Since its introduction, the JEOL ARM series with spherical aberration correction has become the leading atomic resolution microscope used in advanced research. In response to the increased need for high-resolution imaging of materials containing light elements and of specimens susceptible to electron-beam irradiation, JEOL developed the NEOARM atomic resolution analytical electron microscope. The NEOARM enables atomic-resolution imaging at accelerating voltages ranging from 30 kV to 200 kV.
The NEOARM features a unique cold field emission gun (Cold-FEG) as well as a next generation Cs corrector (ASCOR) that compensates for higher order aberrations. An automated aberration correction system incorporates JEOL’s new aberration correction algorithm for fast and precise aberration correction. This system enables higher-throughput atomic-resolution imaging, even at low accelerating voltages. Furthermore, a new STEM detector that provides enhanced contrast of light elements is incorporated as a standard unit. Contrast enhancement of light elements is achieved by a new STEM imaging technique, e-ABF: enhanced ABF, facilitating observation of light-element materials. Cs corrector 'ASCOR' (Advanced STEM Corrector) The ASCOR incorporated in the NEOARM suppresses the six-fold astigmatism that limits resolution after Cs correction. This combination of ASCOR with a Cold-FEG achieves higher resolution than ever, 0.071nm in HAADF-STEM, with unprecedented resolution at low voltages.
Automated aberration correction software JEOL COSMO™ (Corrector System Module) JEOL COSMO™ adopts a new aberration correction algorithm SRAM: Segmented Ronchigram Auto-correlation function Matrix. No special sample is required for aberration correction, leading to high-precision and quick correction of higher order aberrations up to 5th order. This system enables fast processing compared to the conventional correction algorithm, and automates this operation, thus eliminating a complicated correction workflow.
These features enable higher-throughput atomic-resolution imaging. New ABF (Annular Bright Field) detector system The ABF detector is widely used as a technique suitable for high-resolution imaging of light elements.
NEOARM supports enhanced contrast of light elements by a newly-designed ABF imaging technique, e-ABF:enhanced ABF. This capability facilitates atomic-level structure observation of materials containing light elements. Perfect sight detector The perfect sight detector, integrated into NEOARM, uses hybrid scintillators.
This detector enables acquisition of high-contrast and quantitative STEM images over the complete range of accelerating voltage values. Viewing Camera system The Viewing Camera system, intended to be used for remote operation, is an image observation system that uses dual cameras.
This system allows for remote operation in a room separated from the microscope room and also enables the smart exterior designing of the NEOARM.
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Received 4th December 2012, Accepted 11th February 2013 First published on 20th February 2013 Determining the structure of macromolecular samples is vital for understanding and adapting their function. ( ) is widely used to achieve this, but, owing to the weak electron scattering cross-section of carbon, TEM images of macromolecular samples are generally low contrast and low resolution.
Here we implement a fast and practically simple routine to achieve high-contrast imaging of macromolecular samples using exit wave reconstruction (EWR), revealing a new level of structural detail. This is only possible using ultra-low contrast supports such as the graphene oxide (GO) used here and as such represents a novel application of these substrates. We apply EWR on GO membranes to study self-assembled block copolymer structures, distinguishing not only the general morphology or, but also evidence for the substructure ( i.e. The polymer chains) which gives insight into their formation mechanisms and functional properties. Introduction Characterizing the structure of organic macromolecular materials at the sub-nanometer scale is a major challenge that is critical to advancing our understanding of biological processes and capitalizing on the remarkable opportunities afforded by of amphiphilic. A common approach is to use ( ), often with heavy metal staining. Although staining can be used to highlight functionality, it is often required merely to give sufficient contrast.
Macromolecular samples are typically weak phase objects, i.e. Due to their low average atomic number they scatter the wave function of the electron beam changing its phase with little effect on the amplitude. The in-focus depends mainly on the intensity, i.e. On the amplitude, of the wave and hence the observed contrast for such samples is weak. Imaging them on conventional amorphous carbon supports is thus problematic, as contrast from the support usually dominates that from the object under investigation.
