Making the practically impossible “Merely difficult”—Cryogenic FIB lift‐out for “Damage free” soft matter imaging

The preparation of thinned lamellae from bulk samples for transmission electron microscopy (TEM) analysis has been possible in the focussed ion beam scanning electron microscope (FIB‐SEM) for over 20 years via the in situ lift‐out method. Lift‐out offers a fast and site specific preparation method for TEM analysis, typically in the field of materials science. More recently it has been applied to a low‐water content biological sample (Rubino 2012). This work presents the successful lift‐out of high‐water content lamellae, under cryogenic conditions (cryo‐FIB lift‐out) and using a nanomanipulator retaining its full range of motion, which are advances on the work previously done by Rubino (2012). Strategies are explored for maintaining cryogenic conditions, grid attachment using cryo‐condensation of water and protection of the lamella when transferring to the TEM. Microsc. Res. Tech. 79:298–303, 2016. © 2016 Wiley Periodicals, Inc.     

Contact X‐ray microscopy of living cells by using LiF crystal as imaging detector

In this paper, the use of lithium fluoride (LiF) as imaging radiation detector to analyse living cells by single‐shot soft X‐ray contact microscopy is presented. High resolved X‐ray images on LiF of cyanobacterium Leptolyngbya VRUC135, two unicellular microalgae of the genus Chlamydomonas and mouse macrophage cells (line RAW 264.7) have been obtained utilizing X‐ray radiation in the water window energy range from a laser plasma source. The used method is based on loading of the samples, the cell suspension, in a special holder where they are in close contact with a LiF crystal solid‐state X‐ray imaging detector. After exposure and sample removal, the images stored in LiF by the soft X‐ray contact microscopy technique are read by an optical microscope in fluorescence mode. The clear image of the mucilaginous sheath the structure of the filamentous Leptolyngbya and the visible nucleolus in the macrophage cells image, are noteworthiness results. The peculiarities of the used X‐ray radiation and of the LiF imaging detector allow obtaining images in absorption contrast revealing the internal structures of the investigated samples at high spatial resolution. Moreover, the wide dynamic range of the LiF imaging detector contributes to obtain high‐quality images. In particular, we demonstrate that this peculiar characteristic of LiF detector allows enhancing the contrast and reveal details even when they were obscured by a nonuniform stray light.     

Localized grounding, excavation, and dissection using in-situ probe techniques for focused ion beam and scanning electron microscopy: experiments with rock varnish

While investigating rock varnish, we explored novel uses for an in‐situ micromanipulator, including charge collection, sample manipulation, as well as digging and dissection at the micron level. Dual‐beam focused ion beam microscopes (DB‐FIB or FIBSEM) equipped with micromanipulators have proven to be valuable tools for material science, semiconductor research, and product failure analysis. Researchers in many other disciplines utilize the DB‐FIB and micromanipulator for site‐specific transmission electron microscope (TEM) foil preparation. We have demonstrated additional applications for in‐situ micromanipulators. SCANNING 34: 279–283, 2012. © 2012 Wiley Periodicals, Inc.     

Authenticating and distinguishing the eight species of traditional Tibetan medicine “Meiduoluomi” by microscopic technique

Because of the morphological and macroscopic similarity, many species of Erigeron and Aster (Asteraceae) are confusable and usually used under the same name “Meiduoluomi” in traditional Tibetan medicine (TTM). To find an easy, quick, and reliable method to authenticate and distinguish the eight main medicinal plants of these species, the light microscope was used to reveal the morphoanatomic details. The fixed, sectioned, and stained plant materials and epidermis materials were studied by microscopic techniques. The results of the microscopic features are systematically described and illustrated, and comparison parameters are presented. Furthermore, a key to the eight species of “Meiduoluomi” was constructed. Microscopy can be unambiguously used to authenticate and distinguish the eight main species of TTM “Meiduoluomi.” Microsc. Res. Tech., 2009. © 2009 Wiley‐Liss, Inc.     

