1：Experimental Design and Method Selection:

The study used fluorescence anisotropy imaging (FAIM) and defocused single-molecule fluorescence imaging to investigate the orientation and heterogeneity of polydiacetylene fibers. The rationale was to provide detailed qualitative analysis of internal fiber structure based on emission dipole moments.

2：Sample Selection and Data Sources:

Mixed monolayer samples of perfluorotetradecanoic acid (PF) and 10,12-pentacosadiynoic acid (PCDA) were prepared with varying compositions (e.g., 4PF:1PCDA, 5PF:1PCDA). Films were deposited on glass coverslides using Langmuir-Blodgett techniques and photopolymerized with UV illumination.

3：List of Experimental Equipment and Materials:

Chemicals included perfluorotetradecanoic acid and 10,12-pentacosadiynoic acid from Sigma-Aldrich, hexanes and tetrahydrofuran from EMD and Merck EM Science, microscope coverslides from VWR International. Equipment included a Langmuir trough system from KSV Instruments, a plasma cleaner from Harrick Plasma, modified inverted microscopes (Nikon TE2000 and Olympus IX71), EMCCD cameras (Photon MAX 1024 and Photon Max 512 from Princeton Instruments), lasers (532 nm CW diode laser and 473 nm CW diode laser from Shanghai Laser and Optics Century Co.), polarization splitters (Cairn Research), objectives (Nikon Apo 60x NA1.45, Olympus PFO 100x NA1.3), and various optical components from ThorLabs and Chroma Technology.

4：45, Olympus PFO 100x NA3), and various optical components from ThorLabs and Chroma Technology. Experimental Procedures and Operational Workflow:

4. Experimental Procedures and Operational Workflow: Films were prepared by depositing surfactant mixtures on a Langmuir trough, compressing at 10 mm min?1, and transferring to glass substrates at 15 mN m?1. Photopolymerization was done with a UV pen lamp for 10 min. FAIM involved excitation with a 532 nm laser, collecting orthogonal emission polarizations simultaneously, and processing images with MATLAB. Defocused imaging used a 473 nm laser, manual defocusing by ~1 μm, and capturing emission patterns.

5：Data Analysis Methods:

Fluorescence anisotropy maps were generated pixel-by-pixel using a custom MATLAB code with correction factors. Defocused images were analyzed qualitatively for emission patterns indicative of single molecules, and time trajectories were examined for blinking behavior.