1}$
(1)

where, $d_{\text{i}}$ represents the length of the layer with scattering
length density $b_{\rm i}$. The roughness between two layers is given by
$\sigma_{\rm i}$. $\sigma_{\rm i}$ denotes the roughness of a layer $i$ with
the layer above $i-1$. A similar model has been successfully used to fit NR-
curves of pNIPAM nanogels 44, 46.

For the regular microgels, $N$ was chosen equal four to satisfactory fit the
experimental curves. In contrast, good fits of the R(Q)s of monolayer of
ultra-low cross-linked microgels were obtained using three layer.
Additionally, to demonstrate that a Fresnel reflectivity calculation of a slab
model that includes Gaussian error function connecting the layers is valid
even in our case, where the obtained roughness values are of the order of the
layer thicknesses, an alternative model based on a continuous variation of the
SLD profile was used. The SLD profiles were divided into many thin layers (1.5
Å), which sustain the same physical polymer fraction distribution. The results
are compared in the Supplementary Information, Supplementary Figs. 10a-d. In
particular, two sets of data (5 mol% D7 and ULC D3) were fitted with this
alternative method (see Supplementary Information) yielding similar results
and, therefore, validating the findings from the different slab-models used.

###  4.9 Computer simulations

Standard and ULC microgels modeling Individual microgels were obtained by
self-assembling a binary mixture of patchy particles with valence two and four
71 mimicking the NIPAM monomers and the BIS cross-linkers, respectively. The
assembly was carried out through the oxdna simulation package 84. Standard
microgels were created from a total number of monomers $N$ $\approx$ 42000
within a sphere with the radius $Z=100\sigma_{\rm m}$, where $\sigma_{\rm m}$
is the unit of length in simulations. The cross-linkers, whose concentration
was set to be the $5\%$ of the total number of monomers, experienced an
additional designing force during the assembly so that they were more densely
distributed in the center of the particle. The effect of this additional force
has been extensively studied in previous works 85. For ultra-low-cross-linked
(ULC) microgels, we used $N\approx 21000$ and a sphere with $Z=55.5\sigma_{\rm
m}$, as determined from the comparison of the form factors in bulk. In this
case, the number of cross-linkers was set to $0.3\%$ of the total number of
monomers. In both standard and ULC microgels, the assembly was carried out
until $>99.9\%$ of the possible bonds in the network were formed.

At this stage, reversible patchy interactions were made permanent by allowing
the microgel beads to interact via the Kremer-Grest model 86, according to
which all beads interact via the Weeks-Chandler-Anderson (WCA) potential:

$V_{\rm
WCA}(r)=\begin{cases}4\epsilon\left[\left(\frac{\sigma_{m}}{r}\right)^{12}-\left(\frac{\sigma_{m}}{r}\right)^{6}\right]+\epsilon&\text{if
$r\leq 2^{\frac{1}{6}}\sigma_{m}$}\\\ 0&\text{otherwise.}\end{cases}$ (2)

where $\epsilon$ sets the energy scale and $r$ is the distance between two
particles. Connected beads interacted also via the Finitely Extensible
Nonlinear Elastic (FENE) potential,

$V_{\rm FENE}(r)=-\epsilon
k_{F}R_{0}^{2}\ln\left[1-\left(\frac{r}{R_{0}\sigma_{m}}\right)^{2}\right]\text{
if $r<R_{0}\sigma_{m}$,}$ (3)

with $k_{\rm F}=15$ which determines the stiffness of the bond and $R_{\rm
0}=1.5$ is the maximum bond distance.

To account for the responsivity of the microgel at different temperatures,
monomers also interact via an additional potential

$V_{\alpha}(r)=\begin{cases}-\epsilon\alpha&\text{if }r\leq
2^{1/6}\sigma_{m}\\\
\frac{1}{2}\alpha\epsilon\left\\{\cos\left[\gamma{\left(\frac{r}{\sigma_{m}}\right)}^{2}+\beta\right]-1\right\\}&\text{if
}2^{1/6}\sigma_{m}<r\leq R_{0}\sigma_{m}\\\ 0&\text{if
}r>R_{0}\sigma_{m}\end{cases}$ (4)

with $\gamma=\pi\left(\frac{9}{4}-2^{1/3}\right)^{-1}$ and
$\beta=2\pi-\frac{9}{4}\gamma$ 87. $V_{\rm\alpha}$ introduces an effective
attraction among polymer beads, modulated by the parameter $\alpha$, whose
increase allows to mimic the collapse of the microgel observed at high
temperatures.

