MICROSCOPY RESEARCH AND TECHNIQUE 74:559562 (2011)

Introduction: Bio and Nano Imaging and Analysis

Research focusing on the intersection of life sciences and nanotechnology is producing new elds such as nanobiotechnology, nanobiology and nanomedicine. This interaction is occurring in both directions. On one hand, biological molecules can be used to synthesize and organize inorganic functional nanomaterials into well-dened structures (Ma and others, 2008; Sotiropoulou and others, 2008; van Bommel and others, 2003). On the other hand, nanomaterials can be used to study biology and develop biomedicines. For example, labeled nanomaterials can serve as effective longlasting dyes for bio-imaging (Lee and others, 2007; Medintz and others, 2005; Xie and others, 2011) and nanomaterials are ideal drug-carriers for the development of new drug systems (Dobson, 2006; Nakanishi and others, 2009; Portney and Ozkan, 2006). Microscopy is a powerful tool that can image bionanostructures, track nanomaterials in bio-systems, measure physical properties, determine compositions, and even create and manipulate nanostructures. In this special issue on bio-nano imaging and analysis, different microscopy techniques were used to characterize the structures of bionanomaterials, image nanoparticles in biological systems, study biological functions, or manipulate nanostructures (Figure 1). This special issue includes 13 articles from leading scientists in bionanotechnology and the microscopy community and covers the following four interesting topics: 1) use of nanoparticles as imaging probes, 2) manipulation of nanostructures using microscopy, 3) bio-inorganic hybrid nanomaterials imaging, and 4) imaging nanomaterials using uorescence and single-molecule microscopy. In this special issue the rst topic is about the use of nanoparticles as imaging probes. Four frequently used nanoparticles and their principal method of imaging are summarized in table 1. This section includes three review articles and one research article. The three review articles concern recent advancements in the application of gold nanoparticles, magnetic nanoparticles and quantum dots for bio-imaging. The research article presents a special method for labeling axonal transport with quantum dots. Subramanian Tamil Selvan et al, at the Institute of Materials Research and Engineering in Singapore have reviewed the recent advances in the synthesis and application of bimodal magnetic-uorescent probes for bio-imaging (pages 563576). Recent advances in imaging with nanoparticles, which include quantum dots, magnetic nanoparticles, rare-earth doped upconversion uorescent nanoparticles, and multifunctional nanoparticles have been very rapid. These nanoparticles have not only enhanced imaging sensitivity, resolution, and specicity, but they have also allowed for simultaneous multi-targeting, monitoring, and
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WILEY-LISS, INC. DOI 10.1002/jemt.21051 Published online in Wiley Online Library (wileyonlinelibrary.com).

enhanced diagnostics and delivery of therapeutic effects. In the second part of this article, molecular imaging modalities clinically used such as position emission tomography (PET), single photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), optical and and ultrasound microscopy are summarized in a table along with the related imaging methods for the specic nanoparticles. Also described are recent advances in the assemblies of (1) superparamagnetic iron oxide (SPIO)-quantum dot (QD) based magnetic-uorescent probes, (2) SPIOrare-earth (RE) based magnetic-uorescent probes, (3) Gd31-based magnetic contrast agents covalently attached to uorescent probes, and (4) Gd31-based MRI agents and uorescent probes in a single nanomaterial domain. This article should be important to those studying nanomaterials for bio-imaging. The next article by Jesus Ruiz-Cabello from Universidad Complutense de Madrid, Madrid, Spain described use of magnetic iron oxide nanoparticles and gold nanoparticles (GNPs) in MRI imaging and gene therapy (Pages 577591). The magnetic nanoparticles are good contrast enhancement agents in MRI imaging. They can also be magnetically manipulated to guide delivery of genes into target cells for transfection. When GNPs are modied with either Gd supramolecular complexes or iron oxide, the new conjugates can also serve as contrast enhancement agents in MRI. More importantly, they elucidate the conjugation of these nanoparticles with biological molecules such as viruses to form nanobioconjugates. This article summarizes how these nanobioconjugates can improve the performance of the nanoparticles in MRI imaging and gene therapy. This article should be useful for those who are studying magnetic nanoparticles-based MRI imaging and gene delivery. The article by Eliza Hutter and Dusica Maysinger, McGill University in Canada, reviews the unique properties of GNPs and QDs, and how their properties benet cellular and in vivo imaging (pages 592604). GNPs have strong light scattering and surface plasmon enhanced luminescence, both of which can be used for bio-imaging. Light scattering by GNPs is usually visualized by dark-eld microscopy and surface plasmon enhanced luminescence is most commonly monitored by two-photon luminescence microscopy. In the rst part of this issue are described the physicochemical characteristics of GNPs, how to apply GNPs in the imaging of cells and animals, advantages of applying GNPs, and other potential applications of GNPs. QDs, which are stable, highly uorescent, and tunable nanoparticles, can be further functionalized for specic applications. In the second part of this article, the authors reviewed the use of QDs as bio-imaging probes to image protein location at cellular or the intracellular organelle surfaces, to screen cancer markers in biological uids, and to diagnose primary and metastatic tumors in vivo. This article introduces two very useful nanoparticles, luminescent QDs and plasmonic GNPs,

