Effects of preservation on wing morphometry of the little brown bat (Myotis lucifugus)

The effects of formalin fixation and subsequent alcohol preservation on various morphometric variables and their derivatives (lifting surface area, wingspan, mass, aspect ratio, wing loading and minimum power speed) and on the results of procedures that estimate lifting surface area of the little brown bat ( Myotis lucifugus ) are identified and quantified. Statisitical analysis demonstrates that the values of all of the examined morphometric variables depends upon the specimen type from which they are obtained (live animal; freshly killed specimen; immediately following formalin fixation; or after 36 weeks in alcohol). Over the short term, the choice of preservation fluid is ot important with respect to determination of the six variables studied. The fixation positin of the wing is an important factor in the determination of all variables except mass. Although originally suggested for study skins and not fluid-preserved specimens, 'intermediate' and 'extended' wing positions are demonstrably better than the conventional 'compressed' position. The estimation procedures of both Pirlot (1977) and Blood & McFarlane (1988) significantly underestimate analogous lifting surface areas determinedby tracing live bats. Smith & Starrett's (1979) procedure was found to yield accurate estimates occasionally: on live animals and preserved specimens with wings fixed in the extended position. Aldridge's (1988) method also yields accurate estimates of lifting surface area, but is limited to those museum specimens where the live or freshly-killed mass is known. Such conclusions permit recommendation of procedures that minimize changes arising through the fixation and preservation process in fluid-preserved museum specimens when compared to the live animal.     

Imaging the live plant cell

Observing a biological event as it unfolds in the living cell provides unique insight into the nature of the phenomenon under study. Capturing live cell data differs from imaging fixed preparations because living plants respond to the intense light used in the imaging process. In addition, live plant cells are inherently thick specimens containing colored and fluorescent molecules often removed when the plant is fixed and sectioned. For fixed cells, the straightforward goal is to maximize contrast and resolution. For live cell imaging, maximizing contrast and resolution will probably damage the specimen or rapidly bleach the probe. Therefore, the goals are different. Live cell imaging seeks a balance between image quality and the information content that comes with increasing contrast and resolution. That "lousy" live cell image may contain all the information needed to answer the question being posed--provided the investigator properly framed the question and imaged the cells appropriately. Successful data collection from live cells requires developing a specimen-mounting protocol, careful selection and alignment of microscope components, and a clear understanding of how the microscope system generates contrast and resolution. This paper discusses general aspects of modern live cell imaging and the special considerations for imaging live plant specimens.     

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood^[1-2].We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.     

Microscopic optical projection tomography in vivo

We describe a versatile optical projection tomography system for rapid three-dimensional imaging of microscopic specimens in vivo. Our tomographic setup eliminates the in xy and z strongly asymmetric resolution, resulting from optical sectioning in conventional confocal microscopy. It allows for robust, high resolution fluorescence as well as absorption imaging of live transparent invertebrate animals such as C. elegans. This system offers considerable advantages over currently available methods when imaging dynamic developmental processes and animal ageing; it permits monitoring of spatio-temporal gene expression and anatomical alterations with single-cell resolution, it utilizes both fluorescence and absorption as a source of contrast, and is easily adaptable for a range of small model organisms.     

The pros and cons of common actin labeling tools for visualizing actin dynamics during Drosophila oogenesis

Dynamic remodeling of the actin cytoskeleton is required for both development and tissue homeostasis. While fixed image analysis has provided significant insight into such events, a complete understanding of cytoskeletal dynamics requires live imaging. Numerous tools for the live imaging of actin have been generated by fusing the actin-binding domain from an actin-interacting protein to a fluorescent protein. Here we comparatively assess the utility of three such tools – Utrophin, Lifeact, and F-tractin – for characterizing the actin remodeling events occurring within the germline-derived nurse cells during Drosophila mid-oogenesis or follicle development. Specifically, we used the UAS/GAL4 system to express these tools at different levels and in different cells, and analyzed these tools for effects on fertility, alterations in the actin cytoskeleton, and ability to label filamentous actin (F-actin) structures by both fixed and live imaging. While both Utrophin and Lifeact robustly label F-actin structures within the Drosophila germline, when strongly expressed they cause sterility and severe actin defects including cortical actin breakdown resulting in multi-nucleate nurse cells, early F-actin filament and aggregate formation during stage 9 (S9), and disorganized parallel actin filament bundles during stage 10B (S10B). However, by using a weaker germline GAL4 driver in combination with a higher temperature, Utrophin can label F-actin with minimal defects. Additionally, strong Utrophin expression within the germline causes F-actin formation in the nurse cell nuclei and germinal vesicle during mid-oogenesis. Similarly, Lifeact expression results in nuclear F-actin only within the germinal vesicle. F-tractin expresses at a lower level than the other two labeling tools, but labels cytoplasmic F-actin structures well without causing sterility or striking actin defects. Together these studies reveal how critical it is to evaluate the utility of each actin labeling tool within the tissue and cell type of interest in order to identify the tool that represents the best compromise between acceptable labeling and minimal disruption of the phenomenon being observed. In this case, we find that F-tractin, and perhaps Utrophin, when Utrophin expression levels are optimized to label efficiently without causing actin defects, can be used to study F-actin dynamics within the Drosophila nurse cells.     