Behavior at the interface To investigate the behavior of a microgel adsorbed
at an interface, we reproduced the effects of the surface tension by placing a
microgel between two fluids. Such fluids were modeled with soft beads within
the dissipative particle dynamics (DPD) framework 88, 89. The total
interaction force among beads is $\vec{F}_{\rm ij}=\vec{F}^{C}_{\rm
ij}+\vec{F}^{D}_{\rm ij}+\vec{F}^{R}_{\rm ij}$, where:

$\displaystyle\vec{F}^{C}_{ij}$ $\displaystyle=$ $\displaystyle
a_{ij}w(r_{ij})\hat{r}_{ij}$ (5) $\displaystyle\vec{F}^{D}_{ij}$
$\displaystyle=$ $\displaystyle-\gamma
w^{2}(r_{ij})(\vec{v}_{ij}\cdot\vec{r}_{ij})\hat{r}_{ij}$ (6)
$\displaystyle\vec{F}^{R}_{ij}$ $\displaystyle=$ $\displaystyle
2\gamma\frac{k_{B}T}{m}w(r_{ij})\frac{\theta}{\sqrt{\Delta t}}\hat{r}_{ij}$
(7)

where $\vec{F}^{C}_{ij}$ is a conservative repulsive force, with $w(r_{\rm
ij})=1-r_{\rm ij}/r_{\rm c}$ for $r_{\rm ij}<r_{c}$ and $0$ elsewhere,
$\vec{F}^{D}_{\rm ij}$ and $\vec{F}^{R}_{\rm ij}$ are a dissipative and a
random contribution of the DPD, respectively; $a_{\rm ij}$ quantifies the
repulsion between two particles, $\gamma=2.0$ is a friction coefficient,
$\theta$ is a Gaussian random variable with zero average and unit variance,
and $\Delta t=0.002$ is the integration time-step. Following previous works
10, 37, we chose $a_{\rm 11}=a_{\rm 22}=8.8$, $a_{\rm 12}=31.1$, for the
interactions between fluid 1 and fluid 2. Instead, for the monomer-solvent
interactions we chose $a_{\rm m1}=4.5$ and $a_{\rm m2}=5.0$. In this way, we
made fluid 1 the preferred phase for the microgel particle. The cut-off radius
was always set to be $r_{\rm c}=1.9\sigma_{m}$ and the reduced solvent density
$\rho_{\rm DPD}=4.5$. In this way, the total number of particles was about
$2.6\times 10^{6}$ for simulating standard microgels and $\approx 5.3\times
10^{6}$ for ULC microgels. The reduced temperature of the system $T^{*}$ was
fixed to $1$ via the DPD thermostat. We note that by adjusting $V_{\rm\alpha}$
to reproduce the effect of temperature on the microgel, we did not change the
feature of the interface, which remains defined by the DPD parameters listed
above. Simulations were performed with the lammps simulation package 90.

##  5 Data availability

Raw data were generated at the Institute Laue-Langevin (ILL, Grenoble, France)
using the Fluid Interfaces Grazing Angles Reflectometer (FIGARO). The NR raw
data used in this study are available in the ILL Data Portal database under
accession code 10.5291/ILL-DATA.9-11-187191 and 10.5291/ILL-DATA.EASY-46292.
The raw data, associated data, and derived data supporting the results of this
study have been deposited in the RADAR4Chem database under DOI:10.22000/60393
or are available from the corresponding author at the link
http://hdl.handle.net/21.11102/b0e200f4-d196-44bd-874a-2f5f79d22527.

