Confocal microscopy of molecular rotors and bacteria in two diatom aggregates and the mucus layer of an upside-down jellyfish ephyra
Data files
Jul 23, 2026 version files 9.80 GB
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e01_m09_r15.nd2
3.36 GB
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eph_ry3_1_1005.nd2
2.84 GB
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g0030_r15.nd2
67.63 MB
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m0001_r15.nd2
67.63 MB
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PnA_m02_r15T_001.nd2
2.91 GB
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psf.mat
121.92 MB
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README.md
3.01 KB
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um_e01_m09_r15.nd2
419.77 MB
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um_g0030_r15.nd2
8.51 MB
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um_m0001_r15.nd2
8.51 MB
Abstract
Interactions between bacteria and particulate organic matter, algae, coral reefs, fish, plant root systems, animals, and humans occur primarily through a dynamic interface of viscous mucus or mucilage. While mucus influences fundamental rates of bacterial infection, respiration, and carbon and nutrient cycling, our observations of this physical habitat of bacteria are limited by methods that damage the material and obfuscate spatial relationships. We present a technique using confocal microscopy of molecular rotors to reveal the 3D viscous structure of undisturbed mucus and associated bacteria. Quantification of the internal viscosity of mucus from different sources highlights variations in microscale morphologies that structure microbial distributions and ecological interactions. Individual examples of mucus aggregates from cultures of Chaetoceros affinis and Pseudo-nitzschia sp. diatoms exhibit consolidated versus patchy viscous morphologies, respectively, along with distinct patterns of microbial colonization. A viscous mucus layer surrounding an ephyra of the upside-down jellyfish Cassiopea xamachana may maintain a local microbiome while preventing direct contact with the animal. By quantifying the complex “mucoscape” shaping bacteria-organic matter interactions, this method provides a physical context for chemical fluxes and microbial activity in diverse ecosystems.
Dataset DOI: 10.5061/dryad.x3ffbg818
Description of the data and file structure
An example of a Chaetoceros affinis diatom aggregate with colonizing bacteria imaged with the RY3 molecular rotor and SYBR Green I. The images were captured on a Nikon A1R+ confocal microscope and saved in .nd2 format.
Files and variables
File: e01_m09_r15.nd2
Description: A z-stack of the 9th Chaetoceros affinis diatom aggregate imaged using optical configuration 15 during the 1st microscopy session. The 12-bit image stack has pixel dimensions of 1024x1024x50 across 32 channels from 400-720 nm in 10 nm bins.
File: g0030_r15.nd2
Description: A glycerol viscosity standard (~30 mPa.s) imaged alongside aggregates during the microscopy session. This image has the same dimensions as the previous file, except that it is a single plane.
File: m0001_r15.nd2
Description: A Milli-Q water viscosity standard (~1 mPa.s) with the same details as the previous file.
File: um_g0030_r15.nd2
Description: The g0030_r15.nd2 file spectrally unmixed into SYBR Green I, RY3 red, and chlorophyll a components.
File: um_m0001_r15.nd2
Description: The m0001_r15.nd2 file spectrally unmixed into SYBR Green I, RY3 red, and chlorophyll a components.
File: um_e01_m09_r15.nd2
Description: The e01_m09_r15.nd2 file spectrally unmixed into SYBR Green I, RY3 red, and chlorophyll a components.
File: psf.mat
Description: A Matlab data file containing the experimentally-measured point spread function (PSF) of the confocal microscope on which the images were taken. The data file loads two variables, emi (481x481x71 double) is a 3D image of PSF intensities and met (1x1 struct) provides the following metadata: met.zc is the z-plane count, met.wv is the emission wavelength in nm, met.zs (1x71 double) contains the z-plane positions in μm, and met.mpp is the the horizontal x-y plane resolution in μm-per-pixel. This file is loaded by the script rc_a_deconv.m for deconvolution of experimental images (see Code/software below).
File: PnA_m02_r15T_001.nd2
Description: A z-stack of an aggregate from a culture of Pseudo-nitzschia sp. diatoms.
File: eph_ry3_1_1005.nd2
Description: A z-stack of a mucus layer surrounding an ephyra of the upside-down jellyfish Cassiopea xamachana.
Code/software
These files can be viewed using the free Nikon NIS-Elements Viewer or FIJI with BioFormats import. MATLAB scripts for calibrating and analysing experimental microscopy images using the RY3 molecular rotor are available at 10.5281/zenodo.20800236.