Combination of thresholding and fitting methods for measuring nanoparticle sizes and size distributions in (S)TEM

The practical need for a simple and reliable tool for routine size analysis of nanoparticles with diameters down to a few nm embedded in a polymer matrix motivated the development of a new approach. The idea underlying the method proposed in this work is to combine intensity thresholding and contrast fitting procedures in the same software for particle recognition and measurements of sizes and size distributions of nanoparticles in transmission and scanning transmission electron microscopy images. Particle recognition in images is performed in an interactive process of manual setting the numerical threshold level after image preprocessing. We show that fitting the calculated gray level distribution to the real images is able to provide a maximum accuracy in measurements of the particle diameters in contrast to thresholding approaches. The fitting procedure is applied in the vicinity of nanoparticle images with the mass‐thickness, diffraction, and chemical contrast. The grayscale function associated to the nanoparticle thickness is described using polynomial with degree ? 2 and undetermined coefficients. The program for particle detection and size measurement— An alyzer of Na noparticles ( AnNa )—has been written and is described here. It was successfully tested on systems containing Ag nanoparticles grown and stabilized in aqueous solutions of different polymers for biomedical use and is available from the authors.     

Aberration‐corrected STEM for atomic‐resolution imaging and analysis

Aberration‐corrected scanning transmission electron microscopes are able to form electron beams smaller than 100 pm, which is about half the size of an average atom. Probing materials with such beams leads to atomic‐resolution images, electron energy loss and energy‐dispersive X‐ray spectra obtained from single atomic columns and even single atoms, and atomic‐resolution elemental maps. We review briefly how such electron beams came about, and show examples of applications. We also summarize recent developments that are propelling aberration‐corrected scanning transmission electron microscopes in new directions, such as complete control of geometric aberration up to fifth order, and ultra‐high‐energy resolution EELS that is allowing vibrational spectroscopy to be carried out in the electron microscope.     

Mechanisms of radiation damage in beam‐sensitive specimens,for TEM accelerating voltages between 10 and 300 kV

Ionization damage (radiolysis) and knock‐on displacement are compared in terms of scattering cross section and stopping power, for thin organic specimens exposed to the electrons in a TEM. Based on stopping power, which includes secondary processes, radiolysis is found to be predominant for all incident energies (10–300 keV), even in materials containing hydrogen. For conducting inorganic specimens, knock‐on displacement is the only damage mechanism but an electron dose exceeding 1000 C cm^?2 is usually required. Ways of experimentally determining the damage mechanism (with a view to minimizing damage) are discussed. Microsc. Res. Tech., 2012. © 2012 Wiley Periodicals, Inc.     

Combining FIB milling and conventional Argon ion milling techniques to prepare high‐quality site‐specific TEM samples for quantitative EELS analysis of oxygen in molten iron

This paper reports a procedure to combine the focused ion beam micro‐sampling method with conventional Ar‐milling to prepare high‐quality site‐specific transmission electron microscopy cross‐section samples. The advantage is to enable chemical and structural evaluations of oxygen dissolved in a molten iron sample to be made after quenching and recovery from high‐pressure experiments in a laser‐heated diamond anvil cell. The evaluations were performed by using electron energy‐loss spectroscopy and high‐resolution transmission electron microscopy. The high signal to noise ratios of electron energy‐loss spectroscopy core‐loss spectra from the transmission electron microscopy thin foil, re‐thinned down to 40 nm in thickness by conventional Argon ion milling, provided us with oxygen quantitative analyses of the quenched molten iron phase. In addition, we could obtain lattice‐fringe images using high‐resolution transmission electron microscopy. The electron energy‐loss spectroscopy analysis of oxygen in Fe_0.94O has been carried out with a relative accuracy of 2%, using an analytical procedure proposed for foils thinner than 80 nm. Oxygen K‐edge energy‐loss near‐edge structure also allows us to identify the specific phase that results from quenching and its electronic structure by the technique of fingerprinting of the spectrum with reference spectra in the Fe‐O system.     