Recent developments in ultra-low contrast supports have now made it possible to routinely image organic macromolecular samples without staining. For example, and its oxidised derivative graphene oxide (GO) are strong and almost transparent under the electron beam. For macromolecular samples, we have shown that using GO as a support it is possible to unambiguously resolve the morphology of assemblies such as polymersomes without staining, whilst Pantelic et al. Have demonstrated analysis of unstained vitrified biological on GO with high signal to noise ratio (SNR). Pantelic et al. Have also shown that due to their greater crystallinity and lower scattering cross-section supports outperform even GO supports, and demonstrated their application through imaging the periodic structure of the tobacco mosaic virus. As that report exemplified, these supports decrease the noise (contrast in the background support) and thus increase the signal (scattering from the object) to noise ratio.
Coupled with advances in aberration corrected (ac-TEM), which now routinely enables sub-Angstrom resolution imaging of crystalline inorganic materials, this should enable structural determination of macromolecular samples with unprecedented resolution. However, to get sufficient contrast it is typically necessary to image under defocus. This acts like Fresnel diffraction, interfering the scattered and unscattered waves to make phase contrast visible in the bright field TEM image, but in the process the resolution is reduced. This effect is captured in the contrast transfer function (CTF) which defines the information transferred from the object to the image and depends on the microscope and imaging parameters.
The CTF oscillates from positive to negative as a function of spatial frequency, with the position of the oscillations dependent on focus. As the defocus is increased, the point resolution (defined as the first zero in the CTF) decreases. As a result, although the general morphology of the sample at the nanometre scale can be deduced by imaging under defocus, the higher spatial resolution information is generally lost. For a single image, contrast is typically only achieved by sacrificing resolution.
Approaches have been developed to overcome this problem. The use of a Zernike phase plate enables pure phase imaging, dramatically increasing image contrast for weak phase objects. Application of Zernike phase plates to requires advances in instrumentation and additional hardware that have limited its uptake, but it has shown great promise for structural determination of biological specimens at subnanometer resolutions.
To a limited extent, the CTF can be corrected for a single image, as is routinely applied for single particle analysis. However, this cannot recover the missing information from the length scales at which the CTF is zero and for noisy images CTF correction can even amplify the noise. A more sophisticated approach involves focal pair merging whereby information from two images, one near focus and one at large defocus, are combined to reconstruct a higher-contrast and higher-resolution image.
In essence, this is similar to exit wave reconstruction (EWR), a technique developed to maximise the resolution of high resolution (HR)-TEM of inorganic materials. EWR works by acquiring a series of images whilst incrementally changing the focus (a focal series) and then recombining the information from each image to reconstruct the wave function as it left the object under investigation. In the process the image is corrected for aberrations, increasing the resolution that can be achieved. However, the crucial point here is that both the amplitude and phase of the wave function are recovered. EWR has previously been used for recovering phase information, for example as ‘inline electron ’ for studying static magnetic and electric potentials, but has not been demonstrated for macromolecular samples such as the ones investigated here. Here we show that through advances in support material and digital image acquisition and processing, it is now possible to use a fast and practically simple methodology to achieve high-contrast increased resolution imaging of macromolecular samples through exit wave reconstruction. The technique can be implemented without additional hardware on any conventional computer controlled and results in significant improvements in image resolution and contrast for macromolecular objects.
The information transferred is increased through the use of low-voltage. It is important to note that these advances are only possible through the use of ultra-low contrast supports, such as the graphene oxide sample support used here.
We demonstrate the application of exit wave reconstruction through the analysis of two macromolecular structures formed by of, giving information beyond the basic which is normally resolved. Imaging of an archetypal poly(acrylic acid)- b-poly(styrene) polymersome shows evidence for individual polymer chains that form a corona around it, while analysis of a poly( L-lactide) containing cylindrical micelle provides an indication of its formation mechanism. Results and discussion The effects of defocus on image contrast and resolution shows images of a polymersome from the solution assembly of the poly(acrylic acid) 11- b-poly(styrene) 250 (at 0.65 mg mL −1) on a single layer GO support. The images were taken on a JEOL ARM200F operated at 80 kV with spherical aberration corrected to.
