Confocal three dimensional tracking of a single nanoparticle with concurrent spectroscopic readouts

Cang, Hu; Wong, C.; Xu, Chao; Rizvi, Abbas H.; Yang, Haw

doi:10.1063/1.2204652

Cited by 115 publications

(122 citation statements)

References 8 publications

Supporting

Mentioning

119

Contrasting

Order By: Relevance

“…6B the trajectory is plotted as a projection on the x-y plane of the glass slide. Three-dimensional confocal tracking (34) of the single FND is practical if either the bright fluorescence or the strong scattered laser light (35) from the diamond nanoparticle is used as a feedback signal to locate its position in the live cell.…”

Section: Resultsmentioning

confidence: 99%

Characterization and application of single fluorescent nanodiamonds as cellular biomarkers

Lee

Chen

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

804

455

View full text Add to dashboard Cite

Type Ib diamonds emit bright fluorescence at 550 -800 nm from nitrogen-vacancy point defects, (N-V) 0 and (N-V) ؊ , produced by high-energy ion beam irradiation and subsequent thermal annealing. The emission, together with noncytotoxicity and easiness of surface functionalization, makes nano-sized diamonds a promising fluorescent probe for single-particle tracking in heterogeneous environments. We present the result of our characterization and application of single fluorescent nanodiamonds as cellular biomarkers. We found that, under the same excitation conditions, the fluorescence of a single 35-nm diamond is significantly brighter than that of a single dye molecule such as Alexa Fluor 546. The latter photobleached in the range of 10 s at a laser power density of 10 4 W/cm 2 , whereas the nanodiamond particle showed no sign of photobleaching even after 5 min of continuous excitation. Furthermore, no fluorescence blinking was detected within a time resolution of 1 ms. The photophysical properties of the particles do not deteriorate even after surface functionalization with carboxyl groups, which form covalent bonding with polyL-lysines that interact with DNA molecules through electrostatic forces. The feasibility of using surface-functionalized fluorescent nanodiamonds as single-particle biomarkers is demonstrated with both fixed and live HeLa cells.blinking ͉ photobleaching ͉ single-molecule detection ͉ single-particle tracking ͉ live cell O ne of the key avenues to understanding how biological systems function at the molecular level is to probe biomolecules individually and observe how they interact with each other directly in vivo. Laser-induced fluorescence is a technique widely adopted for this purpose owing to its ultrahigh sensitivity and capabilities of performing multiple-probe detection (1-3). However, in applying this technique to imaging and tracking a single molecule or particle in a biological cell, progress is often hampered by the presence of ubiquitous endogenous components such as flavins, nicotinamide adenine dinucleotides, collagens, and porphyrins that produce high fluorescence background signals (4-6). These biomolecules typically absorb light at wavelengths in the range of 300-500 nm and fluoresce at 400-550 nm (Fig. 1). To avoid such interference, a good biological fluorescent probe should absorb light at a wavelength longer than 500 nm and emit light at a wavelength longer than 600 nm, at which the emission has a long penetration depth through cells and tissues (5, 7). Organic dyes and fluorescent proteins are two types of molecules often used to meet such a requirement (1,8,9); however, the detrimental photophysical properties of these molecules, such as photobleaching and blinking, inevitably restrict their applications for long-term in vitro or in vivo observations. Fluorescent semiconductor nanocrystals (or quantum dots), on the other hand, have gained considerable attention in recent years because they hold a number of advantageous features including high photobleaching thresholds a...

show abstract

Section: Resultsmentioning

confidence: 99%

Characterization and application of single fluorescent nanodiamonds as cellular biomarkers

