Techniques to prevent sample surface charging and reduce beam damage effects for SBEM imaging

Sample surface charging and electron beam damage are common problems in scanning electron microscopy. In the case of serial block‐face electron microscopy (SBEM), sample charging prevents or restricts stack acquisition of non-conducting samples in high vacuum; beam damage imposes a lower limit on the cutting thickness (z-resolution). In this dissertation, I present three approaches that aim to overcome these limitations. To solve the charging problem, I implemented and evaluated two charge elimination techniques for SBEM: surface charge neutralization with an ion gun and automated in-chamber specimen coating with an electron beam evaporator. The ion gun method provides only partial charge neutralization in many cases, although high positive ion current densities are achieved. The automated in-chamber coating method, on the other hand, provides reliable charge elimination in all cases and allows the acquisition of SBEM stacks of non-conducting samples in high vacuum. Thin metallic films provide charge elimination for large sample surfaces and high beam currents, and reduce the signal-to-noise ratio less than the widely-used low-vacuum method. To reduce the effects of beam damage – with the aim of allowing thin cutting at higher electron doses – I have explored imaging and cutting at low temperatures (~100 K) using a closed-loop cooling system and a custom-built cryo-microtome. Further experiments will be necessary to quantify potential improvements of 'Cryo-SBEM' over room-temperature SBEM operation

Koalas use a novel vocal organ to produce unusually low-pitched mating calls

SummaryDuring the breeding season, male koalas produce ‘bellow’ vocalisations that are characterised by a continuous series of inhalation and exhalation sections, and an extremely low fundamental frequency (the main acoustic correlate of perceived pitch) [1]. Remarkably, the fundamental frequency (F0) of bellow inhalation sections averages 27.1 Hz (range: 9.8–61.5 Hz [1]), which is 20 times lower than would be expected for an animal weighing 8 kg [2] and more typical of an animal the size of an elephant (Supplemental figure S1A). Here, we demonstrate that koalas use a novel vocal organ to produce their unusually low-pitched mating calls

SBEMimage: Versatile Acquisition Control Software for Serial Block-Face Electron Microscopy

We present SBEMimage, an open-source Python-based application to operate serial block-face electron microscopy (SBEM) systems. SBEMimage is designed for complex, challenging acquisition tasks, such as large-scale volume imaging of neuronal tissue or other biological ultrastructure. Advanced monitoring, process control, and error handling capabilities improve reliability, speed, and quality of acquisitions. Debris detection, autofocus, real-time image inspection, and various other quality control features minimize the risk of data loss during long-term acquisitions. Adaptive tile selection allows for efficient imaging of large tissue volumes of arbitrary shape. The software’s graphical user interface is optimized for remote operation. In its user-friendly viewport, tile grids covering the region of interest to be acquired are overlaid on previously acquired overview images of the sample surface. Images from other sources, e.g., light microscopes, can be imported and superimposed. SBEMimage complements existing DigitalMicrograph (Gatan Microscopy Suite) installations on 3View systems but permits higher acquisition rates by interacting directly with the microscope’s control software. Its modular architecture and the use of Python/PyQt make SBEMimage highly customizable and extensible, which allows for fast prototyping and will permit adaptation to a wide range of SBEM systems and applications

Проект благоустройства овражной территории в пос. Кольцово (г. Новосибирск)

Объект исследования – овражная территория п. Кольцово (г. Новосибирск). Цель работы – разработка проекта благоустройства овражной территории в п. Кольцово (г. Новосибирск). В процессе исследования проводилось: изучение нормативно-правовой базы, исследование особенностей территории, рассмотрены основные положения по разработке проектов благоустройства. В результате исследования разработан проект благоустройства территории.The object of study is the ravine territory of Koltsovo (Novosibirsk). The purpose of the work is the development of a project to improve the ravine territory in Koltsovo (Novosibirsk). In the course of the study, the following was carried out: a study of the regulatory framework, a study of the peculiarities of the territory, the main provisions for the development of improvement projects were considered. As a result of the study, a project was developed to improve the territory

Fast Homogeneous En Bloc Staining of Large Tissue Samples for Volume Electron Microscopy