1 The effect of defocus on imaging. Images of a polyacrylic acid- b-polystyrene polymersome at (a) −955 nm focus, (b) −481 nm focus and (c) 6 nm focus. Inset are sections from their respective fast Fourier transforms. The normalized power spectral density (black) and square of the predicted contrast transfer function (red) for (a)–(c) are given in (d)–(f) respectively.
The was synthesized by the reversible addition fragmentation chain transfer (RAFT), deprotected and then assembled using the switch method as described previously. As shown in these images, and as expected from many previous investigations, this system self-assembles into well-defined polymersomes.
The simplicity of the polymersome structure (a hollow sphere), its nanoscale dimensions and robustness of the morphology under irradiation in the electron beam make it an ideal test object. It also represents an important class of materials: self-assembled block copolymer systems in solution have applications in medicinal chemistry and catalysis. In both cases the primary function of the is to provide a well-defined environment for the encapsulation of an active species ( e.g., imaging agents, reactants, etc.).
The structure of the particles is thus particularly important as it defines their functional properties. The effect of defocus on image resolution and contrast can readily be seen in.
As the defocus is decreased from the most obvious change is the decrease in contrast. The characteristic structure of the polymersome, which has a radius of ∼30 nm with a roughly spherical hollow core of radius ∼10 nm, is readily apparent in the under focus images.
But in the near focus image, the lack of contrast makes it difficult to accurately discern the interface between the polymersome and the support or the boundary of its hollow core. Inset in the images are sections of their fast Fourier transforms (FFTs) which convey the information content in the image as a function of reciprocal length and hence give a clear visualization of the resolution in the image (detail further from the centre in the FFT corresponds to higher spatial resolution in the image). The FFTs have an essentially circular symmetry, as expected for this amorphous material, except for the hexagon of spots due to the single layer of graphene oxide. These GO spots give a useful inbuilt calibration for the images (corresponding to 4.69 nm −1) and it is remarkable that they are seen in the FFT despite no evident -like lattice in the real space image. Plots of the power spectral density are given, with direct comparison to the square of the predicted phase contrast transfer function.
The CTF 2 shows a series of minima, often called Thon rings, which are also evident as dark circles in the FFT. The inner Thon ring defines the point resolution of the image. From the defocus decreases (from −955 nm to −481 nm to 6 nm) with a corresponding increase in the point resolution (from 0.5 nm −1 to 0.7 nm −1 to around 4 nm −1) but with a concomitant decrease in contrast evident from the drop in magnitude of the CTF 2. Note that this dependence of resolution and contrast on focus is a fundamental limit. Although advances in instrumentation reduce the aberrations and hence improve the CTF, the oscillations in the CTF are an inevitable consequence of defocus. Hence for individual images there is a compromise between contrast and resolution.
Rachel K Ore…
High resolution and high contrast through exit wave reconstruction The solution to this problem, as implemented in exit-wave reconstruction, is to acquire a focal series. An algorithm is then used to combine the images to reconstruct the phase of the wave function as it leaves the object under investigation, combining the information from every image. Shows the result of such a reconstruction as operated on a focal series containing 40 images, with a nominal focal step between images of 26 nm. The images in are examples extracted from this focal series (the focal series is given in ESI, S1). The EWR algorithm used here is based on the focal and tilt series reconstruction (FTSR) algorithm developed by Kirkland and co-workers. 3 Comparison of conventional imaging and EWR phase imaging. (a) Near focus, and (b) EWR phase image, for the region of polymersome marked by the dashed box in.
Three levels of hierarchy exist in the polymersomes; monomers, which are reacted together to form the polymers, which are assembled to form the. In only the highest level structure is observable ( i.e.
The size and shape of the nanostructure) whereas the EWR images give evidence for the structure at the next level down of information ( i.e. The individual polymer chains) and consequently the method represents a significant step forward in imaging of self-assembled soft materials. Practical considerations for EWR The robustness of the reconstruction can readily be tested. Reconstructions using non-overlapping subsets of the focal series give the same result (ESI S3), as do different EWR algorithms (ESI S5). A test for consistency can be made by using the reconstructed exit wave to simulate the expected image as a function of focus and the results match the experimental images with a high degree of fidelity (ESI S6). EWR does not require an aberration corrected.
