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siFLIM: single-image frequency-domain FLIM provides fast and photon-efficient lifetime data
Marcel Raspe1, Katarzyna M Kedziora1, Bram van den Broek1
1Department of Cell Biology, The Netherlands Cancer Institute, Amsterdam, the Netherlands.
Nature Methods
|April 19, 2016
Summary
We developed single-image fluorescence lifetime imaging microscopy (siFLIM) for fast, quantitative lifetime imaging. This method uses new cameras for minimal phototoxicity and immunity to cellular movement artifacts.
Area of Science:
- Biophotonics
- Microscopy
- Cellular Imaging
Background:
- Fluorescence lifetime imaging microscopy (FLIM) provides quantitative biological information.
- Traditional FLIM methods can be slow and prone to artifacts from phototoxicity and cellular movement.
Purpose of the Study:
- To develop a novel method for rapid, quantitative FLIM.
- To overcome limitations of existing FLIM techniques, including speed and artifact susceptibility.
Main Methods:
- Developed single-image fluorescence lifetime imaging microscopy (siFLIM).
- Utilized new camera technology to capture two phase-shifted images simultaneously from a single exposure.
- Enabled video-rate data acquisition.
Main Results:
- Achieved quantitative lifetime imaging from a single exposure.
- Demonstrated video-rate imaging capabilities.
- Showcased minimal phototoxicity and bleaching.
- Established inherent immunity to artifacts from rapid cellular movements and signal transients.
Conclusions:
- siFLIM offers a significant advancement for live-cell imaging requiring quantitative fluorescence lifetime data.
- The method provides high-speed, artifact-free lifetime measurements with reduced photodamage.
- siFLIM is a powerful tool for studying dynamic biological processes.
