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Fluorescence detection methods for microfluidic droplet platforms
Published on: December 10, 2011
High resolution detection of fluorescently labeled microorganisms in environmental samples using time-resolved
Russell Connally1, Duncan Veal, James Piper
1Centre for Fluorometric Applications in Biotechnology, Department of Biological Sciences, Macquarie University, Sydney, NSW 2109, Australia. rconnall@ics.mq.edu.au
FEMS Microbiology Ecology
|August 28, 2009
Summary
Time-resolved fluorescence microscopy (TRFM) effectively reduces background autofluorescence in environmental samples. This technique enhances the detection of microbial targets like Giardia cysts, improving contrast significantly.
Area of Science:
- Microbiology
- Microbial Ecology
- Microscopy
Background:
- Fluorescence microscopy is vital for analyzing microbial cells but struggles with autofluorescent environmental samples.
- Autofluorescence obscures target signals, limiting microbial ecology applications.
- Time-resolved fluorescence microscopy (TRFM) minimizes autofluorescence by delaying signal detection, preserving target luminescence.
Purpose of the Study:
- To adapt a commercial fluorescence microscope for time-resolved operation.
- To develop a TRFM method for detecting microorganisms in challenging environmental samples.
- To assess the efficacy of TRFM with a novel immunofluorophore for Giardia cyst detection.
Main Methods:
- Modified a commercial fluorescence microscope with an image-intensified camera and flashlamp for TRFM.
- Utilized a novel immunofluorophore for specific Giardia cyst labeling.
- Applied a 60-microsecond gate delay between excitation and detection.
Main Results:
- Achieved a 30-fold increase in contrast for labeled Giardia cysts compared to conventional methods.
- Successfully detected Giardia cysts in water samples with high autofluorescence.
- Demonstrated the effectiveness of the modified TRFM system.
Conclusions:
- The modified TRFM system offers a cost-effective solution for microbial analysis in complex samples.
- TRFM significantly enhances the detection of specific microorganisms by overcoming autofluorescence.
- This study presents the first report of TRFM for detecting microorganisms in environmental samples.
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