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as bio-imaging agents. Researchers who are trying to apply nanotechnology to develop biomedical imaging techniques will nd these nanoparticles particularly helpful. The next research article is presented by Bianxiao Cui et al, at Stanford University. They selected QDs as the imaging probes for tracing axonal transport (pages 605613). This article presents a method of automatic image analysis for constructing vesicle or cargo trajectories during axonal transport with higher data processing throughput, better spatial resolution, and minimal axon perturbation. They rst transformed the problem of particle tracking in a 3D data set as a curve tracing problem in a 2D spatiotemporal kymograph image. They located the initial seeding points by identifying local intensity maxima within the kymograph which were set as local brightest points. Then they carried out the core tracing algorithm using an implementation of Stegers algorithm, which is based on the direction-dependence of the edge and intensity response functions between a template and the neighborhood of nearby pixels. To achieve high spatial resolution, they rened the position of each trajectory point on the 2D kymograph image by back-projecting the trajectory points located in the kymograph onto the original image data and tting the particle image with a 2D Gaussian function. After all candidate segments were extracted, they selected and connected the segments through an optimization. This article provides a new

Fig. 1. The Microscopy techniques used in this special issue to study bionanomaterials and bionanostructures. [Color gure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

method of automated data analysis for axonal transport processes. The second topic, the manipulation of nanostructures using microscopy, includes one review article presented by Jun Hu et al., Shanghai Institute of Applied Physics in China. Hu reviewed recent ndings on imaging and manipulation of biological macromolecular structures using atomic force microscopy (AFM) (pages 614626). The authors highlighted the study of Ryanodine receptors and the dynamic process of peptide self-assembly by using the high resolution imaging capacity of the AFM. In the second part, they presented a method of cutting, pushing, isolating, and analyzing individual DNA molecules by using AFM. In addition they introduced the vibrating mode scanning polarization force microscopy (VSPFM), which could be used to study the elasticity of individual biomolecules and living cells. This article describes the AFM is a multi-faceted biophysical tool for both imaging and nano-manipulation of biological structures on surfaces. Five articles are included in the third topic of bioinorganic hybrid nano-imaging. Because biomolecules have been frequently used as templates for the formation of inorganic nanomaterials, imaging the resultant bio-inorganic hybrid nanomaterials is an extremely important issue and is also a daunting challenge for microscopists. These ve articles are therefore included in this special issue to highlight the progress made in this research area. These articles cover several microscopy techniques including negative staining, thin-sectioning, cryo-EM, advanced electron microscopies, and transmission X-ray microscopy. The rst article by Cao, Xu, and Mao at University of Oklahoma describes imaging bio-inorganic nanohybrids by using transmission electron microscopy (TEM) as the primary tool (pages 627635). TEM is a popular and relatively simple tool that can offer a direct visualization of the nanomaterials with high resolutions. When TEM is applied to visualize bioinorganic nanohybrids, a treatment of negative staining is necessary due to the presence of biological molecules in the nanohybrids. However, the conventional negative-staining procedure for regular biological samples cannot be directly applied to such bio-inorganic nanohybrids. To image a specic bio-inorganic nanohybrid, negative-staining factors such as negative stain type, working pH, staining time, and drying method, should be identied. Mao et al chose bacteriophagegold nanoparticle hybrids as a model to systemically study the effects of each factor on the negative staining of the nanohybrids. They found the best staining conditions for imaging bacteriophage-gold nanoparticle hybrids and discussed the effects of each factor on the negative staining of nanohybrids. This article should be a helpful guide for scientists choosing the correct

