Study of single-molecule dynamics and reactions with classic light microscopy

Area of Science:

• Biophysics
• Biochemistry
• Microscopy

Background:

• Single-molecule studies are crucial in life sciences, but observing reactions is challenging.
• Traditional methods often require specialized environments (e.g., scanning probe microscopy).
• Classic far-field light microscopy has been underutilized for single-molecule reaction studies.

Purpose of the Study:

• To develop and demonstrate techniques for studying single-molecule reactions using conventional far-field light microscopy.
• To investigate enzyme kinetics and macromolecular processing at the single-molecule level.
• To overcome limitations of existing experimental setups for observing biological reactions.

Main Methods:

• Utilized nonscanning far-field light microscopy for single-molecule observations.
• Employed NADH autofluorescence to study the lactate dehydrogenase 1 (LDH-1) enzyme reaction.
• Applied phase-contrast microscopy to monitor reaction progress via product scattering/absorption.
• Investigated the behavior and enzymatic cutting of fluorescently stained lambda-DNA.

Main Results:

• Successfully monitored the conversion of nicotinamide adenine dinucleotide (NAD+) and lactate to NADH catalyzed by LDH-1.
• Quantified DNA molecule dynamics, including collapse and stretching due to viscosity.
• Directly observed sequence-specific DNA cutting by restriction enzymes in real-time.
• Visualized the theoretical restriction pattern of DNA under direct inspection.

Conclusions:

• Far-field light microscopy is a viable technique for studying single-molecule reactions in biological systems.
• The developed methods enable direct observation and quantification of enzymatic reactions and DNA processing.
• This approach offers a powerful tool for advancing single-molecule biophysics and biochemistry research.