Electrophysiology studies of ion channels, in live filamentous fungi by patch clamp method are not possible due to presence of rigid chitinous cell wall that prevents patch clamp pipette to access the plasma membrane. We present laser nano-surgery of the fungal cell wall that enables patch clamp electrophysiology studies. Similar approaches as one-time reports utilizing nanosecond laser pulses long time ago were not pursued further (Roberts 1997, Véry 1998). Here, we demonstrate reproducible method using femtosecond lasers accompanied by two-photon excitation fluorescence (TPEF) imaging of hyphae (Pajić 2024).

A wild-type strain of filamentous fungus Phycomyces blakesleeanus (Burgeff) [NRRL 1555(-)] were grown on glass coverslips with hand-etched grid, coated with a thin layer of 50% collagen type I as an immobilizer. Home built nonlinear laser scanning microscope (Pajić 2022) utilizing Ti:Sa tunable fs laser was used for TPEF imaging of hyphae and the cell surgery. The latter is enabled with the custom made add-on in software. The dwell time (1s) and the laser power (4-15mW) were set with fixed repetition rate (76MHz), pulse duration (160fs) and laser wavelength (730nm). Coverslip with hyphae is transferred to another microscope setup for patch clamp, consisting of micromanipulators and precise electronics for pA current measurements. Obtained current recordings provide valuable information on fungal cell membrane ionic channels.

Funding: project BioPhysFUN, program GREEN, Science Fund of the Republic of Serbia; Ministry of Science and Technological Development and Inovtions of the Republic of Serbia.

References:

1. Roberts, Stephen K. et al, (1997) ‘Laser Ablation of the Cell Wall and Localized Patch Clamping of the Plasma Membrane in the Filamentous Fungus Aspergillus: Characterization of an Anion-Selective Efflux Channel’, The New Phytologist 137(4) 579–85.
2. Véry AA, Davies JM, (1998) ‘Laser microsurgery permits fungal plasma membrane single-ion-channel resolution at the hyphal tip’, Applied and Environmental Microbiology, 64(4):1569-72.
3.  Pajić Tanja, et al (2024) “In vivo femtosecond laser nanosurgery of the cell wall enabling patch-clamp measurements on filamentous fungi” Microsystems & Nanoengineering 10, 47
4.  Pajić Tanja, et al (2022) “Label-free third harmonic generation imaging and quantification of lipid droplets in live filamentous fungi” Scientific Reports 12, 18760

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Meeting ID: 508 144 0931

Seminar hosts: Neven Šantić i Matija Čulo