Attoliter detection volumes by confocal total-internal-reflection fluorescence microscopy.

Area of Science:

• Microscopy
• Biophysics
• Analytical Chemistry

Background:

• Conventional confocal microscopy has limitations in isolating individual molecules at high analyte concentrations.
• Many biologically relevant processes occur under these high-concentration conditions.
• A smaller detection volume is crucial for accurate molecular analysis.

Purpose of the Study:

• To report a novel confocal total-internal-reflection fluorescence (TIRF) microscope.
• To demonstrate its capability for significantly reduced detection volume.
• To enable precise analysis of single molecules in complex biological environments.

Main Methods:

• Utilized a confocal total-internal-reflection fluorescence (TIRF) microscope.
• Achieved diffraction-limited supercritical focusing and fluorescence collection.
• Employed a parabolic mirror objective for enhanced optical performance.

Main Results:

• Generated a detection volume of less than 5 al (5 x 10^-18 L) at a water-glass interface.
• Achieved a reduction of almost 2 orders of magnitude compared to conventional confocal microscopy.
• Delivered TIRF images with excellent spatial resolution and high signal-to-background ratio for single-molecule detection.

Conclusions:

• The developed TIRF microscope significantly enhances the ability to isolate and detect single molecules.
• This technology is vital for studying biologically relevant processes occurring at high analyte concentrations.
• The system offers superior spatial resolution and signal-to-background ratio, advancing molecular analysis capabilities.