1：Experimental Design and Method Selection:

The study compared four aqueous solution-based tissue-clearing techniques (ClearT2, CUBIC, ScaleS, SeeDB2) for 3D visualization of liposome distribution in mouse lungs. Protocols followed standard methods from literature, involving refractive index matching or hyperhydration mechanisms.

2：Sample Selection and Data Sources:

Male ICR mice (6 weeks old, 26–28 g) were used. Lung samples were prepared by intrapulmonary administration of fluorescent probes (e.g., rhodamine B-labeled liposomes, Texas Red dextran) and intravenous administration of DyLight-conjugated tomato lectin, followed by fixation with paraformaldehyde.

3：List of Experimental Equipment and Materials:

Equipment included a Liquid MicroSprayer? (Model IA-1C, PennCentury, Inc.), laser-scanning confocal microscope (LSM 700, Zeiss), fluorescence zoom microscope (Axio Zoom.V16, Zeiss), microplate reader (Powerscan HT, DS Pharma Biomedical), and Zetasizer Nano ZS (Malvern Instruments Ltd.). Materials included lipids (HEPC, DSPE-PEG2000, DPPE, cholesterol, DCP), fluorescent probes (rhodamine B-DPPE, DiI, DiD, FITC-dextran, Texas Red dextran, DyLight-conjugated tomato lectin), and clearing reagents (e.g., formamide, glycerol, urea, Triton X-100).

4：0). Experimental Procedures and Operational Workflow:

4. Experimental Procedures and Operational Workflow: Mice were administered probes via MicroSprayer or intravenous injection, perfused and fixed with paraformaldehyde, then subjected to tissue-clearing treatments per standard protocols. Clearing was assessed via microscopy, and 3D images were reconstructed from confocal stacks. Liposome stability was tested by incubating FITC-dextran-encapsulated liposomes in clearing reagents and measuring release.

5：Data Analysis Methods:

Fluorescence intensity was measured with a microplate reader, and images were analyzed using confocal microscopy for depth and distribution. Statistical analysis included mean ± SD calculations for multiple replicates.