DOI: 10.5061/dryad.70rxwdc6k
title: Data and Code for: Phospholipase-catalyzed Degradation Drives Domain Morphology and Rheology Transitions in Model Lung Surfactant Monolayers

description: Lung surfactant is inactivated in acute respiratory distress syndrome
 (ARDS) by a mechanism that remains unclear. Phospholipase (PLA2) plays an
 essential role in the normal lipid recycling processes, but is present in
 elevated levels in ARDS, suggesting it plays a role in ARDS
 pathophysiology. PLA2 hydrolyzes lipids such as DPPC—the primary component
 of lung surfactant—into palmitic acid (PA) and lyso-PC (LPC). Because PA
 co-crystallizes with DPPC to form rigid, elastic domains, we hypothesize
 that PLA2-catalyzed degradation establishes a stiff, heterogeneous
 rheology in the monolayer, and suggests a potential mechanical role in
 disrupting lung surfactant function during ARDS. Here we study the
 morphological and rheological changes of DPPC monolayers as they are
 degraded by PLA2 using interfacial microbutton microrheometry coupled with
 fluorescence microscopy.  While degrading, domain morphology
 passes through qualitatively distinct transitions:
  compactification, coarsening, solidification, aggregation,
 network percolation, network erosion, and nucleation of PLA2-rich domains.
 Initially, condensed domains relax to more compact shapes, and coarsen via
 Ostwald ripening and coalescence up until the domains solidify, marked by
 a distinct roughening of domain boundaries that does not relax.
  Domains aggregate and eventually form a percolated network,
 whose elements then erode and whose connections are broken as degradation
 continues.  The relative enzymatic activity of PLA2, set by the
 age of the sample, impacts the order and the duration of morphology
 transitions. The fresher the PLA2, the faster the overall degradation, and
 the earlier the onset of domain solidification: domains solidify before
 aggregating with fresh PLA2 samples, but aggregate and percolate before
 solidification with aged PLA2.  Irrespective of the activity of
 the PLA2, all measured linear viscoelastic surface shear moduli obey the
 same dependence on condensed phase area fraction (log|G*|∝Φ) throughout
 monolayer degradation.  Moreover, the onset of domain
 solidification coincides with the time when the relative surface
 elasticity begins to increase.
description: # Data and Code for: Phospholipase-catalyzed degradation drives domain
 morphology and rheology transitions in model lung surfactant monolayers
 [https://doi.org/10.5061/dryad.70rxwdc6k](https://doi.org/10.5061/dryad.70rxwdc6k) ## Description of the data and file structure The data collected for the associated article includes both interfacial rheology collected using the microbutton microrheometry technique and micrographs collected using fluorescence microscopy. The interfacial rheology and micrograph data describe monolayers of the phospholipid DPPC being actively degraded by the enzyme PLA2. The files included are: * "Figure Data for Dryad" which corresponds to all plotted data in the associated article * Folders for Figure 4, 5, 6, 7, 8 images include the full fluorescence micrographs corresponding to each figure in the associated article * "Degrading monolayer video example" contains a sequence of sped up videos from a single monolayer being actively degraded by PLA2 * "Sample area fraction data", "Area fraction examples", "SurfaceStructuresJFv3", and "SurfaceStructuresTests" are a set demonstrating how the domain area fraction calculations were done in the associated article. "Area fraction examples" contains 2 original example images and the intermediate images output by the Matlab file "SurfaceStructuresTests". The data output by "SurfaceStructuresTests" for the 2 examples and additional calculations are in the "Sample area fraction data" file. "SurfaceStructuresTests" is the main Matlab file and it is dependent on the "SurfaceStructuresJFv3" file. ### Files and variables #### File: Figure\_Data\_for\_Dryad.xlsx **Description:** This file includes the data from every plot in the associated article ##### Variables * Figure 3a: horizontal axis is area per molecule (Angstrom^2) and vertical axis is surface pressure (mN/m) * Figure 3b: horizontal axis is surface pressure (mN/m), left vertical axis is the magnitude of G* (micro-Newtons/m), right vertical axis is surface viscosity (micro-Newton seconds/m) * Figure 3c: horizontal axis is surface pressure (mN/m), vertical axis is delta (radians) * Figure 8a rheology: horizontal axis is time since PLA2 injection (h), vertical axis is the magnitude of G* (micro-Newtons/m) * Figure 8a area fraction:  horizontal axis is time since PLA2 injection (h), vertical axis is area fraction * Figure 8b: horizontal axis is time since PLA2 injection (h), vertical axis is delta (radians) * Figure 9: this contains 5 separate data sets denoted by diamonds, squares, triangles, stars, and circles. The horizontal axis for each data set is the respective area fraction and the vertical axis for each set is the respective magnitude of G* (micro-Newtons/m) * Figure 10: the horizontal axis is time of solidification onset "t solid" (min) and the vertical axis is the time when delta starts decreasing "t delta" (min). Each data point has a top, bottom, left, and right error bar. The values of each error bar signifies the length of each respective error bar. For example, for the x-coordinate equal to 45.5833 min, the left error bar extends 5.96667 min to the left of the point on the x-axis. #### File: Sample\_area\_fraction\_data.xlsx **Description:** This file contains two examples of numerical outputs from the SurfaceStructuresTests.m file. The original images used to create these examples are called "2022.10.04_Series009_t071_ch00" and "2022.10.04_Series009_t362_ch00" ##### Variables * The mask data corresponds to the outputs from the mask creation step in SurfaceStructuresTests.m as well as some calculations. "image series" and "image number" identify the source image. "Mask area" (pixels^2) and "mask perimeter" (pixels) are the outputs from the code after the mask is drawn. "# of pixels" and "image length (microns)" are inputs referring to the dimensions of the original image in pixels and microns. "area outside mask" corresponds to the area of the original image minus the area inside the mask (pixels^2). * The area fraction data corresponds to the final outputs after the SurfaceStructuresJFv3 step in SurfaceStructuresTests.m as well as some calculations. "image series" and "image number" identify the source image. "Area" (pixels^2) is the area of black pixels. "Lsize" is the length of the cropped image (pixels). "sensitivity", "threshold_disk*", *and "threshold_area" are all inputs for a Matlab function embedded in SurfaceStructuresJFv3. "pix" (pixels) and "dim" (microns) are inputs referring to the dimensions of the original image in pixels and microns. "domain area" (pixels) is the total area of condensed domains in the image, which is equal to "area" minus "area outside mask". "area fraction" is the area fraction of the condensed domains in the image, which is equal to "domain area" divided by "mask area". ## Code/software SurfaceStructuresTests.m is the Matlab file written to pull raw fluorescence micrographs from a folder, apply a mask to isolate the region of interest, denoise the image, binarize the image, add up all the black pixels, and export the relevant quantities. This file depends on SurfaceStructuresJFv3.m to perform some of these steps. The ultimate goal of these files are to calculate the area of condensed domains in a region of interest in a fluorescence micrograph. Examples of the data exported by these files and the additional calculations needed to calculate the area of condensed domains are in the "Sample area fraction data" file. The corresponding original images and the intermediate images produced by SurfaceStructuresTests.m are in the "Area fraction examples" folder.