Design and construction of a new temperature-controlled chamber for light and confocal microscopy under monitored conditions: biological application for plant samples

A new light microscope–temperature‐controlled chamber (LM–TCC) has been constructed. The special feature of the light microscope–temperature‐controlled chamber is the Peltier‐element temperature control of a specimen holder for biological samples, with a volume capacity of 1 mL. This system has marked advantages when compared to other approaches for temperature‐controlled microscopy. It works in a temperature range of −10°C to +95°C with an accuracy of ±0.1°C in the stationary phase. The light microscope–temperature‐controlled chamber allows rapid temperature shift rates. A maximum heating rate of 12.9°C min⁻¹ and a maximum cooling rate of 6.0°C min⁻¹ are achieved with minimized overshoots (≤1.9°C). This machinery operates at low cost and external coolants are not required. Especially with samples absorbing irradiation strongly, temperature control during microscopy is necessary to avoid overheating of samples. For example, leaf segments of Ficaria verna exposed to 4500 μmol photons m⁻² s⁻¹ in a standard microscopic preparation show a temperature increase (δT) of 18.0°C, whereas in the light microscope–temperature‐controlled chamber this is reduced to 4°C. The kinetics of microscope‐light induced δT are described and infrared thermography demonstrates the dissipation of the temperature. Chloroplasts of the cold adapted plant Ranunculus glacialis show the tendency to form stroma‐filled protrusions in relation to the exposure temperature. The relative number of chloroplasts with protrusions is reduced at 5°C when compared to 25°C. This effect is reversible. The new light microscope–temperature‐controlled chamber will be useful in a wide range of biological applications where a rapid change of temperature during microscopic observations is necessary or has to be avoided allowing a simulation of ecologically relevant temperature scenarios.     

Application of microscopy for discrimination of eight Swertia species utilized in the traditional Chinese medicine “Qingyedan”

Many Swertia species are utilized as a traditional medicine under the name “Qingyedan” in China, but are easily confused with one another. To distinguish eight Swertia species (S. mileensis, S. cincta, S. patens, S. punicea, S. delavayi, S. nervosa, S. macrosperma, and S. yunnanensis) and to ensure their safety and efficacy, the microscopic and macroscopic characteristics of the roots, stems, leaves, and flowers of them were examined. The results showed that microscopic and macroscopic features helpful for authentication of the eight species were the sinuosity of the anticlinal walls of epidermal cells and presence or absence of hairs on the leaf lamina; presence or absence of V‐shaped fibers and fibers with sinuous abaxial wall in the sepals; shape of epidermal cells and pattern of papillae on hairs on the margin of corolla nectary; distribution of stomata in leaf and sepal epidermises, stone cells in cortex and phloem of roots and in cortex and pith of stems, crystals in parenchymatous cells of mesophyll and stem, stomata size, stem diameter, and 4‐ or 5‐merous flowers, and so on. Two keys to the eight Swertia species based on macroscopic and microscopic characteristics are presented. The study indicates that microscopy and related techniques are convenient, practicable, and can be unambiguously applied for authentication of Swertia species. Microsc. Res. Tech. 76:296–310, 2013. © 2013 Wiley Periodicals, Inc.     

Identification of four Aconitum species used as “Caowu” in herbal markets by 3d reconstruction and microstructural comparison

Authentication is the first priority when evaluating the quality of Chinese herbal medicines, particularly highly toxic medicines. The most commonly used authentication methods are morphological identification and microscopic identification. Unfortunately, these methods could not effectively evaluate some herbs with complex interior structures, such as root of Aconitum species with a circular conical shape and an interior structure with successive changes. Defining the part that should be selected as the standard plays an essential role in accurate microscopic identification. In this study, we first present a visual 3D model of Aconitum carmichaeli Debx. constructed obtained from microscopic analysis of serial sections. Based on this model, we concluded that the point of largest root diameter should be used as the standard for comparison and identification. The interior structure at this point is reproducible and its shape and appearance can easily be used to distinguish among species. We also report details of the interior structures of parts not shown in the 3D model, such as stone cells and cortical thickness. To demonstrate the usefulness of the results from the 3D model, we have distinguished the microscopic structures, at their largest segments, of the other three Aconitum species used for local habitat species of Caowu. This work provides the basis for resolution of some debate regarding the microstructural differences among these species. Thus, we conclude that the 3D model composed of serial sections has enabled the selection of a standard cross‐section that will enable the accurate identification of Aconitum species in Chinese medicine. Microsc. Res. Tech. 78:425–432, 2015. © 2015 Wiley Periodicals, Inc.     

