Cell and tissue manipulation with ultrashort infrared laser pulses in light-sheet microscopy

Gustavo De Medeiros,Stefan Günther,Yannick Schwab,Lars Hufnagel,Nils Norlin,Francesca Peri,Paolo Ronchi,Uros Krzic,Shinya Komoto,Maria Leptin,Dimitri Kromm,Matteo Rauzi,Stefano De Renzis,Emiliano Izquierdo,Balint Balazs

doi:10.1038/s41598-019-54349-x

Abstract

Three-dimensional live imaging has become an indispensable technique in the fields of cell, developmental and neural biology. Precise spatio-temporal manipulation of biological entities is often required for a deeper functional understanding of the underlying biological process. Here we present a home-built integrated framework and optical design that combines three-dimensional light-sheet imaging over time with precise spatio-temporal optical manipulations induced by short infrared laser pulses. We demonstrate their potential for sub-cellular ablation of neurons and nuclei, tissue cauterization and optogenetics by using the Drosophila melanogaster and zebrafish model systems.

Highlights

Three-dimensional live imaging has become an indispensable technique in the fields of cell, developmental and neural biology
To induce multi-photon absorption, high photon densities are necessary. The latter can be achieved by focusing fs laser pulses with high numerical aperture (NA) lenses
For our system we used a 180 fs pulsed IR laser coupled to a 1.1 NA detection objective of our MuVi-SPIM set-up to focus the pulses on the imaging plane

Summary

Introduction

Three-dimensional live imaging has become an indispensable technique in the fields of cell, developmental and neural biology. We present a home-built integrated framework and optical design that combines three-dimensional light-sheet imaging over time with precise spatio-temporal optical manipulations induced by short infrared laser pulses. We demonstrate their potential for sub-cellular ablation of neurons and nuclei, tissue cauterization and optogenetics by using the Drosophila melanogaster and zebrafish model systems. Laser-based manipulation is a useful tool to dissect single axons in developed animals for behavioral studies as demonstrated for instance in early work on laser axotomy in C. elegans[21,22] This technique was used to perturb morphogenetic movements in the developing Drosophila embryo[23].

Methods

Results

Conclusion