Ratiometric Sensing of Redox Environments Inside Individual Carboxysomes Trapped in Solution

Abstract

Diffusion of biological nanoparticles in solution impedes our ability to continuously monitor individual particles and measure their physical and chemical properties. To overcome this, we previously developed the interferometric scattering anti-Brownian electrokinetic (ISABEL) trap, which uses scattering to localize a particle and applies electrokinetic forces that counteract Brownian motion, thus enabling extended observation. Here we present an improved ISABEL trap that incorporates a near-infrared scatter illumination beam and rapidly interleaves 405 and 488 nm fluorescence excitation reporter beams. With the ISABEL trap, we monitored the internal redox environment of individual carboxysomes labeled with the ratiometric redox reporter roGFP2. Carboxysomes widely vary in scattering contrast (reporting on size) and redox-dependent ratiometric fluorescence. Furthermore, we used redox sensing to explore the chemical kinetics within intact carboxysomes, where bulk measurements may contain unwanted contributions from aggregates or interfering fluorescent proteins. Overall, we demonstrate the ISABEL trap’s ability to sensitively monitor nanoscale biological objects, enabling new experiments on these systems.

Exploring masses and internal mass distributions of single carboxysomes in free solution using fluorescence and interferometric scattering in an anti-Brownian trap

“…As with the beads, we deliberately alternated feedback on and off in order to efficiently sample the wide distribution of carboxysomes, but we note that longer trapping times up to minutes is feasible for single particles. 30 , 31 The normalized scatter displays fluctuations expected around each level from position changes, similar to what was observed for polystyrene beads in Figure 2a. The scatter and the fluorescence levels both span large ranges.…”

“…Compared to other light scattering methods, the ISABEL trap has the benefit of simultaneous spectroscopy capabilities over extended time periods (multiple seconds) as a consequence both of the near-infrared scattering wavelength and the extended observation without tethering. We can already measure the redox behavior inside carboxysomes in the ISABEL trap 31 , and have measured detailed photophysical and kinetic parameters inferred from fluorescence in previous ABEL trap studies. 27 , 29 These measurement capabilities could be combined to apply to many types of biological microcompartments with an enclosing shell, loaded cargo, and sequestered internal chemistry.…”

“…The NIR spot at the sample was ~ 500 nm in diameter, and the pitch of the scan pattern was chosen to provide a time-averaged uniform intensity of ~ 250 kW/cm 2 over a 3.6 μm × 3.6 μm region. 31 The back-scattered and reflected light were separated from the pumping beam using a polarizing beam-splitter and quarter-wave plate combination and focused through an iris to block unwanted light onto a large area photodiode (Newport 2031) for interferometric scattering detection. The fluorescence excitation laser at 488 nm follows a path distinct from the AODs and is aligned to overlap with the NIR beam scan pattern using a 775SP dichroic.…”

“…The ISABEL trap experiments were performed on a custom-built optical setup as has been recently described 31 (see also Note S1 and Fig S4). Briefly, a near-IR (NIR) laser at 802 nm is scanned to different x-y positions in the sample plane with a pair of acousto-optic deflectors (AODs) in a 32-point Knight's tour pattern.…”

“…30 In recent advances to the technique we have shifted the high intensity scattering illumination laser to the near-infrared (NIR) and implemented simultaneous interleaved fluorescence excitation and detection of reporters in the visible to yield a more flexible ISABEL trap. 31 This instrument was able to trap single carboxysomes and precisely measure their redox states from fluorescence excitation spectroscopy over ~ 1s.…”