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Real-time background suppression during frequency domain lifetime measurements.
Petr Herman1, Badri P Maliwal, Joseph R Lakowicz
1Center for Fluorescence Spectroscopy, University of Maryland at Baltimore, Department of Biochemistry and Molecular Biology, 725 West Lombard Street, Baltimore, MD 21201, USA.
Analytical Biochemistry
|October 17, 2002
Summary
This study introduces real-time background suppression for biological sample autofluorescence during fluorescence lifetime measurements. This technique improves signal quality by gating detectors and using a reference fluorophore.
Area of Science:
- Biophysics
- Spectroscopy
- Optical Instrumentation
Background:
- Autofluorescence in biological samples complicates sensitive fluorescence measurements.
- Accurate fluorescence lifetime measurements require minimizing background noise.
Purpose of the Study:
- To develop and demonstrate a real-time background suppression method for autofluorescence.
- To enable precise frequency domain fluorescence lifetime measurements in complex biological matrices.
Main Methods:
- Excitation of samples with sub-picosecond pulses.
- Gating the detector for 10-40 ns after excitation to block autofluorescence.
- Utilizing a long-lifetime reference fluorophore for phase modulation measurements.
Main Results:
- Successful suppression of autofluorescence in real-time.
- Demonstrated effectiveness with mixtures of short- and long-lifetime probes.
- Validated the method in human plasma, a sample with high autofluorescence.
Conclusions:
- The developed gating technique effectively suppresses autofluorescence.
- This method enhances the accuracy of frequency domain fluorescence lifetime measurements.
- Applicable to various biological samples, improving diagnostic and research capabilities.