Quantitative histological studies of the variability of Golgi-impregnated cortical neurons in rats and cats]

Quantitative GOLGI-studies are executed about the layer V pyramidal neurons of the albino rat and the cat sensorimotor cortex. To this the length LP of the perikarya, the lenth LAD of the apical main dendrites, the spine-densities (spine-dendrite-quotients) DQ and the length NZ of the spineless ("nude") initial zone of the apical dendrites are measured in several male animals descending of the same litters. The neuronal signs are compared within the specimens of the same age. There are no statistical significant differences (level: 0,05) between these neuronal signs in all specimens of the same age and species. Relating to the examined marks of the cortical pyramids it is allowed to decline the hypothesis of variability between animals (rats or cats respectively) of the same age and sex. By this it is legal and sufficient to examine only one specimen (animal) for each stage of age in histological researches of ontogenetic series. This specimen is representative to this stage of evolution.     

Using Biplanar Fluoroscopy to Guide Radiopaque Vascular Injections: A New Method for Vascular Imaging

Studying vascular anatomy, especially in the context of relationships with hard tissues, is of great interest to biologists. Vascular studies have provided significant insight into physiology, function, phylogenetic relationships, and evolutionary patterns. Injection of resin or latex into the vascular system has been a standard technique for decades. There has been a recent surge in popularity of more modern methods, especially radiopaque latex vascular injection followed by CT scanning and digital “dissection.” This technique best displays both blood vessels and bone, and allows injections to be performed on cadaveric specimens. Vascular injection is risky, however, because it is not a standardizable technique, as each specimen is variable with regard to injection pressure and timing. Moreover, it is not possible to view the perfusion of injection medium throughout the vascular system of interest. Both data and rare specimens can therefore be lost due to poor or excessive perfusion. Here, we use biplanar video fluoroscopy as a technique to guide craniovascular radiopaque latex injection. Cadaveric domestic pigs (Sus scrofa domestica) and white-tailed deer (Odocoileus virginianus) were injected with radiopaque latex under guidance of fluoroscopy. This method was found to enable adjustments, in real-time, to the rate, location, and pressure at which latex is injected in order to avoid data and specimen loss. In addition to visualizing the injection process, this technique can be used to determine flow patterns, and has facilitated the development of consistent markers for complete perfusion.     

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of the Drosophila larva makes it an attractive model organism for live imaging studies. However, an important challenge for live imaging techniques is to noninvasively immobilize and position an animal on the microscope. This protocol presents a simple and easy to use method for immobilizing and imaging Drosophila larvae on a polydimethylsiloxane (PDMS) microfluidic device, which we call the ''larva chip''. The larva chip is comprised of a snug-fitting PDMS microchamber that is attached to a thin glass coverslip, which, upon application of a vacuum via a syringe, immobilizes the animal and brings ventral structures such as the nerve cord, segmental nerves, and body wall muscles, within close proximity to the coverslip. This allows for high-resolution imaging, and importantly, avoids the use of anesthetics and chemicals, which facilitates the study of a broad range of physiological processes. Since larvae recover easily from the immobilization, they can be readily subjected to multiple imaging sessions. This allows for longitudinal studies over time courses ranging from hours to days. This protocol describes step-by-step how to prepare the chip and how to utilize the chip for live imaging of neuronal events in 3^rd instar larvae. These events include the rapid transport of organelles in axons, calcium responses to injury, and time-lapse studies of the trafficking of photo-convertible proteins over long distances and time scales. Another application of the chip is to study regenerative and degenerative responses to axonal injury, so the second part of this protocol describes a new and simple procedure for injuring axons within peripheral nerves by a segmental nerve crush.     