TABLE 1. Nanoparticles as bio-imaging probes, their principal method of imaging, and equipment Nanoparticles used as imaging probes Gold nanoparticles Quantum dots Magnetic nanoparticles Upconversion nanoparticles Imaging Method Surface enhanced Raman spectroscopy Quantum connements Magnetic resonance imaging (MRI) Upconversion luminescence Equipment Con-focal Raman microscope Fluorescence microcope MRI spectrometer Two-photon emission uorescence microscope

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negative staining conditions for imaging their bio-inorganic nanomaterials. The next research article is presented by Qian Wang et al., at the University of South Carolina. The authors have developed a method to characterize the protein distribution in protein-polymer composites (pages 636 641). They assembled poly(4-vinylpyridine) (P4VP) with ferritin proteins into composite spheres and performed TEM thin-sectioning technique to investigate how ferritin particles distributed within the spheres. In the process of thin-sectioning, the choice of xative and xative inltration are crucial steps. They found that when specimens were treated with an optimal procedure for xation and inltration, a round-shaped thin sectioned structure with black and white regions was observed. This article provides a new method to investigate the protein distribution in protein-polymer composites which should be helpful for studying drug and gene delivery as well as developing new delivery vehicles. Bjoern Sander and Monika Golas from Aarhus University in Denmark have reviewed TEM techniques for the visualization of bionanostructures (pages 642663). They describe the sample preparation techniques including conventional negative staining, cryo-negative staining, and unstained cryo-EM as well as the various imaging and image processing methods such as electron crystallography, electron tomography, and single-particle EM. They also discussed how deviations from an ideal symmetry and structural heterogeneity can limit the resolution. This review article suggests that nanoimaging techniques, especially TEM, can not only offer new applications in the eld of nanomaterials, but can also serve as powerful tools for the visualization and analysis of bionanostructures. The next research article presented by Pratibha Gai et al., from the University of York in United Kingdom describes the probing of nanostructures using advanced electron microscopy methods, including aberration-corrected transmission electron microscopy (ACTEM), AC-high angle annular dark eld scanning TEM (AC-HAADF-STEM), AC-energy ltered TEM, electron-stimulated energy dispersive spectroscopy in the AC-(S)TEM and high-resolution TEM (HRTEM) with a scanning tunneling microscopy (STM) holder (pages 664670). They used these techniques to image the presence of single Au atoms on titania surfaces in catalytic reactions, atomic-scale grain boundaries in nanotwinned metals, atomically clean surface in nanoZnO tetrapods, and crystallization and edge reconstruction in grapheme. These advanced electron microscopy methods open up new opportunities for the study of nanomaterials at atomic scales and indicate future direction of electron microscopy development. Joy Andrews et al., at SLAC National Accelerator Laboratory in California introduced full-eld transmission X-ray microscopy (TXM) for nano-imaging of biomaterials (pages 671681). Full-eld TXM is a kind of microscopy that can obtain a 3D view of both subcellular and extensive intercellular structures in biological materials at the nano-scale. They rst described the experimental setup and sample preparation for TXM. Then different capabilities of TXM, including 3D tomography, Zernike phase contrast, quantication of absorption, and chemical identication via X-ray uoMicroscopy Research and Technique

