Robust real-time 3D single-particle tracking using a dynamically moving laser spot

Hou, Shangguo; Lang, Xiaoqi; Welsher, Kevin

doi:10.1364/ol.42.002390

Cited by 58 publications

(91 citation statements)

References 24 publications

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“…Enhanced retention of eLNPs in vesicular compartments likely leads to a higher total cytosolic mRNA over time. Since endosomal escape is a transient event, we deployed three-dimensional (3D)-Dynamic Photon Localization Tracking (3D-DyPLoT) that is capable of measuring intracellular transport of individual nanoparticles in live cells on a millisecond temporal resolution 46,47 . 3D-DyPLoT is a real-time 3D single-particle tracking method that locks onto moving fluorescence targets and tracks their 3D position with high spatiotemporal precision.…”

Section: T O S T E R O L -A R G I N I N E β -S I T O S T E R O L -Amentioning

confidence: 99%

“…High-resolution live-cell imaging. Three-dimensional trajectories of internalized LNPs and eLNPs were collected using 3D-Dynamic Photon Localization Tracking (3D-DyPLoTTo perform 3D-DyPLoT 46,47 , a 640 nm excitation laser (Coherent OBIS,~76 nW at the focus) is focused onto the sample by an objective lens (Zeiss Plan Apo, 100x NA=1.49) and is deflected in a 3D pattern by a 2D electro-optic deflector (ConOptics) and a tunable acoustic gradient (TAG) lens (TAG Optics) 70 . Fluorescence photons from the moving probe are collected by the objective lens and on focused through an emission filter onto a single-photon counting avalanche photodiode (Excelitas SPCM-ARQH-15).…”

Section: Selectionmentioning

confidence: 99%

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Naturally-occurring cholesterol analogues in lipid nanoparticles induce polymorphic shape and enhance intracellular delivery of mRNA

et al. 2020

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Endosomal sequestration of lipid-based nanoparticles (LNPs) remains a formidable barrier to delivery. Herein, structure-activity analysis of cholesterol analogues reveals that incorporation of C-24 alkyl phytosterols into LNPs (eLNPs) enhances gene transfection and the length of alkyl tail, flexibility of sterol ring and polarity due to-OH group is required to maintain high transfection. Cryo-TEM displays a polyhedral shape for eLNPs compared to spherical LNPs, while x-ray scattering shows little disparity in internal structure. eLNPs exhibit higher cellular uptake and retention, potentially leading to a steady release from the endosomes over time. 3D single-particle tracking shows enhanced intracellular diffusivity of eLNPs relative to LNPs, suggesting eLNP traffic to productive pathways for escape. Our findings show the importance of cholesterol in subcellular transport of LNPs carrying mRNA and emphasize the need for greater insights into surface composition and structural properties of nanoparticles, and their subcellular interactions which enable designs to improve endosomal escape.

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Section: T O S T E R O L -A R G I N I N E β -S I T O S T E R O L -Amentioning

confidence: 99%

Section: Selectionmentioning

confidence: 99%

Naturally-occurring cholesterol analogues in lipid nanoparticles induce polymorphic shape and enhance intracellular delivery of mRNA

et al. 2020

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“…Several approaches can be used to extend this method to three dimensions ( Levi et al, 2005 ; McHale et al, 2007 ). We used the TAG lens resonant axial scanner in combination with galvanometric mirrors to create a cylindrical scan pattern ( Figure 1A ), an approach independently developed for molecular tracking ( Hou et al, 2017 ).…”

Section: Resultsmentioning

confidence: 99%

Recording Neural Activity in Unrestrained Animals with Three-Dimensional Tracking Two-Photon Microscopy

et al. 2018

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SUMMARY Optical recordings of neural activity in behaving animals can reveal the neural correlates of decision making, but brain motion, which often accompanies behavior, compromises these measurements. Two-photon point-scanning microscopy is especially sensitive to motion artifacts, and two-photon recording of activity has required rigid coupling between the brain and microscope. We developed a two-photon tracking microscope with extremely low-latency (360 μs) feedback implemented in hardware. This microscope can maintain continuous focus on neurons moving with velocities of 3 mm/s and accelerations of 1 m/s2 both in-plane and axially. We recorded calcium dynamics of motor neurons and inter-neurons in unrestrained freely behaving fruit fly larvae, correlating neural activity with stimulus presentations and behavioral outputs, and we measured light-induced depolarization of a visual interneuron in a moving animal using a genetically encoded voltage indicator. Our technique can be extended to stabilize recordings in a variety of moving substrates.