Structure and biomechanics of culms of Phragmites australis used for reeds of Japanese wind instrument “hichiriki”

Hichiriki is a traditional Japanese double‐reed wind instrument used in Japanese ancient imperial court music, gagaku, which has been performed since the 7th century. The best reeds for hichiriki have been made of culms or stems of Phragmites australis (P. australis) that are harvested from only a limited reed bed at Udono near Kyoto. The aim of this study is to elucidate why the stems from Udono are the best materials for hichiriki reeds. Plant anatomy was examined for choice stems of P. australis grown in different reed beds in Japan as well as morphology, and the local indentation hardness and Young's modulus of tissues on the cross‐sections of some representatives of hichiriki reeds were measured. It is concluded that the good stems for hichiriki reeds have an outer diameter of about 11 mm, a wall thickness of about 1 mm and comparatively homogeneous structure where harder materials, such as epidermis, hypodermis, sclerenchymatous cells, and vascular bundle sheaths with hard walls, are orderly deployed with softer materials such as parenchyma cells and vascular bundles. This structure has smaller differences of hardness and Young's modulus between the hard and soft materials in the reed, providing the best music performance. Microsc. Res. Tech. 78:260–267, 2015. © 2015 Wiley Periodicals, Inc.     

Off-axis electron holography of electrostatic potentials in unbiased and reverse biased focused ion beam milled semiconductor devices

Off‐axis electron holography in the transmission electron microscope (TEM) is used to measure two‐dimensional electrostatic potentials in both unbiased and reverse biased silicon specimens that each contain a single p–n junction. All the specimens are prepared for examination in the TEM using focused ion beam (FIB) milling. The in situ electrical biasing experiments make use of a novel specimen geometry, which is based on a combination of cleaving and FIB milling. The design and construction of an electrical biasing holder are described, and the effects of TEM specimen preparation on the electrostatic potential in the specimen, as well as on fringing fields beyond the specimen surface, are assessed.     

Influence of gallium‐doped zinc‐oxide thickness on polymer light‐emitting diode luminescence efficiency

Conducting atomic force microscopy and scanning surface potential microscopy were used to study the local electrical properties of gallium‐doped zinc oxide (GZO) films prepared by pulsed laser deposition (PLD) on a polyimide (PI) substrate. For a PLD deposition process time of 8 min, the root‐mean‐square roughness, coverage percentage of the conducting regions, and mean work function on the GZO surface were 2.33 nm, 96.6%, and 4.82 eV, respectively. When the GZO/PI substrate was used for a polymer light‐emitting diode (PLED), the electroluminescence intensity increased by nearly 20% compared to a standard PLED, which was based on a commercial‐ITO/glass substrate. Microsc. Res. Tech. 76:783–787, 2013. © 2013 Wiley Periodicals, Inc.     

The effect of a root‐dentin pretreatment technique combining PIPS with MTAD aiming to improve the bond strength of glass fiber post

The aim of this study was to evaluate the combined effect of MTAD and photon‐induced photoacoustic streaming (PIPS) technique on the smear layer removal and the bond strength of glass fiber post. Fifty‐five human mandibular premolars were chosen. After root canal therapy and post space preparation, the teeth were equally divided to five groups according to the methods of root‐dentin pretreatment: G1: distilled water (control); G2: 2.5% NaOCl+17% EDTA; G3: MTAD; G4: PIPS; G5: MTAD+PIPS. One sample was randomly selected from each group, and scanning electron microscope (SEM) was used to observe the microscopic morphology of the coronal, middle and apical level of the root‐dentin. The remaining ten samples from each group were glued to glass fiber posts, and the bond strength was assessed by push‐out test. SEM evaluation showed that the most complete smear layer removal was found in MTAD + PIPS group, especially in coronal third. Push‐out test results exhibited that there was a statistically significant interaction between the experimental groups and root canal third (p < .0005). Significant differences could be found among five groups (p < .05): G5 > G4 > G3 > G2 > G1. G5 had the highest bond strength. In conclusion, post space pretreatments with MTAD or PIPS technique can significantly remove smear layer and improve the bond strength of glass fiber post, and combination of them works best.     

