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Internalization and Observation of Fluorescent Biomolecules in Living Microorganisms via Electroporation
Published on: February 8, 2015
Red light, green light: probing single molecules using alternating-laser excitation
Yusdi Santoso1, Ling Chin Hwang, Ludovic Le Reste
1Department of Physics and Interdisciplinary Research Collaboration in Bionanotechnology, Clarendon Laboratory, University of Oxford, Parks Road, Oxford, UK.
Biochemical Society Transactions
|July 18, 2008
Summary
Single-molecule fluorescence resonance energy transfer (FRET) and alternating-laser excitation (ALEX) spectroscopy reveal biological system dynamics. ALEX enhances FRET by providing structure and stoichiometry data, enabling interaction detection and extending distance measurements.
Area of Science:
- Biophysics
- Molecular Biology
- Biochemistry
Background:
- Single-molecule fluorescence techniques offer deep insights into biological systems.
- Single-molecule Förster Resonance Energy Transfer (smFRET) is crucial for studying molecular structure, interactions, and dynamics.
Purpose of the Study:
- To introduce Alternating Laser Excitation (ALEX) spectroscopy as an advancement over traditional smFRET.
- To demonstrate ALEX's capability in providing simultaneous structural and stoichiometric information.
- To highlight ALEX's utility in detecting molecular interactions and expanding dynamic range.
Main Methods:
- Alternating Laser Excitation (ALEX) spectroscopy combined with confocal microscopy for solution and in-gel studies.
- ALEX coupled with Total Internal Reflection Fluorescence (TIRF) microscopy for surface-immobilized molecule analysis.
Main Results:
- ALEX provides simultaneous information on molecular structure and stoichiometry.
- ALEX enables interaction detection even without FRET signals.
- The method extends the dynamic range for distance measurements using FRET.
Conclusions:
- ALEX spectroscopy significantly enhances single-molecule FRET capabilities.
- The integration of ALEX with microscopy techniques (confocal and TIRF) broadens its applicability.
- ALEX is particularly valuable for studying complex biological interactions, such as protein-nucleic acid interactions.