Atomic force microscope (AFM) combined with the ultramicrotome: a novel device for the serial section tomography and AFM/TEM complementary structural analysis of biological and polymer samples

A new device (NTEGRA Tomo) that is based on the integration of the scanning probe microscope (SPM) (NT‐MDT NTEGRA SPM) and the Ultramicrotome (Leica UC6NT) is presented. This integration enables the direct monitoring of a block face surface immediately following each sectioning cycle of ultramicrotome sectioning procedure. Consequently, this device can be applied for a serial section tomography of the wide range of biological and polymer materials. The automation of the sectioning/scanning cycle allows one to acquire up to 10 consecutive sectioned layer images per hour. It also permits to build a 3‐D nanotomography image reconstructed from several tens of layer images within one measurement session. The thickness of the layers can be varied from 20 to 2000 nm, and can be controlled directly by its interference colour in water. Additionally, the NTEGRA Tomo with its nanometer resolution is a valid instrument narrowing and highlighting an area of special interest within volume of the sample. For embedded biological objects the ultimate resolution of SPM mostly depends on the quality of macromolecular preservation of the biomaterial during sample preparation procedure. For most polymer materials it is comparable to transmission electron microscopy (TEM). The NTEGRA Tomo can routinely collect complementary AFM and TEM images. The block face of biological or polymer sample is investigated by AFM, whereas the last ultrathin section is analyzed with TEM after a staining procedure. Using the combination of both of these ultrastructural methods for the analysis of the same particular organelle or polymer constituent leads to a breakthrough in AFM/TEM image interpretation. Finally, new complementary aspects of the object's ultrastructure can be revealed.     

Descriptive studies on the tongue of two micro‐mammals inhabiting the Egyptian fauna; the Nile grass rat (Arvicanthis niloticus) and the Egyptian long‐eared hedgehog (Hemiechinus auritus)

The current study aimed to describe the anatomical features of the tongues of two micro‐mammals common in the Egyptian fauna; the Nile grass rat (Arvicathis niloticus), and the Egyptian long‐eared hedgehog (Hemiechinus auritus). The tongues of five adult individuals of each species were excised and processed histologically, histochemically, and morphometrically. Statistical analysis comparing the relative tongue length in both species showed that there was a significant difference, which may correlate with the difference in feeding preferences. Grossly, the Nile grass rat has a dorsal lingual prominence with bifurcated apex while, the long‐eared hedgehog has a median slight elevation with rounded apex. Numerous forms of mechanical and gustatory papillae are scattered along the lingual dorsal epithelium. The histochemical detection of keratin by Holland's trichrome stain showed an intense expression in the case of A. niloticus and mild expression in H. auritus. The framework of the tongue (entoglossum) is supported by either a core of cartilage in H. auritus or bone in A. niloticus which incorporated in the lingual root. The lingual glands also showed marked variation, the Nile grass rat exhibit dense populations of mucous‐secreting glands and lesser populations of serous‐secreting glands, the contrary is true in H. auritus. In conclusion, the micro and macro‐anatomical features of the tongues of both species showed adaptive changes to accommodate the feeding lifestyle. Such type of studies using mammals from different phylogenetic traits and almost have different feeding preferences provide answers to many research questions related to tongue evolution among mammalian vertebrates.     

The chemistry,mechanism and function of tricresyl phosphate (TCP) as an anti‐wear lubricant additive

The detailed understanding of the physical and reaction chemistry of engineering lubricants is key to new developments in the future. Here, we draw together the main chemical/engineering literature in the first systematic review of the standard anti‐wear additive used as an aviation lubricant, tricresyl phosphate (TCP), focusing on understanding the links between the surface chemistry, tribology and decomposition of TCP. While there is still debate concerning the mechanism of TCP, it is clear that it is activated by moisture, oxygen or oxidised metal surfaces. Its anti‐wear properties arise from the resulting formation of iron phosphate or polyphosphate on the contact surfaces. Evidence stresses the importance of chemistry at the boundary layer and a mechanism involving pre‐coordination of TCP at the surface, resulting in activation at the P‐centre and subsequent attack by residual H₂O or surface O²⁻. This perspective provides a potential baseline for the development of future phosphorus‐based high‐performance additives. Copyright © 2015 John Wiley & Sons, Ltd.     

Tillandsia stricta Sol (Bromeliaceae) leaves as monitors of airborne particulate matter—A comparative SEM methods evaluation: Unveiling an accurate and odd HP‐SEM method

