Related Experiment Videos
Imaging of single fluorescent molecules using video-rate confocal microscopy
H Tadakuma1, J Yamaguchi, Y Ishihama
1Department of Physics, School of Science and Engineering, Waseda University, 3-4-1 Okubo, Tokyo 169-8555, Japan.
Biochemical and Biophysical Research Communications
|September 14, 2001
Summary
This study demonstrates real-time imaging of single fluorescent molecules using Nipkow disk confocal microscopy. This advanced technique allows observation of kinesin molecular movement in both 2D and 3D environments.
Area of Science:
- Biophysics
- Microscopy
- Molecular Biology
Background:
- Confocal microscopy is crucial for high-resolution imaging.
- Observing single molecules in real-time presents significant technical challenges.
Purpose of the Study:
- To evaluate the performance of Nipkow disk confocal microscopy for imaging single fluorescent molecules at video-rate.
- To assess the applicability of this method for studying molecular events in real-time.
Main Methods:
- Utilized Nipkow disk confocal microscopy to image single fluorescent molecules (kinesin) in aqueous solution.
- Employed tetramethylrhodamine (TMR) and IC5 fluorescent dyes for molecular labeling.
- Investigated fluorophore photodecomposition lifetimes and their dependence on laser power.
Main Results:
- Achieved first-time video-rate imaging of single fluorescent molecules.
- Observed 2D sliding movement of kinesin along microtubules and 3D Brownian motion in solution.
- Determined photodecomposition lifetimes for TMR (~10 s) and IC5 (~2 s) at 0.5 W/mm(2).
- Found fluorescence intensity and photobleaching rate to be proportional to laser power (0.65–3 W/mm(2)).
Conclusions:
- Nipkow disk confocal microscopy enables real-time observation of single molecular dynamics.
- The method is suitable for studying dynamic molecular events within living cells.