## References

  * 1 Van Der Scheer, P., Van De Laar, T., Van Der Gucht, J., Vlassopoulos, D. & Sprakel, J. Fragility and strength in nanoparticle glasses. ACS Nano 11, 6755-6763 (2017).
  * 2 Keidel, R., Ghavami, A., Lugo, D., Lotze, G., Virtanen, O., Beumers, P., Pedersen, J., Bardow, A., Winkler, R. & Richtering, W. Time-resolved structural evolution during the collapse of responsive hydrogels: The microgel-to-particle transition. Science Advances 4, eaao7086 (2018).
  * 3 Brijitta, J. & Schurtenberger, P. Responsive hydrogel colloids: Structure, interactions, phase behavior, and equilibrium and nonequilibrium transitions of microgel dispersions. Current Opinion In Colloid & Interface Science 40, 87-103 (2019).
  * 4 Karg, M., Pich, A., Hellweg, T., Hoare, T., Lyon, L., Crassous, J., Suzuki, D., Gumerov, R., Schneider, S., Potemkin, I. & Richtering, W. Nanogels and microgels: From model colloids to applications, recent developments, and future trends. Langmuir 35, 6231-6255 (2019).
  * 5 Scheffold, F. Pathways and challenges towards a complete characterization of microgels. Nature Communications 11, 1-13 (2020).
  * 6 Geisel, K., Isa, L. & Richtering, W. Unraveling the 3D localization and deformation of responsive microgels at oil/water interfaces: A step forward in understanding soft emulsion stabilizers. Langmuir 28, 15770-15776 (2012).
  * 7 Rey, M., Fernandez-Rodriguez, M., Karg, M., Isa, L. & Vogel, N. Poly-N-isopropylacrylamide nanogels and microgels at fluid interfaces. Accounts Of Chemical Research 53, 414-424 (2020).
  * 8 Destribats, M., Lapeyre, V., Wolfs, M., Sellier, E., Leal-Calderon, F., Ravaine, V. & Schmitt, V. Soft microgels as Pickering emulsion stabilisers: Role of particle deformability. Soft Matter 7, 7689-7698 (2011).
  * 9 Fernandez-Rodriguez, M., Martin-Molina, A. & Maldonado-Valderrama, J. Microgels at interfaces, from mickering emulsions to flat interfaces and back. Advances In Colloid And Interface Science , 102350 (2020).
  * 10 Camerin, F., Fernández-Rodriguez, M., Rovigatti, L., Antonopoulou, M., Gnan, N., Ninarello, A., Isa, L. & Zaccarelli, E. Microgels adsorbed at liquid-liquid interfaces: A joint numerical and experimental study. ACS Nano 13, 4548-4559 (2019).
  * 11 Minato, H., Murai, M., Watanabe, T., Matsui, S., Takizawa, M., Kureha, T. & Suzuki, D. The deformation of hydrogel microspheres at the air/water interface. Chemical Communications 54, 932-935 (2018).
  * 12 Cox, J., Yu, K., Constantine, B., Eisenberg, A. & Lennox, R. Polystyrene- poly (ethylene oxide) diblock copolymers form well-defined surface aggregates at the air/water interface. Langmuir 15, 7714-7718 (1999).
  * 13 Cox, J., Bruce Lennox, R. & Others Compression of polystyrene–poly (ethylene oxide) surface aggregates at the air/water interface. Physical Chemistry Chemical Physics 1, 4417-4421 (1999).
  * 14 Zhang, J. & Pelton, R. Poly (N-isopropylacrylamide) microgels at the air- water interface. Langmuir 15, 8032-8036 (1999).
  * 15 Fameau, A., Carl, A., Saint-Jalmes, A. & Von Klitzing, R. Responsive aqueous foams. ChemPhysChem 16, 66-75 (2015).
  * 16 Wu, D., Mihali, V. & Honciuc, A. pH-responsive pickering foams generated by surfactant-free soft hydrogel particles. Langmuir 35, 212-221 (2018).
  * 17 Horiguchi, Y., Kawakita, H., Ohto, K. & Morisada, S. Temperature-responsive Pickering foams stabilized by poly (N-isopropylacrylamide) nanogels. Advanced Powder Technology 29, 266-272 (2018).
  * 18 Ngai, T., Behrens, S. & Auweter, H. Novel emulsions stabilized by pH and temperature sensitive microgels. Chemical Communications, 331-333 (2005).
  * 19 Brugger, B., Rosen, B. & Richtering, W. Microgels as stimuli-responsive stabilizers for emulsions. Langmuir 24, 12202-12208 (2008).
  * 20 Fujii, S., Read, E., Binks, B. & Armes, S. Stimulus-responsive emulsifiers based on nanocomposite microgel particles. Advanced Materials 17, 1014-1018 (2005).