Lee

Chen

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

804

455

View full text Add to dashboard Cite

show abstract

“…Whereas SPT has made important discoveries that change our view of plasma membrane organization (17,19) and molecular motor dynamics (20), the use of SPT in monitoring ''intracellular'' processes is rather limited because of the lack of three-dimensional (3D) tracking capacity that can follow a single particle inside a live cell for a long period of time. In the past decade, new SPT techniques have been developed to visualize molecular motion in the 3D space (termed 3D-SPT), including multiple imaging planes (21,22), orbital tracking (23)(24)(25), point spread function engineering (26,27), and confocal tracking (28,29). Although allowing for direct observation of transport processes from membrane to cytoplasm, current 3D-SPT methods often suffer from shallow imaging depth (because of the use of one-photon excitation) and limited z-tracking range (e.g., astigmatismbased, nonfeedback tracking systems (27)), which prevent these methods from tracking single molecules inside multicellular models such as spheroids (see our review in (8)).…”

Section: Introductionmentioning

confidence: 99%

Segmentation of 3D Trajectories Acquired by TSUNAMI Microscope: An Application to EGFR Trafficking

Liu

Perillo

Liu

et al. 2016

Biophysical Journal

View full text Add to dashboard Cite

Whereas important discoveries made by single-particle tracking have changed our view of the plasma membrane organization and motor protein dynamics in the past three decades, experimental studies of intracellular processes using singleparticle tracking are rather scarce because of the lack of three-dimensional (3D) tracking capacity. In this study we use a newly developed 3D single-particle tracking method termed TSUNAMI (Tracking of Single particles Using Nonlinear And Multiplexed Illumination) to investigate epidermal growth factor receptor (EGFR) trafficking dynamics in live cells at 16/43 nm (xy/z) spatial resolution, with track duration ranging from 2 to 10 min and vertical tracking depth up to tens of microns. To analyze the long 3D trajectories generated by the TSUNAMI microscope, we developed a trajectory analysis algorithm, which reaches 81% segment classification accuracy in control experiments (termed simulated movement experiments). When analyzing 95 EGF-stimulated EGFR trajectories acquired in live skin cancer cells, we find that these trajectories can be separated into three groups-immobilization (24.2%), membrane diffusion only (51.6%), and transport from membrane to cytoplasm (24.2%). When EGFRs are membrane-bound, they show an interchange of Brownian diffusion and confined diffusion. When EGFRs are internalized, transitions from confined diffusion to directed diffusion and from directed diffusion back to confined diffusion are clearly seen. This observation agrees well with the model of clathrin-mediated endocytosis.

show abstract

“…Experimental demonstrations of closed-loop, singleparticle tracking have included two-dimensional [1,2,3,4] and three-dimensional [5,6] tracking with piezo-electric actuation and two-dimensional control of individual [7,8,9] and multiple [10,11] particles with electrophoretic actuation. Concurrently, theoretical results have been developed for understanding the statistics and performance limits of these new control systems [3,4,12,13,14,15,16,10,11,17].…”

Section: Introductionmentioning

confidence: 99%

Fluctuations in closed-loop fluorescent particle tracking

Berglund¹,

McHale²,

Mabuchi³

2007

Opt. Express

View full text Add to dashboard Cite

Abstract:We present a comprehensive theory of closed-loop particle tracking for calculating the statistics of a diffusing fluorescent particle's motion relative to the tracking lock point. A detailed comparison is made between the theory and experimental results, with excellent quantitative agreement found in all cases. A generalization of the theory of (open-loop) fluorescence correlation spectroscopy is developed, and the relationship to previous results is discussed. Two applications of the statistical techniques are given: a method for determining a tracked particle's localization and an algorithm for rapid particle classification based on real-time analysis of the tracking control signal.

show abstract

Confocal three dimensional tracking of a single nanoparticle with concurrent spectroscopic readouts

Cited by 115 publications

References 8 publications

Characterization and application of single fluorescent nanodiamonds as cellular biomarkers

Characterization and application of single fluorescent nanodiamonds as cellular biomarkers

Segmentation of 3D Trajectories Acquired by TSUNAMI Microscope: An Application to EGFR Trafficking

Fluctuations in closed-loop fluorescent particle tracking

Contact Info

Product

Resources

About