Phase contrast without phase plates and phase rings—optical solutions for improved imaging of phase structures

Using the optical methods described, phase specimens can be observed with a modified light microscope in enhanced clarity, purified from typical artifacts which are apparent in standard phase contrast illumination. In particular, haloing and shade‐off are absent, lateral and vertical resolution are maximized and the image quality remains constant even in problematic preparations which cannot be well examined in normal phase contrast, such as specimens beyond a critical thickness or covered by obliquely situated cover slips. The background brightness and thus the range of contrast can be continuously modulated and specimens can be illuminated in concentric‐peripheral, axial or paraxial light. Additional contrast effects can be achieved by spectral color separation. Normal glass or mirror lenses can be used; they do not need to be fitted with a phase plate or a phase ring. The methods described should be of general interest for all disciplines using phase microscopy. Microsc. Res. Tech., 76:1050–1056, 2013. © 2013 Wiley Periodicals, Inc.     

High-resolution compact X-ray microscopy

We demonstrate compact full‐field soft X‐ray transmission microscopy with sub 60‐nm resolution operating at λ= 2.48 nm. The microscope is based on a 100‐Hz regenerative liquid‐nitrogen‐jet laser‐plasma source in combination with a condenser zone plate and a micro‐zone plate objective for high‐resolution imaging onto a 2048 × 2048 pixel CCD detector. The sample holder is mounted in a helium atmosphere and allows imaging of both dry and wet specimens. The microscope design enables fast sample switching and the sample can be pre‐aligned using a visible‐light microscope. High‐quality images can be acquired with exposure times of less than 5 min. We demonstrate the performance of the microscope using both dry and wet samples.     

Precise observation of C. elegans dynamic behaviours under controlled thermal stimulus using a mobile phone–based microscope

We investigated the temperature‐dependent locomotion of Caenorhabditis elegans by using the mobile phone–based microscope. We developed the customized imaging system with mini incubator and smartphone to effectively control the thermal stimulation for precisely observing the temperature‐dependent locomotory behaviours of C. elegans. Using the mobile phone–based microscope, we successfully followed the long‐term progress of specimens of C. elegans in real time as they hatched and explored their temperature‐dependent locomotory behaviour. We are convinced that the mobile phone–based microscope is a useful device for real time and long‐term observations of biological samples during incubation, and can make it possible to carry out live observations via wireless communications regardless of location. In addition, this microscope has the potential for widespread use owing to its low cost and compact design.     

Microscopic study on the concretion of ceramics in the “Nanhai I” shipwreck of China,Southern Song Dynasty (1,127–1,279 A.D.)

“Nanhai I” shipwreck of China Southern Song Dynasty is the oldest and the most integrally preserved shipwreck in the world. The related conservation and archeological research have caught great attention of different experts all over the world. In this study, different types of concretion covered on the surface of the ceramics in “Nanhai I” shipwreck were analyzed by X‐ray diffractometer, micro‐Raman spectrometer, and scanning electron microscope equipped with energy dispersive spectroscopy. Based on the analyses, we found that the grey concretion was mainly composed of quartz, aragonite, and calcite while the reddish concretion was mainly composed of pyrite and quartz. Our study indicated that the formation process of the grey concretion probably included the crystallization and transformation of aragonite, while the corrosion of iron implements and crystallization of pyrite were highly involved in the formation of reddish concretion.     

A novel pixellated solid‐state photon detector for enhancing the everhart–thornley detector

This article presents a pixellated solid‐state photon detector designed specifically to improve certain aspects of the existing Everhart–Thornley detector. The photon detector was constructed and fabricated in an Austriamicrosystems 0.35 µm complementary metal‐oxide‐semiconductor process technology. This integrated circuit consists of an array of high‐responsivity photodiodes coupled to corresponding low‐noise transimpedance amplifiers, a selector‐combiner circuit and a variable‐gain postamplifier. Simulated and experimental results show that the photon detector can achieve a maximum transimpedance gain of 170 dBΩ and minimum bandwidth of 3.6 MHz. It is able to detect signals with optical power as low as 10 nW and produces a minimum signal‐to‐noise ratio (SNR) of 24 dB regardless of gain configuration. The detector has been proven to be able to effectively select and combine signals from different pixels. The key advantages of this detector are smaller dimensions, higher cost effectiveness, lower voltage and power requirements and better integration. The photon detector supports pixel‐selection configurability which may improve overall SNR and also potentially generate images for different analyses. This work has contributed to the future research of system‐level integration of a pixellated solid‐state detector for secondary electron detection in the scanning electron microscope. Microsc. Res. Tech. 76:648–652, 2013. © 2013 Wiley Periodicals, Inc.     

