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Updated: Jul 11, 2026
Imaging Dpp Release from a Drosophila Wing Disc
Published on: October 30, 2019
Localized multiphoton photoactivation of paGFP in Drosophila wing imaginal discs
Periklis Pantazis1, Marcos González-Gaitán
1Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany.
Abstract:
In biological imaging of fluorescent molecules, multiphoton laser scanning microscopy (MPLSM) has become the favorite method of fluorescence microscopy in tissue explants and living animals. The great power of MPLSM with pulsed lasers in the infrared wavelength lies in its relatively deep optical penetration and reduced ability to cause potential nonspecific phototoxicity. These properties are of crucial importance for long time-lapse imaging. Since the excited area is intrinsically confined to the high-intensity focal volume of the illuminating beam, MPLSM can also be applied as a tool for selectively manipulating fluorophores in a known, three-dimensionally defined volume within the tissue. Here we introduce localized multiphoton photoactivation (MP-PA) as a technique suitable for analyzing the dynamics of photoactivated molecules with three-dimensional spatial resolution of a few micrometers. Short, intense laser light pulses uncage photoactivatable molecules via multiphoton excitation in a defined volume. MP-PA is demonstrated on photoactivatable paGFP in Drosophila wing imaginal discs. This technique is especially useful for extracting quantitative information about the properties of photoactivatable fusion proteins in different cellular locations in living tissue as well as to label single or small patches of cells in tissue to track their subsequent lineage.
Insights
Localized multiphoton photoactivation (MP-PA) enables precise 3D analysis of photoactivatable molecules in living tissues. This advanced microscopy technique offers high spatial resolution for studying cellular dynamics and lineage tracing.
Area of Science:
- Biological Imaging
- Microscopy Techniques
- Cellular Dynamics
Background:
- Multiphoton laser scanning microscopy (MPLSM) is favored for deep tissue imaging in vivo due to its optical penetration and low phototoxicity.
- MPLSM's confined excitation volume allows for targeted manipulation of fluorophores within tissues.
- Long-term, high-resolution imaging is critical for understanding dynamic biological processes.
Purpose of the Study:
- Introduce localized multiphoton photoactivation (MP-PA) for analyzing photoactivated molecule dynamics.
- Demonstrate MP-PA's capability for high-resolution, 3D spatial analysis in living tissues.
- Provide a method for quantitative analysis of photoactivatable proteins and cell lineage tracing.
Main Methods:
- Utilized multiphoton laser scanning microscopy (MPLSM) with pulsed infrared lasers.
- Developed localized multiphoton photoactivation (MP-PA) using short, intense laser pulses.
- Applied MP-PA to photoactivatable paGFP in Drosophila wing imaginal discs for demonstration.
Main Results:
- MP-PA achieved three-dimensional spatial resolution of a few micrometers.
- Successfully demonstrated photoactivation of paGFP in a defined volume within Drosophila tissues.
- Enabled quantitative analysis of photoactivatable fusion protein properties in cellular locations.
Conclusions:
- MP-PA is a powerful technique for studying molecular dynamics in living tissues with high spatial precision.
- This method facilitates quantitative analysis of photoactivatable proteins and cellular lineage tracing.
- MP-PA enhances the utility of multiphoton microscopy for advanced biological research.
