1：Experimental Design and Method Selection:

Longitudinal observational study using catheter-based 3-D OCT imaging to monitor biofilm formation in ETTs of mechanically ventilated patients at 24-hour intervals until extubation. Attenuation coefficient analysis was employed to differentiate biofilm from mucus based on optical scattering properties.

2：Sample Selection and Data Sources:

Six intubated critical care patients from the ICU of Carle Foundation Hospital, selected based on requiring mechanical ventilation for over 24 hours. Informed consent was obtained from guardians.

3：List of Experimental Equipment and Materials:

Commercial catheter-based OCT system (Model C7XR, St. Jude Medical, Inc.), OCT catheters (Dragonfly model C7, St. Jude Medical, Inc.), fixative solution (2.0% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M Na-Cacodylate buffer), disinfection solution (Revital-Ox Resert, 4455-AW), fluorescence stains (FilmTracer FM 1-43 and SYPRO Ruby, Invitrogen), scanning electron microscope (FEG 450, FEI Quanta), fluorescence microscope (Axiovert 200, Zeiss).

4：0% paraformaldehyde and 5% glutaraldehyde in 1 M Na-Cacodylate buffer), disinfection solution (Revital-Ox Resert, 4455-AW), fluorescence stains (FilmTracer FM 1-43 and SYPRO Ruby, Invitrogen), scanning electron microscope (FEG 450, FEI Quanta), fluorescence microscope (Axiovert 200, Zeiss). Experimental Procedures and Operational Workflow:

4. Experimental Procedures and Operational Workflow: A sterile OCT catheter was inserted through the suctioning port into the ETT, and radial 3-D OCT imaging was performed over a pullback distance of 5.4 cm. Images were acquired every 24 hours until extubation. After extubation, ETTs were fixed and processed for Gram staining, fluorescence staining, and SEM imaging. Data analysis included pixel-wise attenuation coefficient calculation and statistical comparison using Welch's t-test.

5：4 cm. Images were acquired every 24 hours until extubation. After extubation, ETTs were fixed and processed for Gram staining, fluorescence staining, and SEM imaging. Data analysis included pixel-wise attenuation coefficient calculation and statistical comparison using Welch's t-test. Data Analysis Methods:

5. Data Analysis Methods: Attenuation coefficients were derived from OCT intensity images using a custom algorithm. Statistical analysis was performed in MATLAB to compare biofilm-positive and negative groups based on relative attenuation coefficient (RAC) values.