CT imaging of wet specimens from a pathology museum: How to build a "virtual museum" for radiopathological correlation teaching.

X-rays and CT have been used to examine specimens such as human remains, mummies and formalin-fixed specimens. However, CT has not been used to study formalin-fixed wet specimens within their containers. The purpose of our study is firstly to demonstrate the role of CT as a non-destructive imaging method for the study of wet pathological specimens and secondly to use the CT data as a method for teaching pathological and radiological correlation. CT scanning of 31 musculoskeletal specimens from a pathology museum was carried out. Images were reconstructed using both soft-tissue and bone algorithms. Further processing of the data produced coronal and sagittal reformats of each specimen. The container and storage solution were manually removed using Volume Viewer Voxtool software to produce a 3D reconstruction of each specimen. Photographs of each specimen (container and close-up) were displayed alongside selected coronal, sagittal, 3D reconstructions and cine sequences in a specially designed computer program. CT is a non-destructive imaging modality for building didactic materials from wet specimens in a Pathology Museum, for teaching radiological and pathological correlation.     

Development of a small-scale rotary lobe-pump cell culture model for examining cell damage in large-scale N-1 seed perfusion process

Perfusion technology has been identified as a process improvement capable of eliminating some of the constraints in cell culture and allows for high cell densities and viabilities. However, when implementing this N-1 seed perfusion platform in large-scale manufacturing, unexpected cell damage was observed as early as Day 1. Given that the shear rate within recirculation hollow fibers was normalized and aligned correctly across bench, pilot, and manufacture scale, the primary mitigation was placed on the rotary lobe pump. Lowering the pump rate in manufacture scale successfully alleviated the cell damage. To understand the source of cell damage within the pump, a small-scale rotary lobe-pump robustness model was developed. Testing different pump flow rates and back pressures, it was concluded that high back pressure can cause cell damage. The back pressure within the system can cause back flow and high shear within small clearances inside the pump, which lead to the primary cell damage observed at a large scale. This shear level can be significantly higher than the shear in the hollow fiber. This pump robustness model can be utilized to aid the perfusion skid design, including pump operation efficiency and cell shear sensitivity. Methods to reduce the back pressure and cell shearing were determined to better predict manufacturing performance in the future.     

Transient state imaging of live cells using single plane illumination and arbitrary duty cycle excitation pulse trains

We demonstrate the applicability of Single Plane Illumination Microscopy to Transient State Imaging (TRAST), offering sensitive microenvironmental information together with optical sectioning and reduced overall excitation light exposure of the specimen. The concept is verified by showing that transition rates can be determined accurately for free dye in solution and that fluorophore transition rates can be resolved pixel‐wise in live cells. Furthermore, we derive a new theoretical framework for analyzing TRAST data acquired with arbitrary duty cycle pulse trains. By this analysis it is possible to reduce the overall measurement time and thereby enhance the frame rates in TRAST imaging. (© 2014 WILEY‐VCH Verlag GmbH &Co. KGaA, Weinheim)     

High-resolution imaging of the microbial cell surface

Microorganisms, or microbes, can function as threatening pathogens that cause disease in humans, animals, and plants; however, they also act as litter decomposers in natural ecosystems. As the outermost barrier and interface with the environment, the microbial cell surface is crucial for cell-to-cell communication and is a potential target of chemotherapeutic agents. Surface ultrastructures of microbial cells have typically been observed using scanning electron microscopy (SEM) and atomic force microscopy (AFM). Owing to its characteristics of low-temperature specimen preparation and superb resolution (down to 1 nm), cryo-field emission SEM has revealed paired rodlets, referred to as hydrophobins, on the cell walls of bacteria and fungi. Recent technological advances in AFM have enabled high-speed live cell imaging in liquid at the nanoscale level, leading to clear visualization of cell-drug interactions. Platinum-carbon replicas from freeze-fractured fungal spores have been observed using transmission electron microscopy, revealing hydrophobins with varying dimensions. In addition, AFM has been used to resolve bacteriophages in their free state and during infection of bacterial cells. Various microscopy techniques with enhanced spatial resolution, imaging speed, and versatile specimen preparation are being used to document cellular structures and events, thus addressing unanswered biological questions.     