rescence and X-ray absorption near-edge structure imaging, were discussed and compared based on the results from the imaging of biological materials such as microorganisms, bone, and plants. They discussed the complementarities of TXM with other imaging methods.. This review article is informative to those who are new to the eld of TXM. There are three articles in the fourth topic about uorescence and single-molecule microscopy. The rst research article is about using internal reection uorescence microscopy to study protein adsorption. The second article compares AFM, cryo-TEM, and uorescence microscopy on imaging complicated biological nanostructures. The last one is about the study of protein assemblies using tapping-mode AFM. The rst research article in this series by Yanmei Wang et al. at Washington University in Missouri imaged the interactions of streptavidin molecules with hydrophobic fused-silica surfaces by single molecule uorescence imaging (pages 682687). Controlled surface adsorption of proteins is important for protein-based sensors and protein microarrays. It is necessary to identify and quantify mechanisms for protein adsorption to surfaces. Wang et al used internal reection uorescence microscopy to record the interaction of a single molecule, streptavidin-Cy3 and streptavidin-Alexa555, with silica surfaces in real time. They observed three different surface adsorptions which are deposition-process-associated irreversible adsorption, reversible adsorption caused by direct interaction, and non-adsorption. The results indicate that both regulating postdeposition protein-surface interactions and the deposition process should be taken into consideration in the study of protein-surface adsorption. In the next review article, Victoria Birkedal et al. from Aarhus University in Denmark investigated the complex self-assembled 3D DNA nanostructures by using three single molecule microscopy techniques, which are AFM, cryogenic TEM, and uorescence spectroscopy and microscopy (pages 688698). AFM imaged DNA self-assembled products (DNA sheets, opened DNA box, and closed DNA box) under liquid buffer conditions, and this method produced new information about the assembly and exibility of the DNA box. Cryo-TEM showed unique insights into the structural details of the 3D DNA box. FRET (Fluorescence Resonance Energy Transfer) spectroscopy provided valuable information on the lid opening process. The combination of several single molecule microscopy techniques generated more comprehensive information and provided a detailed understanding of the architecture, assembly, and functionality of biological nanostructures. The last article presented by Jayne Garno et al. from Louisiana State University is about the study of amyloid peptide assemblies using tapping-mode AFM (pages 699708). The aggregation of amyloid peptides into oligomeric and ber assemblies can be a precursor of Alzheimers disease. The continuous monitoring of such aggregation is important for a better understanding of the early accumulation of amyloid. They imaged the assembly stages of bril formation which include the formation of seed nanoparticles, protobrils, development of mature brils, densely branched network

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assemblies, and bril bundles. This article demonstrates that characterizations using AFM are necessary for tracking the evolution of biological nanostructures during time-dependent studies. From these original research and review articles in this special issue, it is evident that microscopy techniques have become one of the most important tools in the characterization of bio-nanomaterials and each technique has its unique advantages and disadvantages for imaging bio-, nano- or hybrid materials. It is therefore better for a researcher to know the principles and applications of a variety of microscopy techniques in this special issue and choose one or more of them for their specic needs. With the rapid development of microscopy techniques for the imaging of bio-nanomaterials, more and more functional bio-nanostructures will be created for practical applications in the near future. At the same time, more novel functional nanomaterials will be developed to serve as novel probes for researchers to develop new microscopy techniques for bio-imaging. As a result, these developments will no doubt advance the elds of nanobiotechnology, nanobiology and nanomedicine. ACKNOWLEDGMENTS I am very grateful to all the contributors in this special issue. I would also like to thank Prof. George Ruben who has given me encouragement and comments during the development of this special issue. Without their contributions, this special issue would be impossible.

CHUANBIN MAO (Guest Editor) Department of Chemistry & Biochemistry Stephenson Life Sciences Research Center University of Oklahoma, Norman

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Dobson J. 2006. Gene therapy progress and prospects: magnetic nanoparticle-based gene delivery. Gene Therapy 13:283287. Lee JH, Huh YM, Jun Y, Seo J, Jang J, Song HT, Kim S, Cho EJ, Yoon HG, Suh JS, Cheon J. 2007. Articially engineered magnetic nanoparticles for ultra-sensitive molecular imaging. Nature Medicine 13:9599. Ma N, Sargent EH, Kelley SO. 2008. Biotemplated nanostructures: directed assembly of electronic and optical materials using nanoscale complementarity. Journal of Materials Chemistry 18:954964. Medintz IL, Uyeda HT, Goldman ER, Mattoussi H. 2005. Quantum dot bioconjugates for imaging, labelling and sensing. Nature Materials 4:435446. Nakanishi M, Inoh Y, Kitamoto D, Furuno T. 2009. Nano vectors with a biosurfactant for gene transfection and drug delivery. Journal of Drug Delivery Science and Technology 19:165169. Portney NG, Ozkan M. 2006. Nano-oncology: drug delivery, imaging, and sensing. Analytical and Bioanalytical Chemistry 384:620630. Sotiropoulou S, Sierra-Sastre Y, Mark SS, Batt CA. 2008. Biotemplated nanostructured materials. Chemistry of Materials 20:821834. van Bommel KJC, Friggeri A, Shinkai S. 2003. Organic templates for the generation of inorganic materials. Angewandte ChemieInternational Edition 42:980999. Xie W, Qiu PH, Mao CB. 2011. Bio-imaging, detection and analysis by using nanostructures as SERS substrates. Journal of Materials Chemistry 21:51905202.

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