Showing posts with label microscope. Show all posts
Showing posts with label microscope. Show all posts

Wednesday, June 29, 2011

Researchers develop biological circuit components, new microscope technique for measuring them


Penn researchers develop biological circuit components, new microscope technique for measuring them

 


Rendering of protein assemblies under an atomic force microscope. Credit: Reprinted with permission from "Direct Probe of Molecular Polarization in De Novo Protein-Electrode Interfaces," Kendra Kathan-Galipeau, Sanjini Nanayakkara, Paul A. OBrian, Maxim Nikiforov, Bohdana M. Discher, Dawn A. Bonnell, ACS Nano, Copyright 2011 American Chemical Society


Electrical engineers have long been toying with the idea of designing biological molecules that can be directly integrated into electronic circuits. University of Pennsylvania researchers have developed a way to form these structures so they can operate in open-air environments, and, more important, have developed a new microscope technique that can measure the electrical properties of these and similar devices.


The research was conducted by Dawn Bonnell, Trustee Chair Professor and director of the Nano/Bio Center, graduate students Kendra Kathan-Galipeau and Maxim Nikiforov and postdoctoral fellow Sanjini Nanayakkara, all of the Department of Materials Science and Engineering in Penn?s School of Engineering and Applied Science. They collaborated with assistant professor Bohdana Discher of the Department of Biophysics and Biochemistry at Penn?s Perelman School of Medicine and Paul A. O?Brien, a graduate student in Penn's Biotechnology Masters Program.


Their work was published in the journal. The development involves artificial proteins, bundles of peptide helices with a photoactive molecule inside. These proteins are arranged on electrodes, which are common feature of circuits that transmit electrical charges between metallic and non-metallic elements. When light is shined on the proteins, they convert photons into electrons and pass them to the electrode.


?It's a similar mechanism to what happens when plants absorb light, except in that case the electron is used for some chemistry that creates energy for the plant,? Bonnell said. ?In this case, we want to use the electron in electrical circuits.? 


Similar peptide assemblies had been studied in solution before by several groups and had been tested to show that they indeed react to light. But there was no way to quantify their ambient electrical properties, particularly capacitance, the amount of electrical charge the assembly holds.


?It?s necessary to understand these kinds of properties in the molecules in order to make devices out of them. We've been studying silicon for 40 years, so we know what happens to electrons there,? Bonnell said.  ?We didn?t know what happens to electrons on dry electrodes with these proteins; we didn't even know if they would remain photoactive when attached to an electrode.? 


Designing circuits and devices with silicon is inherently easier than with proteins. The electrical properties of a large chunk of a single element can be measured and then scaled down, but complex molecules like these proteins cannot be scaled up. Diagnostic systems that could measure their properties with nanometer sensitivity simply did not exist.


The researchers therefore needed to invent both a new way of a measuring these properties and a controlled way of making the photovoltaic proteins that would resemble how they might eventually be incorporated into devices in open-air, everyday environments, rather than swimming in a chemical solution.  


To solve the first problem, the team developed a new kind of atomic force microscope technique, known as torsional resonance nanoimpedance microscopy. Atomic force microscopes operate by bringing an extremely narrow silicon tip very close to a surface and measuring how the tip reacts, providing a spatial sensitivity of a few nanometers down to individual atoms.  


?What we've done in our version is to use a metallic tip and put an oscillating electric field on it. By seeing how electrons react to the field, we?re able to measure more complex interactions and more complex properties, such as capacitance,? Bonnell said.


Bohdana Discher?s group designed the self-assembling proteins much as they had done before but took the additional step of stamping them onto sheets of graphite electrodes.  This manufacturing principle and the ability to measure the resulting devices could have a variety of applications.  


?Photovoltaics ? solar cells ? are perhaps the easiest to imagine, but where this work is going in the shorter term is biochemical sensors,? Bonnell said. 


Instead of reacting to photons, proteins could be designed to produce a charge when in the presence of a certain toxins, either changing color or acting as a circuit element in a human-scale gadget.  

Tuesday, February 1, 2011

New microscope reveals ultrastructure of cells


New microscope reveals ultrastructure of cells

New microscope reveals ultrastructure of cells

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This is a slice through the nucleus of a mouse adenocarcinoma cell showing the nucleolus and the membrane channels running across the nucleus; taken by X-ray nanotomography. Credit: HZB


German researchers at Helmholtz-Zentrum Berlin have developed a new X-ray nanotomography microscope. Using their new system, they can reveal the structures on the smallest components of mammalian cells in three dimensions.



For the first time, there is no need to chemically fix, stain or cut cells in order to study them. Instead, whole living cells are fast-frozen and studied in their natural environment. The new method delivers an immediate 3-D image, thereby closing a gap between conventional microscopic techniques.


The new microscope delivers a high-resolution 3-D image of the entire cell in one step. This is an advantage over electron microscopy, in which a 3-D image is assembled out of many thin sections. This can take up to weeks for just one cell. Also, the cell need not be labelled with dyes, unlike in fluorescence microscopy, where only the labelled structures become visible. The new X-ray microscope instead exploits the natural contrast between organic material and water to form an image of all cell structures. Dr. Gerd Schneider and his microscopy team at the Institute for Soft Matter and Functional Materials have published their development in Nature Methods.


New microscope reveals ultrastructure of cells
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This is a conventional TEM image of a stained thin section. Credit: HZB

With the high resolution achieved by their microscope, the researchers, in cooperation with colleagues of the National Cancer Institute in the USA, have reconstructed mouse adenocarcinoma cells in three dimensions. The smallest of details were visible: the double membrane of the cell nucleus, nuclear pores in the nuclear envelope, membrane channels in the nucleus, numerous inva­ginations of the inner mitochondrial membrane and inclusions in cell organelles such as lysosomes. Such insights will be crucial for shedding light on inner-cellular processes: such as how viruses or nanoparticles penetrate into cells or into the nucleus, for example.

This is the first time the so-called ultrastructure of cells has been imaged with X-rays to such precision, down to 30 nanometres. Ten nanometres are about one ten-thousandth of the width of a human hair. Ultrastructure is the detailed structure of a biological specimen that is too small to be seen with an optical microscope.


Researchers achieved this high 3-D resolution by illuminating the minute structures of the frozen-hydrated object with partially coherent light. This light is generated by BESSY II, the synchrotron source at HZB. Partial coherence is the property of two waves whose relative phase undergoes random fluctuations which are not, however, sufficient to make the wave completely incoherent. Illumination with partial coherent light generates significantly higher contrast for small object details compared to incoherent illumination. Combining this approach with a high-resolution lens, the researchers were able to visualize the ultrastructures of cells at hitherto unattained contrast.


The new X-ray microscope also allows for more space around the sample, which leads to a better spatial view. This space has always been greatly limited by the setup for the sample illumination. The required monochromatic X-ray light was created using a radial grid and then, from this light, a diaphragm would select the desired range of wavelengths. The diaphragm had to be placed so close to the sample that there was almost no space to turn the sample around. The researchers modified this setup: Monochromatic light is collected by a new type of condenser which directly illuminates the object, and the diaphragm is no longer needed. This allows the sample to be turned by up to 158 degrees and observed in three dimensions. These developments provide a new tool in structural biology for the better understanding of the cell structure.