High-contrast single-particle tracking by selective focal plane illumination microscopy

Area of Science:

• Biophysics
• Microscopy
• Optical Engineering

Background:

• Wide-field single molecule microscopy is crucial for analyzing biological dynamics.
• Out-of-focus fluorescence limits its application in extended 3D systems.

Purpose of the Study:

• To develop a high-resolution selective plane illumination microscope (SPIM) to address out-of-focus fluorescence.
• To demonstrate SPIM's capability for analyzing fast, dynamic processes in biological specimens.

Main Methods:

• Construction of a high-resolution SPIM instrument.
• Theoretical calculations and quantitative measurements of light sheet thickness and axial point spread function.
• Direct comparison with epi-illumination using model samples.

Main Results:

• Achieved illumination light sheet thickness of 1.7 microm (543 nm) and 2.0 microm (633 nm).
• Obtained an axial point spread function FWHM of 1.13 microm.
• Demonstrated superior performance of SPIM over epi-illumination for samples with background fluorescence.

Conclusions:

• SPIM effectively solves the out-of-focus fluorescence problem in wide-field microscopy.
• The developed SPIM setup is suitable for visualizing and tracking single quantum dots and dynamic biological processes.