Out-of-laboratory control screening of growth mediums used by sanitary service laboratories for isolation of pathogenic enteric bacteria from fecal specimens

The aim of the study was to test the selective-differential plating mediums used for isolation of Salmonella and Shigella for routine stool specimens examination for epidemiological and sanitary purpose. Three plates of any such medium used in the laboratories in 37 Sanitary Service Stations in Poland were obtained. The specimens of Mac Conkey Lactose Bile Salt Agar and SS Agar were obtained from all laboratories, Hektoen Enteric medium from 8 and EosinMethylene Blue Agar from only one laboratory. The desiccated substrates of these mediums originated from 11 manufactures. The mediums were inoculated by "drops" method. The five control strains of selected taxons were chosen from National Institute of Hygiene strains collection. The quality of growth was evaluated by comparison with the growth on two control mediums: the general outlook of bacterial colonies, size and number of cfu/ml was taken under consideration. It was found that the results were satisfying for Hektoen medium, Levine and all but two Mac Conkey's medium specimens. The results of growth on SS medium were much worse: only on 10 specimens out of 39 checked supported properly the growth of all the five control strains. On 18 specimens the growth appeared after 48 hours of incubation and the size of colonies was too small to be isolated. On 11 there was no growth on 1, 2 or 3 control strains. The need of systematic extra-laboratory control of plating mediums used for examination stool specimen was shown.     

Cranial anatomy,taxonomic implications and palaeopathology of an Upper Jurassic Pliosaur (Reptilia: Sauropterygia) from Westbury,Wiltshire, UK

Abstract: Complete skulls of giant marine reptiles of the Late Jurassic are rare, and so the discovery of the 1.8‐m‐long skull of a pliosaur from the Kimmeridge Clay Formation (Kimmeridgian) of Westbury, Wiltshire, UK, is an important find. The specimen shows most of the cranial and mandibular anatomy, as well as a series of pathological conditions. It was previously referred to Pliosaurus brachyspondylus, but it can be referred reliably only to the genus Pliosaurus, because species within the genus are currently in need of review. The new specimen, together with another from the same locality, also referred to P. brachyspondylus, will be crucial in that systematic revision, and it is likely that the genus Pliosaurus will be found to include several genera. The two Westbury Pliosaurus specimens share many features, including the form of the teeth, but marked differences in the snout and parietal crest suggest sexual dimorphism; the present specimen is probably female. The large size of the animal, the extent of sutural fusion and the pathologies suggest this is an ageing individual. An erosive arthrotic condition of the articular glenoids led to prolonged jaw misalignment, generating a suite of associated bone and dental pathologies. Further damage to the jaw joint may have been the cause of death.     

A morphometric analysis of extant and early miocene fossil hominoid maxillo-dental specimens

It is demonstrated in this paper that before we can hope to formulate phylogenetic relationships between and amongst fossil hominoid material it is first necessary to sex the material accurately. In order to determine whether the morphological and morphometrical variability seen in fossil specimens is due to sexual or inter species dimorphism, it is necessary to calibrate fossil specimens against extant hominoid species' morphologies. Only after fossil specimens have been sexed is it possible to differentiate between morphologies that are related to sex and those that are species specific. This will help reduce fossil misallocation. A morphometric analysis of extant and fossilProconsul hominoid material is presented. Each fossil specimen has been sexed according to symplesiomorphic sex morphologies as defined in this paper. After the fossil specimens have been sexed they are analyzed using multivariate statistics. The identification of differing sex patterns within the specimens examined here suggests that a new species ofProconsul may have to be considered.     

Assessment of Cardiac Function and Myocardial Morphology Using Small Animal Look-locker Inversion Recovery (SALLI) MRI in Rats

Small animal magnetic resonance imaging is an important tool to study cardiac function and changes in myocardial tissue. The high heart rates of small animals (200 to 600 beats/min) have previously limited the role of CMR imaging. Small animal Look-Locker inversion recovery (SALLI) is a T1 mapping sequence for small animals to overcome this problem ¹. T1 maps provide quantitative information about tissue alterations and contrast agent kinetics. It is also possible to detect diffuse myocardial processes such as interstitial fibrosis or edema ^1-6. Furthermore, from a single set of image data, it is possible to examine heart function and myocardial scarring by generating cine and inversion recovery-prepared late gadolinium enhancement-type MR images ¹.The presented video shows step-by-step the procedures to perform small animal CMR imaging. Here it is presented with a healthy Sprague-Dawley rat, however naturally it can be extended to different cardiac small animal models.     

