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Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy
Published on: January 18, 2017
Active wide-field illumination for high-throughput fluorescence lifetime imaging
Optics Letters
|October 2, 2013
Summary
Active wide-field illumination (AWFI) enhances fluorescence lifetime imaging sensitivity for weak signals. This method improves accuracy and speed across samples with varying fluorescence intensities.
Area of Science:
- Biophotonics
- Fluorescence Imaging
- Spectroscopy
Background:
- Wide-field fluorescence lifetime imaging offers fast, large-area sample analysis.
- Low sensitivity to weak fluorescence signals limits current wide-field techniques.
Purpose of the Study:
- To develop an active wide-field illumination (AWFI) strategy.
- To enhance sensitivity and accuracy in fluorescence lifetime imaging.
Main Methods:
- Implemented an active wide-field illumination (AWFI) strategy.
- Optimized spatial intensity for fluorescence signal acquisition.
- Validated with multiwell plate samples and a cell-based assay.
Main Results:
- AWFI accurately estimated lifetimes across a two-order-of-magnitude concentration range.
- Demonstrated successful quantitative Förster resonance energy transfer (FRET) lifetime measurements.
- Achieved enhanced accuracy and high acquisition speed for diverse fluorescence intensities.
Conclusions:
- AWFI overcomes sensitivity limitations in wide-field fluorescence lifetime imaging.
- The method enables accurate, high-speed lifetime measurements in challenging samples.
- AWFI is suitable for quantitative lifetime-based assays with large intensity variations.