  * 21 Bochenek, S., McNamee, C., Kappl, M., Butt, H. & Richtering, W. Interactions between a responsive microgel monolayer and a rigid colloid: from soft to hard interfaces. Physical Chemistry Chemical Physics 23, 16754-16766 (2021).
  * 22 Nerapusri, V., Keddie, J., Vincent, B. & Bushnak, I. Swelling and deswelling of adsorbed microgel monolayers triggered by changes in temperature, pH, and electrolyte concentration. Langmuir 22, 5036-5041 (2006).
  * 23 Schmidt, S., Zeiser, M., Hellweg, T., Duschl, C., Fery, A. & Möhwald, H. Adhesion and mechanical properties of PNIPAM microgel films and their potential use as switchable cell culture substrates. Advanced Functional Materials 20, 3235-3243 (2010).
  * 24 Cors, M., Wiehemeier, L., Hertle, Y., Feoktystov, A., Cousin, F., Hellweg, T. & Oberdisse, J. Determination of internal density profiles of smart acrylamide-based microgels by small-angle neutron scattering: A multishell reverse monte carlo approach. Langmuir 34, 15403-15415 (2018).
  * 25 Schulte, M., Bochenek, S., Brugnoni, M., Scotti, A., Mourran, A. & Richtering, W. Stiffness tomography of ultra-soft nanogels by atomic force microscopy. Angewandte Chemie International Edition 60, 2280-2287 (2021).
  * 26 Richtering, W. Responsive emulsions stabilized by stimuli-sensitive microgels: emulsions with special non-Pickering properties. Langmuir 28, 17218-17229 (2012).
  * 27 Lefroy, K., Murray, B. & Ries, M. Advances in the use of microgels as emulsion stabilisers and as a strategy for cellulose functionalisation. Cellulose 28, 647-670 (2021).
  * 28 Stock, S. & Klitzing, R. Microgels at droplet interfaces of water-in-oil emulsions-challenges and progress. Current Opinion In Colloid & Interface Science, 101561 (2022).
  * 29 Nguyen, B., Wang, W., Saunders, B., Benyahia, L. & Nicolai, T. pH-responsive water-in-water Pickering emulsions. Langmuir 31, 3605-3611 (2015).
  * 30 Monteux, C., Marliere, C., Paris, P., Pantoustier, N., Sanson, N. & Perrin, P. Poly (N-isopropylacrylamide) microgels at the oil- water interface: Interfacial properties as a function of temperature. Langmuir 26, 13839-13846 (2010).
  * 31 Faulde, M., Tonn, J. & Jupke, A. Microgels for the intensification of liquid-liquid extraction processes–feasibility and advantages. Chemical Engineering & Technology 43, 137-142 (2020).
  * 32 Mehrabian, H., Snoeijer, J. & Harting, J. Desorption energy of soft particles from a fluid interface. Soft Matter 16, 8655-8666 (2020).
  * 33 Rey, M., Fernández-Rodriguez, M., Steinacher, M., Scheidegger, L., Geisel, K., Richtering, W., Squires, T. & Isa, L. Isostructural solid–solid phase transition in monolayers of soft core–shell particles at fluid interfaces: structure and mechanics. Soft Matter 12, 3545-3557 (2016).
  * 34 Bochenek, S., Scotti, A., Ogieglo, W., Fernández-Rodríguez, M., Schulte, M., Gumerov, R., Bushuev, N., Potemkin, I., Wessling, M., Isa, L. & Richtering, W. Effect of the 3D swelling of microgels on their 2D phase behavior at the liquid–liquid interface. Langmuir 35, 16780-16792 (2019).
  * 35 Harrer, J., Rey, M., Ciarella, S., Löwen, H., Janssen, L. & Vogel, N. Stimuli-responsive behavior of PNiPAm microgels under interfacial confinement. Langmuir 35, 10512-10521 (2019).
  * 36 Bochenek, S., Scotti, A. & Richtering, W. Temperature-sensitive soft microgels at interfaces: air–water versus oil–water. Soft Matter 17, 976-988 (2021).
  * 37 Camerin, F., Gnan, N., Ruiz-Franco, J., Ninarello, A., Rovigatti, L. & Zaccarelli, E. Microgels at interfaces behave as 2D elastic particles featuring reentrant dynamics. Physical Review X 10, 031012 (2020).
  * 38 Schulte, M., Scotti, A., Brugnoni, M., Bochenek, S., Mourran, A. & Richtering, W. Tuning the structure and properties of ultra-low cross-linked temperature-sensitive microgels at interfaces via the adsorption pathway. Langmuir 35, 14769-14781 (2019).
