1：Experimental Design and Method Selection:

The study used fluorescence lifetime imaging microscopy (FLIM) to measure the fluorescence lifetimes of NADH and FAD in live and fixed cells. A double-exponential decay model was employed for data fitting, with fixed lifetimes for free states based on aqueous solutions.

2：Sample Selection and Data Sources:

Human cervical cell line (HeLa) cells and human pulmonary adenocarcinoma (A549) cells were cultured and fixed with paraformaldehyde. Cells were mounted with neutral balsam or glycerol and measured over 0 to 14 days. Cell cycle was controlled using colchicine or serum starvation.

3：List of Experimental Equipment and Materials:

Equipment includes a laser scanning confocal microscope (Olympus FV300/IX 71), picosecond lasers (BDL-405-SMC and BDL-488-SMN), photomultiplier tube (PMC-100-1), and TCSPC system (SPC-150). Materials include DMEM and RPMI-1640 media, fetal bovine serum, paraformaldehyde, PBS, neutral balsam, glycerol, NADH, FAD, colchicine.

4：0). Materials include DMEM and RPMI-1640 media, fetal bovine serum, paraformaldehyde, PBS, neutral balsam, glycerol, NADH, FAD, colchicine. Experimental Procedures and Operational Workflow:

4. Experimental Procedures and Operational Workflow: Cells were fixed, washed, mounted, and stored. FLIM images were acquired using specific excitation and detection filters. Data were collected pixel-by-pixel, with multiple areas imaged per sample to avoid photobleaching.

5：Data Analysis Methods:

Time-decay data were fitted with multi-exponential models using SPCImage software. Weighted mean lifetimes and contributions of free and bound states were calculated, with goodness-of-fit assessed using χ2 values.