Airborne particulate matter (PM) has been included among the most important air pollutants by governmental environment agencies and academy researchers. The use of terrestrial plants for monitoring PM has been widely accepted, particularly when it is coupled with SEM/EDS. Herein, Tillandsia stricta leaves were used as monitors of PM, focusing on a comparative evaluation of Environmental SEM (ESEM) and High‐Pressure SEM (HPSEM). In addition, specimens air‐dried at formaldehyde atmosphere (AD/FA) were introduced as an SEM procedure. Hydrated specimen observation by ESEM was the best way to get information from T. stricta leaves. If any artifacts were introduced by AD/FA, they were indiscernible from those caused by CPD. Leaf anatomy was always well preserved. PM density was determined on adaxial and abaxial leaf epidermis for each of the SEM proceedings. When compared with ESEM, particle extraction varied from 0 to 20% in air‐dried leaves while 23–78% of particles deposited on leaves surfaces were extracted by CPD procedures. ESEM was obviously the best choice over other methods but morphological artifacts increased in function of operation time while HPSEM operation time was without limit. AD/FA avoided the shrinkage observed in the air‐dried leaves and particle extraction was low when compared with CPD. Structural and particle density results suggest AD/FA as an important methodological approach to air pollution biomonitoring that can be widely used in all electron microscopy labs. Otherwise, previous PM assessments using terrestrial plants as biomonitors and performed by conventional SEM could have underestimated airborne particulate matter concentration.     

A new HPF specimen carrier adapter for the use of high‐pressure freezing with cryoscanning electron microscope: two applications: stearic acid organization in a hydroxypropyl methylcellulose matrix and mice myocardium

Cryogenic transmission electron microscopy of high‐pressure freezing (HPF) samples is a well‐established technique for the analysis of liquid containing specimens. This technique enables observation without removing water or other volatile components. The HPF technique is less used in scanning electron microscopy (SEM) due to the lack of a suitable HPF specimen carrier adapter. The traditional SEM cryotransfer system (PP3000T Quorum Laughton, East Sussex, UK; Alto Gatan, Pleasanton, CA, USA) usually uses nitrogen slush. Unfortunately, and unlike HPF, nitrogen slush produces water crystal artefacts. So, we propose a new HPF specimen carrier adapter for sample transfer from HPF system to cryogenic‐scanning electronic microscope (Cryo‐SEM). The new transfer system is validated using technical two applications, a stearic acid in hydroxypropyl methylcellulose solution and mice myocardium. Preservation of samples is suitable in both cases. Cryo‐SEM examination of HPF samples enables a good correlation between acid stearic liquid concentration and acid stearic occupation surface (only for homogeneous solution). For biological samples as myocardium, cytoplasmic structures of cardiomyocyte are easily recognized with adequate preservation of organelle contacts and inner cell organization. We expect this new HPF specimen carrier adapter would enable more SEM‐studies using HPF.     

Quantitative evaluation of boron neutron capture therapy (BNCT) drugs for boron delivery and retention at subcellular‐scale resolution in human glioblastoma cells with imaging secondary ion mass spectrometry (SIMS)

Boron neutron capture therapy (BNCT) of cancer depends on the selective delivery of a sufficient number of boron‐10 (¹⁰B) atoms to individual tumour cells. Cell killing results from the ¹⁰B (n, α)⁷Li neutron capture and fission reactions that occur if a sufficient number of ¹⁰B atoms are localized in the tumour cells. Intranuclear ¹⁰B localization enhances the efficiency of cell killing via damage to the DNA. The net cellular content of ¹⁰B atoms reflects both bound and free pools of boron in individual tumour cells. The assessment of these pools, delivered by a boron delivery agent, currently cannot be made at subcellular‐scale resolution by clinically applicable techniques such as positron emission tomography and magnetic resonance imaging. In this study, a secondary ion mass spectrometry based imaging instrument, a CAMECA IMS 3f ion microscope, capable of 500 nm spatial resolution was employed. Cryogenically prepared cultured human T98G glioblastoma cells were evaluated for boron uptake and retention of two delivery agents. The first, L‐p‐boronophenylalanine (BPA), has been used clinically for BNCT of high‐grade gliomas, recurrent tumours of the head and neck region and melanomas. The second, a boron analogue of an unnatural amino acid, 1‐amino‐3‐borono‐cyclopentanecarboxylic acid (cis‐ABCPC), has been studied in rodent glioma and melanoma models by quantification of boron in the nucleus and cytoplasm of individual tumour cells. The bound and free pools of boron were assessed by exposure of cells to boron‐free nutrient medium. Both BPA and cis‐ABCPC delivered almost 70% of the pool of boron in the free or loosely bound form to the nucleus and cytoplasm of human glioblastoma cells. This free pool of boron could be easily mobilized out of the cell and was in some sort of equilibrium with extracellular boron. In the case of BPA, the intracellular free pool of boron also was affected by the presence of phenylalanine in the nutrient medium. This suggests that it might be advantageous if patients were placed on a low phenylalanine diet prior to the initiation of BNCT. Since BPA currently is used clinically for BNCT, our observations may have direct relevance to future clinical studies utilizing this agent and provides support for individualized treatment planning regimens rather than the use of fixed BPA infusion protocols.