  * 39 Scotti, A., Bochenek, S., Brugnoni, M., Fernandez-Rodriguez, M., Schulte, M., Houston, J., Gelissen, A., Potemkin, I., Isa, L. & Richtering, W. Exploring the colloid-to-polymer transition for ultra-low crosslinked microgels from three to two dimensions. Nature Communications 10, 1418 (2019).
  * 40 Vialetto, J., Camerin, F., Grillo, F., Ramakrishna, S., Rovigatti, L., Zaccarelli, E. & Isa, L. Effect of internal architecture on the assembly of soft particles at fluid interfaces. ACS Nano 15, 13105-13117 (2021).
  * 41 Ciarella, S., Rey, M., Harrer, J., Holstein, N., Ickler, M., Lowen, H., Vogel, N. & Janssen, L. Soft particles at liquid interfaces: From molecular particle architecture to collective phase behavior. Langmuir 37, 5364-5375 (2021).
  * 42 Menath, J., Eatson, J., Brilmayer, R., Andrieu-Brunsen, A., Buzza, D. & Vogel, N. Defined core–shell particles as the key to complex interfacial self-assembly. Proceedings Of The National Academy Of Sciences 118 (2021).
  * 43 Grillo, F., Fernandez-Rodriguez, M., Antonopoulou, M., Gerber, D. & Isa, L. Self-templating assembly of soft microparticles into complex tessellations. Nature 582, 219-224 (2020).
  * 44 Zielińska, K., Sun, H., Campbell, R., Zarbakhsh, A. & Resmini, M. Smart nanogels at the air/water interface: structural studies by neutron reflectivity. Nanoscale 8, 4951-4960 (2016).
  * 45 Richardson, R., Pelton, R., Cosgrove, T. & Zhang, J. A Neutron reflectivity study of poly (N-isopropylacrylamide) at the air- water interface with and without sodium dodecyl sulfate. Macromolecules 33, 6269-6274 (2000).
  * 46 Zielińska, K., Campbell, R., Zarbakhsh, A. & Resmini, M. Adsorption versus aggregation of NIPAM nanogels: new insight into their behaviour at the air/water interface as a function of concentration. Physical Chemistry Chemical Physics 19, 17173-17179 (2017).
  * 47 Mourran, A., Wu, Y., Gumerov, R., Rudov, A., Potemkin, I., Pich, A. & Möller, M. When colloidal particles become polymer coils. Langmuir 32, 723-730 (2016).
  * 48 Schmidt, S., Liu, T., Rütten, S., Phan, K., Möller, M. & Richtering, W. Influence of microgel architecture and oil polarity on stabilization of emulsions by stimuli-sensitive core–shell poly (N-isopropylacrylamide-co-methacrylic acid) microgels: Mickering versus Pickering behavior? Langmuir 27, 9801-9806 (2011).
  * 49 M. Stieger, W. Richtering, S. Pedersen, J. & P. Lindner Small-angle neutron scattering study of structural changes in temperature sensitive microgel colloids. The Journal Of Chemical Physics 120, 6197-6206 (2004).
  * 50 Pelton, R. & Chibante, P. Preparation of aqueous latices with N-isopropylacrylamide. Colloids And Surfaces 20, 247-256 (1986).
  * 51 Gao, J. & Frisken, B. Cross-linker-free N-isopropylacrylamide gel nanospheres. Langmuir 19, 5212-5216 (2003).
  * 52 Brugnoni, M., Nickel, A., Kröger, L., Scotti, A., Pich, A., Leonhard, K. & Richtering, W. Synthesis and structure of deuterated ultra-low cross-linked poly (N-isopropylacrylamide) microgels. Polymer Chemistry 10, 2397-2405 (2019).
  * 53 Houston, J. E., Fruhner, L., de la Cotte, A., Rojo González, J., Petrunin, A., Gasser, U., Schweins, R., Allgaier, J., Richtering, W., Fernandez-Nieves, A. & Scotti A. Resolving the different bulk moduli within individual soft nanogels using small-angle neutron scattering. Science Advances 8, eabn6129 (2022).
  * 54 Islam, M., Nguyen, R. & Lyon, L. Emergence of non-hexagonal crystal packing of deswollen and deformed ultra-soft microgels under osmotic pressure control. Macromolecular Rapid Communications 42, 2100372 (2021).
  * 55 Scotti, A., Houston, J., Brugnoni, M., Schmidt, M., Schulte, M., Bochenek, S., Schweins, R., Feoktystov, A., Radulescu, A. & Richtering, W. Phase behavior of ultrasoft spheres show stable bcc lattices. Physical Review E 102, 052602 (2020).
