Bioimaging at the Nanoscale: Protein Induced Conformational Changes in DNA Arrays
M. Selim Ünlü
Boston University
Abstract:
A novel application of optical interference to fluorescence microscopy promises nanometer resolution in biological imaging. We have developed spectral self-interference fluorescent microscopy (SSFM) that maps the spectral oscillations emitted by fluorophores located above a reflecting surface into a precise position determination. In contrast to earlier fluorescence interference microscopy techniques that rely on intensity variation of total emission, SSFM utilizes the spectral information and provides nm accuracy with a single measurement. Using monolayers of proteins as well as single and double stranded DNA we have demonstrated sub nanometer axial height determination for thin layers of fluorophores. Using SSFM, we have estimated the shape of coiled single-stranded DNA, the average tilt of double-stranded DNA of different lengths, and the amount of hybridization [1]. The determination of DNA conformations on surfaces and hybridization behavior provide information required to move DNA interfacial applications forward and thus impact emerging clinical and biotechnological fields. Recently, we have also applied SSFM to study the conformational changes of polymers [2] and DNA-protein complexes. [3]
REFERENCES
[1] L. Moiseev, et al., "DNA Conformation on Surfaces Measured by Fluorescence Self-Interference," PNAS, Vol. 103, pp. 2623–2628, (2006)
[2] A. Yalcin et al., “Direct Observation of Conformation of a Polymeric Coating with Implications in Microarray Applications,” Anal. Chem., Vol. 81, pp. 625-630, (2009)
[3] P. S. Spuhler, J. Knezevic, A. Yalcin, Q. Bao, E. Pringsheim, P. Dröge, U. Rant, and M. S. Ünlü, "Platform for in situ real-time measurement of protein-induced conformational changes of DNA ," PNAS Vol. 107 (4), pp. 1397-1401 (2010)
Microbial Forensics: Microanalysis of the 2001 Anthrax Letter Attacks
Joseph R. Michael and Paul G. Kotula
Sandia National Laboratories
Abstract:
The anthrax letter attacks of 2001, which immediately followed the attack on the World Trade Center, killed 5 people and sickened 17 others, caused disruptions in the mail system, temporarily shut down congress and required extensive clean up of contaminated facilities. The anthrax letter attacks of 2001 presented new and unique challenges to the agencies involved in the investigation and has resulted in the development of a new science called “Microbial forensics”. One important aspect of the investigation was to determine any unique attributes of the attack materials. Electron microscopy and other microanalytical techniques were employed to provide information concerning the true nature of the attack materials. This talk will first review the anthrax attacks and then will discuss how a laboratory known for materials science got involved in the investigation. The electron microscopy performed at Sandia National Laboratories in support of the investigation will be discussed in detail as will the experimental results from the Bacillus anthracis spores contained in the Leahy, Daschle and New York Post letter materials.
Targeted Delivery of Multicomponent Cargos to Cancer via Nanoporous Particle-Supported Lipid Bilayers
Carlee Ashley
Sandia National Laboratories, Livermore
Abstract:
Targeted delivery of drugs encapsulated within nanocarriers can potentially ameliorate a number of problems exhibited by conventional ‘free’ drugs, including poor solubility, limited stability, rapid clearing, and, in particular, lack of selectivity, which results in non-specific toxicity to normal cells and prevents the dose escalation necessary to eradicate diseased cells. We have, therefore, developed porous nanoparticle-supported lipid bilayers (protocells), a new class of targeted nanocarrier that synergistically combines properties of both liposomes and nanoporous particles to simultaneously address multiple challenges associated with targeted delivery, including specificity, stability, and a high capacity for chemically disparate cargos. Protocells modified with a targeting peptide that binds to human hepatocellular carcinoma (HCC) exhibit a 10,000-fold greater affinity for HCC than for hepatocytes, endothelial cells, and immune cells. Furthermore, protocells can be loaded with multicomponent cargos, including combinations of therapeutic (drugs, siRNA, plasmids, and toxins) and diagnostic (quantum dots and magnetic particles) agents and modified to promote endosomal escape and nuclear accumulation of selected cargos. Due to the enormous capacity of the high-surface-area nanoporous core combined with the enhanced targeting efficacy enabled by the protocell’s fluid supported lipid bilayer, a single protocell loaded with a drug cocktail can kill a drug-resistant HCC cell, representing a 106-fold improvement over comparable liposomes.
Chemical Imaging for Cancer Pathology
Rohit Bhargava
University of Illinois
Abstract:
Chemical imaging is an emerging modality that can provide molecular information without dyes, probes or human interpretation. In one implementation of chemical imaging, spectroscopy is used to measure both intrinsic molecular composition and structure of man-made and natural materials. Computer algorithms then convert the rich data into information. Here, we first describe the development of mid-infrared spectroscopic imaging instrumentation, associated analytical methods and applications of this new technology. We especially focus on an application that seeks to provide input to the research and clinical efforts in the analysis of human tissues for cancer. Three levels of applications will be discussed for prostate and breast tissue - for the researcher, for clinical application and for predictive medicine. The information can be used as stand-alone diagnostic information or as an adjunct to help pathology laboratories make rapid and efficient decisions. The molecular underpinnings of the spectroscopic information are explored. Next, we describe a new paradigm in making ultrasensitive surface enhanced Raman probes that can result in substantial molecular information to be easily added to biomedical studies or clinical assays. The development and potential use of these probes for predictive medicine will be discussed in the context of label-free imaging. Finally, we describe how a parallel effort in fundamental theory and latest advances in instrumentation can lead to opening of new scientific avenues.
