Supplementary Material
Domain Imaging and Probe Partitioning. We use the imaging mode of our microscope to
directly image the domain size and probe partitioning. To do this we remove the first mirror in
our beam path after the Cassegrain objective and focus the fluorescence onto a CCD camera. In
Figure S1 we demonstrate the imaging of the coexistence regime of DPPC for pressures 3.5, 5, 7
and 10 mN/m; we find average domain sizes of 6, 9 , 11, 20 µm and area fraction of the LC
phase of 7, 12, 34, 75% respectively. We were not able to directly calculate the partition
coefficient through use of the quantum yield due to an inability to directly measure the steady
state absorption spectra of the monolayer. One estimate to quantifying the partition coefficient
within the DPPC films is to take the ratio of the fluorescence intensity in the LC to that of the LE
phase.1 Using this method we find that for the coexistence regime of DPPC at pressure 7 mN/m
we have a partition coefficient of a least 95% to the LE phase. For DMPC at all pressures, our
fluorescence microscopy displays a uniform intensity and featureless film indicating a single
fluid phase of uniform fluorophore distribution. Another more quantitative approach is to
measure partitioning through changes in the fluorophore lifetime as it partitions to alternate
phases.2 The presence of a multiexponental fluorescence decay would indicate multiple
fluorophore populations. We observe a single lifetime decay of our fluorescent probe (see
below), indicating the probe partitions to only the LE phase, consistent with observations in the
literature.3
FIG. S1. Fluorescence microscopy images for DPPC labeled with 1 mol% NBD-PC for various
pressures across the phase coexistence regime.
Measures of NBD-PC Fluorescence Properties. We have measured the steady state
fluorescence spectrum of DMPC and DPPC monolayers labeled with 1% mole NBD-PC at
surface pressures across the entire range of pressures explored. Figure S2a displays data for
DMPC layers at the surface pressure of 3.5 mN/m, 10 mN/m and 20 mN/m. The steady state
fluorescence was also measured for DPPC at the coexistence pressures explored in this study
(Figure S1b). We measure the fluorescence in-situ using an Ocean Optics QE65000-FL
Fluorescence Spectrometer. Fluorescence was collected through the Cassegrain objective, then
focused onto a 600 µm diameter fiber optic which is connected directly to the spectrometer.
Fluorescence is collected for 2 seconds and the data represent the average of 5 scans. Emission
is detected at 543 nm for all the data sets, consistent with the emission maximum observed for
NBD-PC in the literature.3 This consistency in fluorescence maximum is one indication that the
probe maintains a well anchored position through the compression range observed. If there were
a large change in location of the probe within the layer we would expect a large shift in solvent
polarity and solvent accessibility of the probe.
FIG. S2. Steady state fluorescence of a) DMPC with 1% mole NBD-PC at surface pressures of
3.5,10 and 20 mN/m b) DPPC with 1% mole NBD-PC at surface pressures of 3.5,5 and 7 mN/m.
This observation coupled with a single lifetime decay near 6 ns (Figure S3) ensures that the
observed dynamics is due to a change in the local microviscosity and not in a change in probe
geometry or position. We measured the lifetime of our probe by exciting at the magic angle
(54.7˚) and recording the fluorescent transients at 525nm, the values obtained are consistent with
those observed for NBD in the literature.3
FIG. S3. Fluorescence lifetime measurements for NBD-PC at 1 mol% in a DMPC monolayer at two
pressures 3.5 mN/m and 20 mN/m. We observe a single decay at 6 ns.
Additional Dynamic Fitting Analysis. The anisotropy decay is analyzed with both stretched
exponential and Maximum Entropy Method (MEM).4, 5 MEM output analysis is shown in Figure
S4 for both DMPC and DPPC. DMPC dynamic distributions exhibit considerable heterogeneity
(Figure 3 right panel) as would be expected for highly correlated motion. These dynamic clusters
are not a phase separation as this would be apparent in both the isotherm and in the fluorescent
imaging. However they are transient density fluctuations caused by increases in correlated
motion. This dynamic heterogeneity is not readily apparent in the LE phase of DPPC. The
dynamic distributions of MEM analysis of DPPC indicate very little variability in the dynamic
distribution throughout the coexistence regime, Figure 3 lower right panel. The distributions are
similar to the low pressure liquid phase of DMPC, indicating the low density LE phase behaves
as a highly dynamic liquid. These results are consistent with the stretched exponential fitting.
Either model provides the same interpretation, however the stretch exponential model can
adequately describe the observed dynamics with the fewest number of free parameters.
FIG. S4. Maximum Entropy Method analysis of DMPC at surface pressures of 3.5,10, 20 mN/m and
DPPC at pressures 5 mN/m. The graphs represent the output generated by the algorithm from the
Steinbach group at the NIH;6 the curve displayed is the recommended fit. The program plots the
residual on the left, data plus fit in the center, and the fractional amplitudes versus the Log of the
rotational correlation time on the far right panel.
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