  * 56 Scotti, A. Characterization of the volume fraction of soft deformable microgels by means of small-angle neutron scattering with contrast variation. Soft Matter 17, 5548-5559 (2021)
  * 57 Scotti, A., Denton, A., Brugnoni, M., Houston, J., Schweins, R., Potemkin, I. & Richtering, W. Deswelling of microgels in crowded suspensions depends on cross-Link density and architecture. Macromolecules 52, 3995-4007 (2019).
  * 58 Flory, P. & Rehner, J. Statistical mechanics of cross-linked polymer networks I. rubberlike elasticity. The Journal Of Chemical Physics 11, 512-520 (1943).
  * 59 Flory, P. & Rehner, J. Statistical mechanics of cross-linked polymer networks II. swelling. The Journal Of Chemical Physics 11, 521-526 (1943).
  * 60 Lopez, C. & Richtering, W. Does Flory-Rehner theory quantitatively describe the swelling of thermoresponsive microgels? Soft Matter 13, 8271-8280 (2017).
  * 61 Shirota, H., Kuwabara, N., Ohkawa, K. & Horie, K. Deuterium isotope effect on volume phase transition of polymer gel: Temperature dependence. The Journal Of Physical Chemistry B 103, 10400-10408 (1999).
  * 62 Shirota, H. & Horie, K. Deuterium isotope effect on swelling process in aqueous polymer gels. Chemical Physics 242, 115-121 (1999).
  * 63 Cors, M., Wiehemeier, L., Oberdisse, J. & Hellweg, T. Deuteration-induced volume phase transition temperature shift of PNIPMAM microgels. Polymers 11, 620 (2019).
  * 64 Buratti, E., Tavagnacco, L., Zanatta, M., Chiessi, E., Buoso, S., Franco, S., Ruzicka, B., Angelini, R., Orecchini, A., Bertoldo, M. & Others The role of polymer structure on water confinement in poly (N-isopropylacrylamide) dispersions. Journal Of Molecular Liquids 355, 118924 (2022).
  * 65 Maestro, A. & Gutfreund, P. In situ determination of the structure and composition of Langmuir monolayers at the air/water interface by neutron and X-ray reflectivity and ellipsometry. Advances In Colloid And Interface Science 293, 102434 (2021).
  * 66 Geisel, K., Henzler, K., Guttmann, P. & Richtering, W. New insight into microgel-stabilized emulsions using transmission x-ray microscopy: nonuniform deformation and arrangement of microgels at liquid interfaces. Langmuir 31, 83-89 (2014).
  * 67 Destribats, M., Lapeyre, V., Sellier, E., Leal-Calderon, F., Schmitt, V. & Ravaine, V. Water-in-oil emulsions stabilized by water-dispersible poly (N-isopropylacrylamide) microgels: Understanding anti-Finkle behavior. Langmuir 27, 14096-14107 (2011).
  * 68 Geisel, K., Rudov, A., Potemkin, I. & Richtering, W. Hollow and core–shell microgels at oil–water interfaces: Spreading of soft particles reduces the compressibility of the monolayer. Langmuir 31, 13145-13154 (2015).
  * 69 Kleinschmidt, D., Nothdurft, K., Anakhov, M., Meyer, A., Mork, M., Gumerov, R., Potemkin, I., Richtering, W. & Pich, A. Microgel organocatalysts: Modulation of reaction rates at liquid–liquid interfaces. Materials Advances 1, 2983-2993 (2020).
  * 70 Romeo, G., Imperiali, L., Kim, J., Fernández-Nieves, A. & Weitz, D. Origin of de-swelling and dynamics of dense ionic microgel suspensions. The Journal Of Chemical Physics 136, 124905 (2012).
  * 71 Gnan, N., Rovigatti, L., Bergman, M. & Zaccarelli, E. In silico synthesis of microgel particles. Macromolecules 50, 8777-8786 (2017).
  * 72 Scotti, A., Brugnoni, M., Lopez, C., Bochenek, S., Crassous, J. & Richtering, W. Flow properties reveal the particle-to-polymer transition of ultra-low crosslinked microgels. Soft Matter 16, 668-678 (2020).
  * 73 Conley, G., Nöjd, S., Braibanti, M., Schurtenberger, P. & Scheffold, F. Supeomeoresolution microscopy of the volume phase transition of pNIPAM microgels. Colloids And Surfaces A: Physicochemical And Engineering Aspects 499, 18-23 (2016).
  * 74 Andronov, L., Orlov, I., Lutz, Y., Vonesch, J. & Klaholz, B. ClusterViSu, a method for clustering of protein complexes by Voronoi tessellation in super-resolution microscopy. Scientific Reports 6, 1-9 (2016).