Fixation and staining of large tissue samples are critical for the acquisition of volumetric electron microscopic image datasets and the subsequent reconstruction of neuronal circuits. Efficient protocols exist for the staining of small samples, but uniform contrast is often difficult to achieve when the sample diameter exceeds a few hundred micrometers. Recently, a protocol (BROPA, brain-wide reduced-osmium staining with pyrogallol-mediated amplification) was developed that achieves homogeneous staining of the entire mouse brain but requires very long sample preparation times. By exploring modifications of this protocol we developed a substantially faster procedure, fBROPA, that allows for reliable high-quality staining of tissue blocks on the millimeter scale. Modifications of the original BROPA protocol include drastically reduced incubation times and a lead aspartate incubation to increase sample conductivity. Using this procedure, whole brains from adult zebrafish were stained within 4 days. Homogenous high-contrast staining was achieved throughout the brain. High-quality image stacks with voxel sizes of 10 × 10 × 25 nm3 were obtained by serial block-face imaging using an electron dose of ~15 e−/nm2. No obvious reduction in staining quality was observed in comparison to smaller samples stained by other state-of-the-art procedures. Furthermore, high-quality images with minimal charging artifacts were obtained from non-neural tissues with low membrane density. fBROPA is therefore likely to be a versatile and efficient sample preparation protocol for a wide range of applications in volume electron microscopy

Do red deer stags (Cervus elaphus) use roar fundamental frequency (F0) to assess rivals?

It is well established that in humans, male voices are disproportionately lower pitched than female voices, and recent studies suggest that this dimorphism in fundamental frequency (F0) results from both intrasexual (male competition) and intersexual (female mate choice) selection for lower pitched voices in men. However, comparative investigations indicate that sexual dimorphism in F0 is not universal in terrestrial mammals. In the highly polygynous and sexually dimorphic Scottish red deer Cervus elaphus scoticus, more successful males give sexually-selected calls (roars) with higher minimum F0s, suggesting that high, rather than low F0s advertise quality in this subspecies. While playback experiments demonstrated that oestrous females prefer higher pitched roars, the potential role of roar F0 in male competition remains untested. Here we examined the response of rutting red deer stags to playbacks of re-synthesized male roars with different median F0s. Our results show that stags’ responses (latencies and durations of attention, vocal and approach responses) were not affected by the F0 of the roar. This suggests that intrasexual selection is unlikely to strongly influence the evolution of roar F0 in Scottish red deer stags, and illustrates how the F0 of terrestrial mammal vocal sexual signals may be subject to different selection pressures across species. Further investigations on species characterized by different F0 profiles are needed to provide a comparative background for evolutionary interpretations of sex differences in mammalian vocalizations

Ultraviolet Irradiation Induces the Accumulation of Chondroitin Sulfate, but Not Other Glycosaminoglycans, in Human Skin

Ultraviolet (UV) light alters cutaneous structure and function. Prior work has shown loss of dermal hyaluronan after UV-irradiation of human skin, yet UV exposure increases total glycosaminoglycan (GAG) content in mouse models. To more fully describe UV-induced alterations to cutaneous GAG content, we subjected human volunteers to intermediate-term (5 doses/week for 4 weeks) or single-dose UV exposure. Total dermal uronyl-containing GAGs increased substantially with each of these regimens. We found that UV exposure substantially increased dermal content of chondroitin sulfate (CS), but not hyaluronan, heparan sulfate, or dermatan sulfate. UV induced the accumulation of both the 4-sulfated (C4S) and 6-sulfated (C6S) isoforms of CS, but in distinct distributions. Next, we examined several CS proteoglycan core proteins and found a significant accumulation of dermal and endothelial serglycin, but not of decorin or versican, after UV exposure. To examine regulation in vitro, we found that UVB in combination with IL-1α, a cytokine upregulated by UV radiation, induced serglycin mRNA in cultured dermal fibroblasts, but did not induce the chondroitin sulfate synthases. Overall, our data indicate that intermediate-term and single-dose UVB exposure induces specific GAGs and proteoglycan core proteins in human skin in vivo. These molecules have important biologic functions and contribute to the cutaneous response to UV

Perception of Male Caller Identity in Koalas (Phascolarctos cinereus): Acoustic Analysis and Playback Experiments

The ability to signal individual identity using vocal signals and distinguish between conspecifics based on vocal cues is important in several mammal species. Furthermore, it can be important for receivers to differentiate between callers in reproductive contexts. In this study, we used acoustic analyses to determine whether male koala bellows are individually distinctive and to investigate the relative importance of different acoustic features for coding individuality. We then used a habituation-discrimination paradigm to investigate whether koalas discriminate between the bellow vocalisations of different male callers. Our results show that male koala bellows are highly individualized, and indicate that cues related to vocal tract filtering contribute the most to vocal identity. In addition, we found that male and female koalas habituated to the bellows of a specific male showed a significant dishabituation when they were presented with bellows from a novel male. The significant reduction in behavioural response to a final rehabituation playback shows this was not a chance rebound in response levels. Our findings indicate that male koala bellows are highly individually distinctive and that the identity of male callers is functionally relevant to male and female koalas during the breeding season. We go on to discuss the biological relevance of signalling identity in this species' sexual communication and the potential practical implications of our findings for acoustic monitoring of male population levels