Alignment with sub‐pixel accuracy for images of multi‐modality microscopes using automatic calibration

A biological specimen is often imaged with various imaging modalities, and it is crucial that such images are well aligned to best reveal physiological structures and functions of the specimen for in‐depth analyses. In this paper, we present a methodology for automatic calibration of multiple optical imaging modalities within the x–y detector plane using a custom chrome‐on‐glass target and an automatic and accurate registration algorithm. The target contains lines crossing at random angles, and our method of registration is based on the alignment of salient features extracted from the lines within the individual images. Once spatial relationships are found between the various detectors and applied to the resultant images, no further registration is required for all static samples, and the registered images serve as the starting point for registration of dynamic samples, where the remaining misalignment is caused by sample movement. We have validated our algorithm with 40 inter‐modal and 30 intra‐modal image pairs, and the success rates are 95 and 100%, respectively, with sub‐pixel accuracy. This methodology is widely applicable to any multi‐modal microscope that combines a number of imaging modalities on a common platform assuming images of the target can be obtained.     

Ultrastructural and three‐dimensional study of post‐LASIK ectasia cornea

INTRODUCTION: Post‐laser in situ keratomileusis (LASIK) corneal ectasia is a serious late postoperative complication. Here, we report the ultrastructural features of the post‐LASIK cornea of two patients. METHODS: Two normal corneas (age 24 and 37 years old) and two post‐LASIK ectaic corneas from two patients (A and B) were studied. The “patient A” (age 27 years) underwent penetrating keratoplasty and “patient B” (age 31 years) underwent deep‐anterior lamellar keratoplasty. The excised corneas were processed for light and electron microscopy. A total of 120 images for three‐dimensional (3D) reconstruction were taken by using the software “Recorder” and using a bottom mounted camera “Quemesa” attached to a JOEL 1400 transmission electron microscope. The 3D images were constructed using “Visual Kai” software. RESULTS: In the post‐LASIK cornea, the hemidesmosomes, the basement membrane, and Bowman”s layer were abnormal. The stromal lamellae were thin and disorganized. The collagen fibrils (CFs) diameter and interfibrillar spacing had decreased. Aggregated microfibrils were present in the Bowman's layer and all parts of the stroma. A large number of microfilaments were present at the detachment end of the flap and residual stroma. The 3D images showed the presence of collagen microfibrils and proteoglycans (PGs) within the CF of the normal and post‐LASIK cornea. The collagen microfibrils and PGs within the CFs had degenerated in the post‐LASIK cornea. CONCLUSION: Collagen microfibrils and PGs within the CFs were degenerated, leading to the degeneration of CFs, followed by the disorganization of lamellae in post‐LASIK cornea. The CFs diameter and interfibrillar spacing decreased. Microsc. Res. Tech. 77:91–98, 2014. © 2013 Wiley Periodicals, Inc.     

Thermostatic tissue platform for intravital microscopy: 'the hanging drop' model

Intravital microscopy imposes the particular problem of the combined control of the body temperature of the animal and the local temperature of the observed organ or tissues. We constructed and tested, in the rat ileum microcirculation preparation, a new organ‐support platform. The platform consisted of an organ bath filled with physiological solution, and contained a suction tube, a superfusion tube, an intestine‐support hand that was attached to a micromanipulator and a thermometer probe. To cover the intestine we used a cover glass plate with a plastic ring glued on its upper surface. After a routine procedure (anaesthesia, monitoring and surgery), the intestine segment (2–3 cm long) was gently exteriorized and placed on the ‘hand’ of the organ support. A small part of the intestine formed a small ‘island’ in the bath that was filled with physiological salt solution. The cover glass was secured in place. The physiological salt solution from the superfusion tube, which was pointed to the lower surface of the cover glass, formed a ‘hanging drop’. The objective of the microscope was then immersed into distilled water that was formed by the cover glass plastic ring. The ‘hanging drop’ technique prevented any tissue quenching, ensured undisturbed microcirculation, provided for stable temperature and humidity, and permitted a clear visual field.