Applications of High Resolution FIB Nanotomography for Materials and Life Sciences
Jason Huang
Carl Zeiss NTS, LLC.
Abstract:
3-dimensional (3D) imaging capability at the nanoscale has emerged as an important and powerful characterization technique in a wide variety of applications ranging from nanomaterials analysis in materials science to biological imaging in life science. Existing 3D imaging techniques, such as X-ray tomography, TEM tomography and Atom probe tomography all have their advantages and disadvantages when it comes to characterization at the nanoscale. While X-ray tomography can investigate relatively large (100 µm and more in lateral dimension) structures in a non-destructive way, it lacks the resolution to define nanoscale features. Both TEM and Atom probe tomographic techniques require intensive sample preparation, and the size limits imposed on the specimen exclude many interesting scientific topics. For example, a biological cell typically ranges from a few microns to tens of microns in diameter, which are beyond the penetration depth of a typical high energy beam in TEM (80-300 keV). Nanotomography based on serial FIB-SEM imaging has therefore become a viable imaging solution to fill the gap between the resolution and the specimen sizes.
FIB nanotomography involves fully automated, concerted operations of the milling ion beam and the imaging e-beam. While this technique has been proposed for almost a decade, there are still challenges to fully utilize it as a mainstream tomography technique. For example, a medium size biological cell, or a metallurgical structure, on the order of 40-60 µm in diameter, could routinely demand days of unattended serial milling and imaging to scan through the volume of interest. Drift of specimen position, focus and stigmation could be potential issues for such long runs. And, even with the large field of view they may require, the smallest features of interest could down to a few nanometers in size, such as the lipid bilayer in the cell membrane or a gas-permeable nanopores in a shale rock.
In this paper, several application examples from life science and materials science will be presented. Energy Selective Backscatter imaging will be discussed as a unique solution for life science applications. Simultaneously acquisition of multiple signals from materials science samples will also be discussed.
Nanobioscience In Life Sciences And Medicine: Emerging Needs For High Precision Imaging and Metrology Roadmaps
Frederic Zenhausern
University of Arizona
Abstract:
Today, advances in engineering show the potential of nanotechnology in biomedical research but the translation to the clinics is still slow and hindered by the lack of high precision biometrology, scale-up developments, quality standards and regulatory considerations. The necessity for standard testing requirements has been reported in the areas of DNA microarrays and bio-imaging, but there are still important questions to be addressed within the perspective of complex multiple measurements. This paper will discuss the development of scanning probe techniques, DNA microarrays and microfluidic devices in life sciences and medicine towards their clinical assessments. However, the promising commercialization of nanomedicine products and their clinical adoption will rely on well-characterized standard measurements for which future technology roadmaps will be necessary.
Atmospheric Scanning Electron Microscope: Integrating Optical and Electron Microscopy for the Observation of Biological Systems
Donna Guarrera
JEOL USA, Inc.
Abstract:
D. Guarrera; JEOL USA, Inc.
H. Nishiyama, M. Suga, K. Teramoto; JEOL Ltd.
C. Sato; National Institute of Advanced Industrial Science and Technology
Both optical microscopy and electron microscopy are powerful tools in a scientist’s arsenal for studying biological systems. As you consider any technology there are advantages and disadvantages. In optical microscopy, with the exception of the new ‘super resolution’ light microscopy techniques such as PALM and STORM, image resolution is limited due to the diffraction limit of light (~100nm). Electron microscopy enables high resolution imaging at the nanometer scale; however, the sample must be observed in a vacuum and therefore can require extensive sample preparation. This sample preparation can also affect the morphology of the sample that you are trying to study.
To take advantage of both technologies and overcome some of the limitations with sample preparation in scanning electron microscopy, an instrument has been developed which couples both techniques. This instrument integrates a scanning electron microscope (SEM) with a wide field optical microscope. The key to the design of this instrument is that the sample is always open to atmospheric temperature and pressure. Lengthy dehydration steps or cryo preparation can be avoided with biological systems. The optical microscope and SEM are situated on the same axis to enable direct correlation between the images. Since the sample is always open to the atmosphere, this instrument is well suited to the observation of dynamic processes or experiments.
This instrument will be described in more detail. Examples in cell biology and biological systems will be shared as well as a few unique materials applications. The examples will highlight the open configuration of the instrument, speed of sample preparation, for high throughput imaging at resolutions greater than typical wide field optical microscopy.
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