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“…3D-SMART. 3D Single-Molecule Active Real-time Tracking (3D-SMART) was achieved by applying these optimized parameters to the previously reported 3D-DyPLoT 25,32 . The general outline is shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Feedback is applied using either piezoelectric nanopositioners or galvo mirrors 18 , meaning the target molecule or particle is followed at very high speed, rather than being physically confined. Pioneering work has shown that RT-3D-SPT can lock-on to a wide range of targets, from gold nanoparticles 19, 20 and single quantum dots 21-23 all the way to viruses [24][25][26] , and is easily extended to live cell tracking 27, 28 . This is because the "confinement" is not a physical impediment to the target, but rather a high-speed chase which follows the particle without perturbation.While the pioneering work on RT-3D-SPT by several groups occurred more than a decade ago, the extension of this promising method to real-time 3D single molecule tracking (RT-3D-SMT) has been limited ( Supplementary Table 1).…”

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confidence: 99%

Real-time 3D Single Molecule Tracking

Hou

Exell²,

Welsher³

2020

Preprint

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To date, single molecule studies have been reliant on tethering or confinement to achieve long duration and high temporal resolution measurements. Here, we present a 3D single-molecule active real-time tracking method (3D-SMART) which is capable of "locking on" to single fluorophores in solution for minutes at a time with photon limited temporal resolution. As a demonstration, 3D-SMART was applied to actively track single Atto 647N fluorophores in 90% glycerol solution with an average duration of ~16 s at count rates of ~10 kHz. Active feedback tracking was further applied to single proteins and nucleic acids, directly measuring the diffusion coefficient of various lengths (99 to 1385 bp) of single DNA molecules at speeds up to 10 µm 2 /s. In addition, 3D-SMART was able to quantify to occupancy of single Spinach2 RNA aptamers and capture active transcription on single freely diffusing DNA. 3D-SMART represents a critical step towards the untethering of single molecule spectroscopy.which may critically rely on the crowded and non-equilibrium surroundings of the cellular interior.Several methods have sought to overcome this need to tether molecules, including liposomes 9 , convex lens induced confinement (CLIC) 10, 11 and the anti-Brownian electrokinetic (ABEL) trap [12][13][14][15][16] . Although each of these methods remove the tether, they still restrict the molecule in an isolated environment and cannot continuously monitor unbound diffusing molecules, such as an enzyme freely diffusing through the cellular interior.A group of methods with the promise to break this tether is real-time 3D single particle tracking (RT-3D-SPT) 17 . RT-3D-SPT acquires high speed position measurement of a diffusing particle and implements a closed feedback loop to hold the rapidly moving target effectively "locked" in the observation volume. Feedback is applied using either piezoelectric nanopositioners or galvo mirrors 18 , meaning the target molecule or particle is followed at very high speed, rather than being physically confined. Pioneering work has shown that RT-3D-SPT can lock-on to a wide range of targets, from gold nanoparticles 19, 20 and single quantum dots 21-23 all the way to viruses [24][25][26] , and is easily extended to live cell tracking 27, 28 . This is because the "confinement" is not a physical impediment to the target, but rather a high-speed chase which follows the particle without perturbation.While the pioneering work on RT-3D-SPT by several groups occurred more than a decade ago, the extension of this promising method to real-time 3D single molecule tracking (RT-3D-SMT) has been limited ( Supplementary Table 1). The first reported attempt at RT-3D-SPT was by Werner and coworkers, who used a tetrahedral detection pattern to achieve real-time tracking of single Cy5-dUTP molecules in 92 wt% glycerol 27 . While truly a groundbreaking achievement for RT-3D-SMT, the trajectories were limited to several 100 ms. The same group followed up two years later, showing the ability to disentangle the oligomerization states ...

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Robust real-time 3D single-particle tracking using a dynamically moving laser spot

Cited by 58 publications

References 24 publications

Naturally-occurring cholesterol analogues in lipid nanoparticles induce polymorphic shape and enhance intracellular delivery of mRNA

Naturally-occurring cholesterol analogues in lipid nanoparticles induce polymorphic shape and enhance intracellular delivery of mRNA

Recording Neural Activity in Unrestrained Animals with Three-Dimensional Tracking Two-Photon Microscopy

Real-time 3D Single Molecule Tracking

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