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On a conventional TEM operated under high resolution bright field imaging conditions EWR has an even greater effect, correcting some of the microscope aberrations and hence increasing the resolution further as well as restoring the high contrast of the phase image. The high magnification of a polyacrylic acid- b-polystyrene polymersome (as in ) on graphene oxide shown in was taken on a Jeol 2100 with LaB 6 filament operated at 200 kV. Shows a near focus image (−12 nm defocus), one of a focal series of 20 images with focal step of 34 nm. The amplitude and phase images from EWR of the focal series are given in respectively. There are clear similarities in the fine structure of the polymersome resolved in the phase image here compared to that obtained from the aberration corrected in, although it is also readily apparent that the low voltage aberration corrected images result in greater information transfer to the EWR images. 4 EWR of a polymersome on GO using a conventional TEM at 200 kV. (a) Near focus image (focus −12 nm), (b) EWR amplitude image and (c) EWR phase image.
Advances in digital acquisition and processing now make EWR a fast and relatively simple technique. For this work, acquisition of the focal series has been automated by a script written for Gatan's Digital (DMG) software, the most common TEM image acquisition software, and takes around a minute for a typical 20 image sequence. During this acquisition the sample exposure time need only be a few seconds, the majority of the time is the latency in taking the images from the camera. We have found that there is no need to go to large defocus; provided the SNR in the images is sufficient to align the images, focal steps of around 25 nm starting near focus give good results. We have increased the speed of the EWR algorithm and integrated a version into DMG so that a basic reconstruction can be performed in the microscope software in a few minutes immediately after image acquisition (ESI S5). This rapid acquisition and reconstruction allows EWR analysis to be used as a routine technique. The full reconstruction, including additional refinements of the image series alignment and determination of focal drift which are not accounted for in the rapid EWR algorithm, has also been accelerated by utilising general-purpose computing on graphics processing units so that it takes between 1 and 15 seconds for a full FTSR reconstruction depending on the number of images and pixels.
The EWR algorithms require some prior information from the specific microscope being used (for example the modulation transfer function of the camera, the aberrations and the minimum focal increment), but this information need only be acquired once and subsequently acquiring the focal series requires no more expertise than conventional imaging. Most reconstruction algorithms are valid only in the linear imaging regime ( i.e. Thin samples with limited scattering).
However, reconstructions with more complete algorithms such as the maximum likelihood method (MAL), which are valid even for significant non-linear contributions, provide similar results (ESI S5). This indicates that EWR is valid for these comparatively large macromolecular samples, however, for these thicker amorphous samples interpreting the contrast is complicated. In a similar way, the sample support also plays a critical role. The importance of an ultra-low contrast support In recreating the phase image through EWR, the contrast and resolution at which the support is imaged is also increased. As a result, in order to resolve a weak phase object, EWR places strict requirements on the thickness and structure of the support. Compares images of a poly(lactide) containing cylindrical micelle, which was prepared as previously reported, on an amorphous carbon support and a GO support. In the near focus image of the cylindrical micelle on amorphous carbon, the cylindrical micelle shows little contrast with respect to the background, indicating a low SNR as expected.
This is one of a focal series of 20 images with 26 nm focal step. The amplitude image from the EWR of this series, shows similarly low contrast to the near focus image. The structure and contrast in the amorphous carbon support dominates the phase image, making it difficult to discern where the cylindrical micelle is and almost impossible to separate the structure of the micelle from that of the support. Poly(acrylic acid) 11- b-poly(styrene) 250 (PS 250- b-PAA 11) polymersomes. Detailed synthesis and methods were as previously reported.
(PS 250- b-PAA 11) was synthesized by reversible addition chain transfer (RAFT) of followed by chain extension with. Trifluoro acetic acid was used to deprotect the tert-butyl groups resulting in the PS 250- b-PAA 11.
PS 250- b-PAA 11 was then assembled into polymersomes by the switch method, using and as the common and selective respectively. The solution was then extensively dialyzed against using 3.5 kDa molecular weight cut off tubing.
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