Defining safe operational conditions for two-photon imaging with rhodamine B and SYTO 9 dual-staining

Two-photon microscopy (TPM) frequently employs dual-staining techniques for simultaneous visualization of cellular structure and viability. Despite widespread use, the safety of combining Rhodamine B (RB) and SYTO 9 dyes under 920 nm excitation has not been thoroughly assessed. This study systematically evaluated cytotoxicity, sensitization, and dermal irritation of RB (20 μg/mL) and SYTO 9 (2.7 μg/mL) under typical TPM conditions. BJ fibroblasts and THP-1 monocytic cells were exposed to dye-laser treatments (6-60 mW/cm² for 10 min), followed by CCK-8 viability assays and sensitization evaluation via modified human Cell Line Activation Test (h-CLAT). For dermal irritation assessment, BALB/c mice underwent topical application of dyes and laser exposure, followed by histological and macroscopic evaluations. Results showed maintenance of cell viability (>70 %) across all laser intensities, with no indication of sensitization, as evidenced by low CD54 and CD86 expression, and negligible skin irritation (Primary Irritation Index ≤0.5). Histological analysis confirmed no significant inflammatory or structural damage. These comprehensive findings establish an ISO-compliant safety threshold, confirming that RB and SYTO 9 dual-staining under 920 nm TPM conditions pose no significant biological risks.