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.
Cryogenic single‐particle photoluminescence (PL) spectroscopy has been used with great success to directly observe the heterogeneous photophysical states present in a population of luminescent particles. Cryogenic electron tomography provides complementary nanometer scale structural information to PL spectroscopy, but the two techniques have not been correlated due to technical challenges. Here, we present a method for correlating single‐particle information from these two powerful microscopy modalities. We simultaneously observe PL brightness, emission spectrum, and in‐plane excitation dipole orientation of CdSSe/ZnS quantum dots suspended in vitreous ice. Stable and fluctuating emitters were observed, as well as a surprising splitting of the PL spectrum into two bands with an average energy separation of 80 meV. In some cases, the onset of the splitting corresponded to changes in the in‐plane excitation dipole orientation. These dynamics were assigned to structures of individual quantum dots and the excitation dipoles were visualized in the context of structural features.
Carboxysomes are
self-assembled bacterial microcompartments that
facilitate carbon assimilation by colocalizing the enzymes of CO2 fixation within a protein shell. These microcompartments
can be highly heterogeneous in their composition and filling, so measuring
the mass and loading of an individual carboxysome would allow for
better characterization of its assembly and function. To enable detailed
and extended characterizations of single nanoparticles in solution,
we recently demonstrated an improved interferometric scattering anti-Brownian
electrokinetic (ISABEL) trap, which tracks the position of a single
nanoparticle via its scattering of a near-infrared beam and applies
feedback to counteract its Brownian motion. Importantly, the scattering
signal can be related to the mass of nanoscale proteinaceous objects,
whose refractive indices are well-characterized. We calibrate single-particle
scattering cross-section measurements in the ISABEL trap and determine
individual carboxysome masses in the 50–400 MDa range by analyzing
their scattering cross sections with a core–shell model. We
further investigate carboxysome loading by combining mass measurements
with simultaneous fluorescence reporting from labeled internal components.
This method may be extended to other biological objects, such as viruses
or extracellular vesicles, and can be combined with orthogonal fluorescence
reporters to achieve precise physical and chemical characterization
of individual nanoscale biological objects.
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