1：Experimental Design and Method Selection:

The study used a 532 nm laser for simultaneous photoreduction and Raman spectroscopy of single red blood cells (RBCs) to detect changes in hemoglobin states due to chlorpyrifos exposure. A 1064 nm laser was used for optical trapping to manipulate cells. Atomic force microscopy (AFM) was employed to observe membrane damage.

2：Sample Selection and Data Sources:

RBCs were obtained from healthy human blood donors, separated and washed with phosphate buffer saline (PBS), and suspended in buffer with bovine serum albumin (BSA). Chlorpyrifos, H2O2, and 3-Aminotriazole were added at various concentrations for incubation.

3：List of Experimental Equipment and Materials:

Equipment includes a frequency-doubled Nd:YVO4 laser (Verdi-5, Coherent Inc.), inverted microscope (Axiovert 135TV, Carl Zeiss), spectrograph (Shamrock SR-303i, Andor Corp.), CCD camera (iDus 401-BRDD, Andor Corp.), Nd:YAG laser (Compass, Coherent Inc.), Ti:Sapphire laser (Mira, Coherent Inc.), absorption spectrophotometer (Cintra), AFM (NT-MDT, SOLVER-PRO, Russia), and various chemicals (e.g., chlorpyrifos from Sigma Aldrich). Materials include polystyrene particles for calibration, glutaraldehyde for fixation, and fluorescent probes.

4：Experimental Procedures and Operational Workflow:

RBCs were exposed to chlorpyrifos concentrations (1 ppb to 1000 ppm) and incubated. Raman spectra were recorded using 532 nm and 785 nm lasers at specific powers and exposure times. AFM imaging was performed on fixed cells. Additional experiments involved H2O2 and 3-Aminotriazole exposures.

5：Data Analysis Methods:

Raman spectra were analyzed by comparing peak intensities, Lorentzian peak fitting, and difference spectra. AFM images were used to assess morphological changes. Statistical analysis involved mean spectra from multiple cells.