  * 75 Rubin-Delanchy, P., Burn, G., Griffié, J., Williamson, D., Heard, N., Cope, A. & Owen, D. Bayesian cluster identification in single-molecule localization microscopy data. Nature Methods 12, 1072-1076 (2015).
  * 76 Huang, S., Gawlitza, K., Klitzing, R., Gilson, L., Nowak, J., Odenbach, S., Steffen, W. & Auernhammer, G. Microgels at the water/oil interface: In situ observation of structural aging and two-dimensional magnetic bead Microrheology. Langmuir 32, 712-722 (2016).
  * 77 Fernandez-Rodriguez, M., Antonopoulou, M. & Isa, L. Near-zero surface pressure assembly of rectangular lattices of microgels at fluid interfaces for colloidal lithography. Soft Matter 17, 335-340 (2021).
  * 78 Kralchevsky, P. & Nagayama, K. Capillary interactions between particles bound to interfaces, liquid films and biomembranes. Advances In Colloid And Interface Science 85, 145-192 (2000).
  * 79 Vialetto, J., Nussbaum, N., Bergfreund, J., Fischer, P. & Isa, L. Influence of the interfacial tension on the microstructural and mechanical properties of microgels at fluid interfaces. Journal Of Colloid And Interface Science 608, 2584-2592 (2022).
  * 80 Gutfreund, P., Saerbeck, T., Gonzalez, M., Pellegrini, E., Laver, M., Dewhurst, C. & Cubitt, R. Towards generalized data reduction on a chopper-based time-of-flight neutron reflectometer. Journal Of Applied Crystallography 51, 606-615 (2018).
  * 81 Richard, D., Ferrand, M. & Kearley, G. Lamp, the large array manipulation program. J. Neutron Res 4, 33-39 (1996).
  * 82 Parratt, L. Surface studies of solids by total reflection of X-Rays. Phys. Rev. 95, 359-369 (1954).
  * 83 Nelson, A. Co-refinement of multiple-contrast neutron/X-ray reflectivity data using MOTOFIT. Journal Of Applied Crystallography 39, 273-276 (2006).
  * 84 Rovigatti, L., Šulc, P., Reguly, I. & Romano, F. A comparison between parallelization approaches in molecular dynamics simulations on GPUs. Journal Of Computational Chemistry 36, 1-8 (2015).
  * 85 Ninarello, A., Crassous, J., Paloli, D., Camerin, F., Gnan, N., Rovigatti, L., Schurtenberger, P. & Zaccarelli, E. Modeling microgels with a controlled structure across the volume phase transition. Macromolecules 52, 7584-7592 (2019).
  * 86 Kremer, K. & Grest, G. Dynamics of entangled linear polymer melts: A molecular-dynamics simulation. The Journal Of Chemical Physics 92, 5057-5086 (1990).
  * 87 Soddemann, T., Dünweg, B. & Kremer, K. A generic computer model for amphiphilic systems. The European Physical Journal E 6, 409-419 (2001).
  * 88 Groot, R. & Warren, P. Dissipative particle dynamics: Bridging the gap between atomistic and mesoscopic simulation. J. Chem. Phys. 107, 4423-4435 (1997).
  * 89 Camerin, F., Gnan, N., Rovigatti, L. & Zaccarelli, E. Modelling realistic microgels in an explicit solvent. Scientific Reports 8, 1-12 (2018).
  * 90 Plimpton, S. Fast parallel algorithms for short-range molecular dynamics. Journal Of Computational Physics 117, 1-19 (1995).
  * 91 Bochenek, S., Gutfreund P., Lima, L., Maestro, A., Richtering, W., Schmidt, M. M., Scotti, A. Thermo-responsive microgels at air-water interfaces: specular neutronreflectometry study to obtain out-fo-plane density profiles. Institut Laue-Langevin Data Portal (ILL Data Portal), doi:10.5291/ILL-DATA.9-11-1871 (2018).
  * 92 Bochenek, S., Maestro, A., Scotti, A. Deuterated microgels at the liquid-air interface: Effect of crosslinking and temperature. (Continuation of experiment 9-11-1871). Institut Laue-Langevin Data Portal (ILL Data Portal), doi:10.5291/ILL-DATA.EASY-462 (2019).
  * 93 Bochenek, S., Camerin, F., Zaccarelli, E., Maestro, A., Schmidt, M. M., Richtering, W., Scotti, A. Dataset: In-situ study of the impact of temperature and architecture on the interfacial structure of thermo-responsive microgels. RADAR4Chem, doi:10.22000/603 (2022).

