1：Experimental Design and Method Selection:

The experiment is designed to validate the theoretical prediction of optimal laser power ratio for SNR in SRS microscopy. The method involves Stimulated Raman Loss (SRL) microscopy with shot noise limited detection, using amplitude modulation and lock-in detection to measure the modulation transfer between laser beams.

2：Sample Selection and Data Sources:

Samples include 2 μm polystyrene beads in water and adult C. Elegans roundworms. Polystyrene beads are used for controlled validation, and C. Elegans for biological relevance. Data are collected from regions of interest in images.

3：List of Experimental Equipment and Materials:

Equipment includes an 80 MHz laser (Lumera Plecter Duo), Optical Parametric Oscillator (APE Levante Emerald), acousto-optic modulator (Crystal Technology 3080194), Zeiss laser scanning microscope (LSM 7MP) with 32× objective (C-Achroplan W, NA=0.85), Si photodiode (Thorlabs DET36A), home-built transimpedance amplifier (using Texas Instruments OPA656), and lock-in amplifier (Stanford Research Systems SR844). Materials include half-wave plates, polarizing beamsplitters, optical filters, glass slides, coverslips, polystyrene beads, C. Elegans worms, M9 buffer, levamisole, and agarose.

4：85), Si photodiode (Thorlabs DET36A), home-built transimpedance amplifier (using Texas Instruments OPA656), and lock-in amplifier (Stanford Research Systems SR844). Materials include half-wave plates, polarizing beamsplitters, optical filters, glass slides, coverslips, polystyrene beads, C. Elegans worms, M9 buffer, levamisole, and agarose. Experimental Procedures and Operational Workflow:

4. Experimental Procedures and Operational Workflow: The set-up involves overlapping pump (816.7 nm) and Stokes (1064 nm) beams in space and time, modulating the Stokes beam at 3.636 MHz, focusing on the sample with the microscope objective, detecting the pump beam with the photodiode, amplifying the signal with the transimpedance amplifier, and demodulating with the lock-in amplifier. Images are recorded at specific pixel dwell times and frames averaged.

5：7 nm) and Stokes (1064 nm) beams in space and time, modulating the Stokes beam at 636 MHz, focusing on the sample with the microscope objective, detecting the pump beam with the photodiode, amplifying the signal with the transimpedance amplifier, and demodulating with the lock-in amplifier. Images are recorded at specific pixel dwell times and frames averaged. Data Analysis Methods:

5. Data Analysis Methods: SNR is calculated as signal divided by the sum of electronic and shot noise. Data points are mean intensities from regions of interest, with error bars as standard deviations. Theoretical curves are fitted to data, and improvements in SNR are quantified.