Lens-free shadow image based high-throughput continuous cell monitoring technique

A high-throughput continuous cell monitoring technique which does not require any labeling reagents or destruction of the specimen is demonstrated. More than 6000 human alveolar epithelial A549 cells are monitored for up to 72h simultaneously and continuously with a single digital image within a cost and space effective lens-free shadow imaging platform. In an experiment performed within a custom built incubator integrated with the lens-free shadow imaging platform, the cell nucleus division process could be successfully characterized by calculating the signal-to-noise ratios (SNRs) and the shadow diameters (SDs) of the cell shadow patterns. The versatile nature of this platform also enabled a single cell viability test followed by live cell counting. This study firstly shows that the lens-free shadow imaging technique can provide a continuous cell monitoring without any staining/labeling reagent and destruction of the specimen. This high-throughput continuous cell monitoring technique based on lens-free shadow imaging may be widely utilized as a compact, low-cost, and high-throughput cell monitoring tool in the fields of drug and food screening or cell proliferation and viability testing.     

The Leeuwenhoek lecture 2006. Microscopy goes cold: frozen viruses reveal their structural secrets

The electron microscope provides a powerful tool for investigating the structure of biological complexes such as viruses. A modern instrument is fully capable of atomic resolution on suitable non-biological specimens, but biological materials are difficult to preserve, owing to their fragility, and to image, owing to their radiation, sensitivity. The act of imaging the specimen severely damages it. Originally, samples were prepared by staining with a heavy metal salt, which provides a stable specimen but limits the amount of details that can be retrieved. Now particulate specimens, such as viruses, are prepared by rapid freezing of unstained material and observed in a frozen state with low doses of electrons. The resulting images require extensive computer processing to extract fully detailed three-dimensional information about the specimen. The whole process is referred to as single-particle electron cryomicroscopy. Using this approach, the structure of the human hepatitis B virus core was solved at the level of the protein fold. By comparing maps of RNA- and DNA-containing cores, it was possible to propose a model for the maturation and control of the envelopment of the virus during assembly. These examples show that cryomicroscopy offers great potential for understanding the structure and function of complex biological assemblies.     

Retrospective: radiation damage and its associated "information limitations"

The fact that radiation damage would limit the usefulness of electron microscopy with biological specimens was a concern in the earliest days of the field. Good estimates of what that limitation must be can be made by using Rose's empirical relationship between the inherent image contrast, the exposure used to record an image, and the smallest feature size that is detectable. Such estimates show that it is necessary to average many images in order to obtain statistically well-defined data at high resolution. Structures are now routinely obtained by averaging large numbers of shot-noise limited images, and some of these extend to atomic resolution. The signal level in current images is nevertheless far below what physics would allow it to be. A possible explanation is that beam-induced movement limits the quality of images recorded by electron microscopy. For specimens embedded in vitreous ice, beam-induced movement can even be severe enough to limit the resolution achieved during tomographic reconstruction. The fact that very high-quality images can nevertheless be obtained, although only unpredictably, suggests that it may be possible to devise new techniques of specimen preparation and/or data collection that at least partially overcome beam-induced movement. If so, the need for image averaging would be correspondingly reduced.     

Measurement of oxidative stress in stroke-prone spontaneously hypertensive rat brain using in vivo electron spin resonance spectroscopy

A number of researchers have reported that free radicals generated in the brain are involved in various brain dysfunctions, including ischemia-reperfusion injury, brain tumors, and neurodegenerative diseases. It has been reported that the spin probe MC-PROXYL can penetrate the blood-brain barrier and can be useful for evaluating oxidative stress in the brain. Preliminary comparisons were made by ESR imaging of the heads of live mice and isolated rat brains using the spin probe MC-PROXYL and the blood-brain-barrier impermeable probe carbamoyl-PROXYL. The results showed that MC-PROXYL, but not carbamoyl-PROXYL, was widely distributed in the brain. These methods were also applied for the imaging of brains from stroke-prone spontaneously hypertensive rats (SHRSPs). The rapid decay of 2D ESR images of MC-PROXYL in isolated SHRSP-brain was observed, compared to Wistar-Kyoto rats (WKYs), using the ESR imaging system. Furthermore, we provide evidence, by using L-band ESR non-invasively, that the decay rate of MC-PROXYL in the head region is faster in live SHRSPs than in live WKYs. Taken together, the high oxidative stress sustained by oxygen radical generation in SHRSPs may cause the alteration of MC-PROXYL metabolism in the brain. Our results suggest that in vivo ESR could be applied to the assessment of antioxidant effects on oxidative stress in the brain in animal disease models, such as the SHRSP.