Intravital multiphoton photoconversion with a cell membrane dye

Photoconversion, an irreversible shift in a fluorophore emission spectrum after light exposure, is a powerful tool for marking cellular and subcellular compartments and tracking their dynamics in vivo. This paper reports on the photoconversion properties of Di-8-ANEPPS, a commercially available membrane dye. When illuminated with near-infrared femtosecond laser pulses, Di-8-ANEPPS undergoes multiphoton photoconversion as indicated by the supralinear dependence of the conversion rate ρ_pc on the incident power (), and by the ability to photoconvert a thin optical section in a three-dimensional matrix. The characteristic emission spectrum changed from red to blue, and ratiometric analysis on single cells in vitro revealed a 65-fold increase in the blue to red wavelength ratio after photoconversion. The spectral shift is preserved in vivo for hours, making Di-8-ANEPPS a useful dye for intravital cell marking and tracking applications.

The color of the moon can change during a lunar eclipse so can certain fluorescent dyes when light is shone on them. The changes in fluorescence emission spectrum, termed photoconversion, can be highly controlled, thus making this process a useful tool tostudy biological process in vivo at the cellular and subcellular levels. This Letter describes intravital multiphoton photoconversion of Di-8-ANEPPS, a membrane dye, in murine bone marrow.

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