In vivo femtosecond laser nanosurgery of the cell wall enabling patch-clamp measurements on filamentous fungi

Studying the membrane physiology of filamentous fungi is key to understanding their interactions with the environment and crucial for developing new therapeutic strategies for disease-causing pathogens.However,their plasma membrane has been inaccessible for a micron-sized patch-clamp pipette for pA current recordings due to the rigid chitinous cell wall.Here,we report the first femtosecond IR laser nanosurgery of the cell wall of the filamentous fungi,which enabled patch-clamp measurements on protoplasts released from hyphae.A reproducible and highly precise(diffraction-limited,submicron resolution)method for obtaining viable released protoplasts was developed.Protoplast release from the nanosurgery-generated incisions in the cell wall was achieved from different regions of the hyphae.The plasma membrane of the obtained protoplasts formed tight and high-resistance(GΩ)contacts with the recording pipette.The entire nanosurgical procedure followed by the patch-clamp technique could be completed in less than 1 hour.Compared to previous studies using heterologously expressed channels,this technique provides the opportunity to identify new ionic currents and to study the properties of the ion channels in the protoplasts of filamentous fungi in their native environment.