1：Experimental Design and Method Selection:

The study uses fluorescence lifetime imaging microscopy (FLIM) to analyze redox states in HeLa cells treated with doxorubicin. It employs two-photon excitation for NAD(P)H and FAD, and three-photon for Tryptophan, with FLIM data fitting using a two-component incomplete model.

2：Sample Selection and Data Sources:

HeLa cervical cancer cells were cultured in DMEM with 10% cosmic calf serum, plated on cover slips, and treated with 1 μM doxorubicin. Data were collected from 51 cells at control and post-treatment time points (15, 30, 45, 60 minutes).

3：List of Experimental Equipment and Materials:

Includes a Zeiss LSM-780 NLO microscope, Chameleon Vision-II laser, HPM-100-40 detectors, SPC-150 cards, and various filters. Materials include DMEM, cosmic calf serum, doxorubicin, and cover slips.

4：Experimental Procedures and Operational Workflow:

Cells were imaged before and after treatment at identical fields-of-view. FLIM images were acquired at 740 nm for NAD(P)H and Trp (60s), and 890 nm for FAD (60s), with temperature maintained at 37°C. Data were processed using ImageJ/FIJI and custom Python code for segmentation and analysis.

5：Data Analysis Methods:

FLIM parameters (lifetimes, fractions) were extracted, and statistical analysis included Pearson correlation coefficients, means, medians, histograms, and P-values with significance level α=0.05.