Chapter four - Detecting and Tracking Nonfluorescent Nanoparticle Probes in Live Cells

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Abstract

Precisely imaging and tracking dynamic biological processes in live cells are crucial for both fundamental research in life sciences and biomedical applications. Nonfluorescent nanoparticles are emerging as important optical probes in live-cell imaging because of their excellent photostability, large optical cross sections, and low cytotoxicity. Here, we provide a review of recent development in optical imaging of nonfluorescent nanoparticle probes and their applications in dynamic tracking and biosensing in live cells. A brief discussion on cytotoxicity of nanoparticle probes is also provided.

Introduction

Studying dynamic biological processes at the single molecule level inside live cells is a challenging yet rewarding task in life science. Recent advances in single molecule and single particle techniques gave great opportunities to examine time-dependent biological processes of target molecules, one at a time, in unprecedented detail (Joo et al., 2008, Saxton and Jacobson, 1997). In particular, the transient intermediates and individual time-dependent pathways that are difficult, if not impossible, to measure in ensemble experiments can be resolved.

To date, the majority of single molecule fluorescence experiments are limited to in vitro studies due to high fluorescence background and high bleaching propensity of fluorophores in the cellular environment. However, the fast development of nanoparticle probes greatly advanced the study of dynamic biological processes (Levi and Gratton, 2007, Miura, 2005, Saxton and Jacobson, 1997). Of particular interest are nonfluorescent nanoparticles. They have large cross sections so that they can be localized with high spatial precision and temporal resolution. Compared to single fluorophores or fluorophore-doped nanoparticles, nonfluorescent nanoparticle probes have excellent photostability, allowing them to be dynamically tracked for arbitrarily long time without blinking or bleaching. As no fluorophore is required to be present in the particle, these nonfluorescent nanoparticles are usually nontoxic. Combined with far-field optical imaging techniques, which require no contact thus minimum interference with the biological processes, nonfluorescent nanoparticles are suitable for visualizing dynamic processes in live cells. In addition, nanoparticles can serve as carriers for functional molecules. They can be manufactured as multifunctional centers. For these reasons, nanoparticles have found widespread applications in live cells including in vivo biosensing, targeted drug/gene delivery, biomedical diagnostics and therapies, etc. (Love et al., 2008, Murphy et al., 2008, Sperling et al., 2008, Wax and Sokolov, 2009).

In this chapter, we provide a review on recent technological development in detecting and tracking nonfluorescent nanoparticle probes in live cells, and the achievements in biological studies. Nanoparticles doped with fluorescent dyes, quantum dots, and particles giving surface-enhanced Raman scattering are not covered in this chapter. Interested readers are referred to other chapters in this volume or recent reviews (Levi and Gratton, 2007, Michalet et al., 2005, Miura, 2005).

Section snippets

Techniques and Tools

The detection and tracking of individual nanoparticles with a size of 1  100 nm require high-sensitivity imaging techniques. The detection schemes reported in recent literature include both existing techniques that are tailored and new techniques to fulfill this special requirement.

Single particle tracking and single particle orientation and rotational tracking

Diffusion of membrane proteins and lipids is essential to many membrane processes including signal transduction, domain formation, protein sorting, protein–lipid complex formation, etc. (Dix and Verkman, 2008, Fan et al., 2010, Kahya and Schwille, 2006, Niemela et al., 2010). Tracking individual lipid or protein molecule movement on cell membranes either with fluorescent molecular probes (Douglass and Vale, 2005, Groc et al., 2004, Kusumi et al., 2005, Murase et al., 2004, Ohsugi et al., 2006,

Cytotoxicity of Nanoparticle Probes

With the increasingly fast development of nanotechnology, human and environmental exposure to engineered nanomaterials becomes inevitable. However, the consequences of the application of engineered nanomaterials are not well understood, and the public are becoming increasingly concerned about the potential toxicity on human health and environmental sustainability. It is important that the nanotoxicology research uncovers how physiochemical properties of nanomaterials influence their toxicity so

Conclusions and Future Perspective

The research at the interface of live-cell imaging and the optical detection of nonfluorescent nanoparticles has been particularly active in recent years. The number of fundamental biological studies using nonfluorescent techniques should continue to increase during the next few years. Due to the high cellular autofluorescence background, single-molecule experiments in live cells remain challenging. The large optical cross sections and nontoxic nature of gold nanoparticles make them ideal

Acknowledgments

This work was supported by start-up funds from Iowa State University and North Carolina State University and a grant from U.S. Department of Energy, Office of Basic Energy Sciences, Division of Chemical Sciences, Geosciences, and Biosciences through the Ames Laboratory. The Ames Laboratory is operated for the U.S. Department of Energy by Iowa State University under contract no. DE-AC02-07CH11358.

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