##  6 Acknowledgements

The authors thank Yuri Gerelli for valuable discussions and Monia Burgnoni for
synthesis of the deuterated microgels. SB, MMS, WR and AS acknowledge funding
from the Deutsche Forschungsgemeinschaft within SFB 985 ”Functional Microgels
and Microgel Systems”, projects A3 and B8. FC and EZ acknowledge financial
support from the European Research Council (Consolidator Grant 681597, MIMIC).
This work is based upon NR experiments performed at the Institute Laue-
Langevin (ILL, Grenoble, France) using the Fluid Interfaces Grazing Angles
Reflectometer (FIGARO). This work is partially based on SANS experiments
performed at the D11 instrument at the Institut Laue-Langevin (ILL), Grenoble,
France and at the KWS-2 instrument operated by JCNS at the Heinz Maier-
Leibnitz Zentrum (MLZ), Garching, Germany.

##  7 Author contributions statement

W.R., A.S., E.Z., F.C., and S.B. designed this study. A.S., M.S., A.M., and
S.B. performed the NR measurements. A.S., A.M. and S.B. designed the model for
the NR data. A.M. and S.B. analyzed the NR data. S.B. synthesized and
characterized the hydrogenated microgels. S.B. performed Langmuir-Blodgett and
AFM measurements. S.B. analyzed the AFM data. F.C. performed the computer
simulations. All authors participated in discussing the results, writing,
finalizing, and revising the manuscript.

##  8 Competing interests statement

The authors declare no competing interests.