Ratiometric Sensing of Redox Environments Inside Individual Carboxysomes Trapped in Solution.

Area of Science:

• Biophysics
• Nanotechnology
• Biochemistry

Background:

• Biological nanoparticle diffusion in solution limits continuous monitoring and property measurement.
• The interferometric scattering anti-Brownian electrokinetic (ISABEL) trap was previously developed to overcome these limitations by localizing particles and counteracting Brownian motion.

Purpose of the Study:

• To present an improved ISABEL trap with enhanced illumination and fluorescence excitation capabilities.
• To demonstrate the trap's utility in monitoring the internal redox environment of individual carboxysomes using a ratiometric redox reporter.

Main Methods:

• Incorporation of a near-infrared scatter illumination beam.
• Rapid interleaving of 405 and 488 nm fluorescence excitation reporter beams.
• Labeling carboxysomes with the ratiometric redox reporter roGFP2 for monitoring internal redox state.

Main Results:

• Carboxysomes exhibit significant variation in scattering contrast (indicating size) and redox-dependent ratiometric fluorescence.
• The ISABEL trap successfully monitored the internal redox environment of individual carboxysomes.
• Redox sensing revealed chemical kinetics within intact carboxysomes, avoiding artifacts from aggregates or interfering proteins.

Conclusions:

• The improved ISABEL trap offers sensitive monitoring of nanoscale biological objects.
• This technology enables novel experiments on the chemical kinetics and properties of individual biological nanoparticles.
• The ISABEL trap advances the study of complex biological systems at the nanoscale.