Biological nano-objects rapidly diffuse in the solution phase, impeding our efforts to monitor their physical and chemical properties for extended periods of time. To overcome this, we developed the Interferometric Scattering Anti-Brownian ELectrokinetic (ISABEL) Trap, which counteracts Brownian motion for an extended time by tracking a particle’s location via its scattering and rapidly applying positional feedback with electrokinetic forces. Recently, we improved the flexibility of these experiments by shifting the scattering detection beam to the near-infrared and opening the visible region for flexible and specific fluorescence measurements. These capabilities allow us to monitor the physical and chemical properties of the carboxysome, a ~100nm bacterial microcompartment responsible for CO2 fixation by the enzyme Rubisco. With the ISABEL trap, we can rapidly interleave 405 and 488 nm fluorescence excitation beams to measure the redox properties inside individual carboxysomes using the redox reporting GFP mutant, roGFP2. The capabilities provided by the ISABEL trap allow us to design solution-phase single-particle experiments for a variety of biological nanoscale objects.
Identifying biomolecules of interest in cryogenic electron tomography (CET) reconstructions is made challenging by the lack of non-perturbative and specific labeling methods compatible with CET. Combining fluorescence and CET is a promising approach to overcome this labeling limitation. However, diffraction-limited fluorescence data has insufficient resolution to provide clear labeling in the crowded cellular environment. Super-resolution fluorescence techniques achieve resolution on the tens of nanometers scale, making them compatible with the length scales of interest in labeling biomolecules in CET. I will discuss the development of a cryogenic single-molecule based super-resolution imaging approach that achieves an average localization precision of less than ten nanometers and is compatible with the latest CET methods.
Abstract: Precision spectroscopic measurements of particles in free solution are greatly aided by use of an Anti-Brownian ELectrokinetic (ABEL) trap, which counteracts the effects of diffusion by means of closed-loop feedback to hold a single particle in a diffraction-limited spot for extended-time measurements. Generally, fluorescent emission from the trapped object is used to produce position estimates for the feedback circuit. However, many objects of interest may fluoresce only dimly (such as native fluorescence from a pigment-protein complex) or intermittently (such as a quantum dot). Here we report the development and demonstration of a new trapping modality that incorporates interferometric scattering to produce particle position estimates, called the Interferometric Scattering Anti-Brownian ELectrokinetic (ISABEL) trap. Using the ISABEL trap, we are able to completely decouple trapping from fluorescence detection, permitting trapping of dim and completely dark nanoparticles.
Anti-Brownian traps enable the measurement of single particles in free solution for long times by actively applying feedback forces based on an observed particle position to counteract Brownian motion. However, current implementations of anti-Brownian traps generally rely on fluorescence emission to detect a particle’s position. This reliance on fluorescence causes particles to be lost from the trap when they enter a fluorescence dark state by blinking or bleaching. Thus, there is a need for non-fluorescent methods of tracking for such traps. Scattered light provides a stable signal free of blinking and bleaching, but is very weak for small particles. However, interferometric scattering, a method of collecting the weak scattered field from a particle and interfering it with a strong reference field reflected from a nearby interface, allows particles to be tracked with sufficient speed and sensitivity. We combine interferometric scattering with our existing anti- Brownian electrokinetic (ABEL) trap to create the interferometric scattering anti-Brownian electrokinetic (ISABEL) trap. This technique enables the trapping of single nanoparticles in free solution for extended durations regardless of fluorescence blinking or bleaching. We verify the scaling of the interferometric scattering signal with the diameter of the particle for gold nanoparticles as small as 20 nm. We also demonstrate the measurement of the fluorescence brightness signal of fluorescent beads as they photobleach, while continuing to trap them with the scattering signal. The ISABEL trap extends the ability of anti-Brownian traps to new samples and new measurements across multiple scientific communities.
Single-molecule superresolution methods enable imaging of specifically-labeled biological samples with structures on length scales below the diffraction limit of visible light. Imaging samples at cryogenic temperatures (77 K) significantly reduces photobleaching, allowing more photons to be collected per emitter and thus improving the localization precision. Cryogenic single-molecule imaging also facilitates correlative imaging with cryogenic electron tomography (cryoET), which provides images of whole biological cells with high-resolution cellular contrast. Combining these two techniques by performing optical imaging under conditions that do not damage the sample for cryoET allows the combination of the high sensitivity and specificity from single-molecule fluorescence with the cellular context from cryoET. In this work, we use PAmKate, a red photoactivatable fluorescent protein, to perform cryogenic single-molecule imaging of proteins in the model organism Caulobacter crescentus at 77 K with sufficiently low illumination powers to prevent damage of the cryogenic sample. The enhanced number of photons detected allows localization precision to be improved to values below 10 nm.
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