Looking at tardigrades in a new light: using epifluorescence to interpret structure

Perry, Emma S.; Miller, William R.; Lindsay, Sara M.

doi:10.1111/jmi.12190

Cited by 19 publications

(9 citation statements)

References 21 publications

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“…Phase contrast microscopy may represent an alternative for very flat specimens showing small halos with this system, especially if objectives of highest magnifications are used (Noyes 1982;Disney 1989;Noyes & Polaszek 1989;Neuhaus 1993, figs 6 and 11;Lillo et al 2010). More recently, tardigrade and mite autofluorescing cuticular structures have been investigated with the help of epifluorescence using UV light (330-485 nm) and confocal laser scanning microscopy using blue light (405 nm), respectively (Chetverikov 2012;Perry et al 2015). Whereas gum-chloral media resulted in a good image quality of internal genitalia and external morphological characters of mites, staining a specimens with iodine or mounting in media containing poly(vinyl alcohol) lead to a severe reduction of the autofluorescent signal (Chetverikov 2012).…”

Section: Microscope Equipmentmentioning

confidence: 99%

Collection management and study of microscope slides: Storage, profiling, deterioration, restoration procedures, and general recommendations

Neuhaus

Schmid

Riedel

2017

Zootaxa

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A wide range of aspects concerning microscope slides, their preparation, long-time storage, curatorial measures in collections, deterioration, restoration, and study is summarized based on our own data and by analyzing more than 600 references from the 19th century until 2016, 15 patents, and about 100 Materials Safety Data Sheets. Information from systematic zoology, conservation sciences, chemistry, forensic sciences, pathology, paleopathology, applied sciences like food industry, and most recent advances in digital imaging are put together in order to obtain a better understanding of which and possibly why mounting media and coverslip seals deteriorate, how slides can be salvaged, which studies may be necessary to identify a range of ideal mounting media, and how microscope studies can benefit from improvements in developmental biology and related fields. We also elaborate on confusing usage of concepts like that of maceration and of clearing. The chemical ingredients of a range of mounting media and coverslip seals are identified as much as possible from published data, but this information suffers in so far as the composition of a medium is often proprietary of the manufacturer and may vary over time. Advantages, disadvantages, and signs of deterioration are documented extensively for these media both from references and from our own observations. It turns out that many media degrade within a few years, or decades at the latest, except Canada balsam with a documented life-time of 150 years, Euparal with a documented life-time of 50 years, and glycerol-paraffin mounts sealed with Glyceel, which represents almost the only non-deteriorating and easily reversible mount. Deterioration reveals itself as a yellowing in natural resins and as cracking, crystallization, shrinkage on drying or possibly on loss of a plasticizer, detachment of the coverslip, segregation of the ingredients in synthetic polymers, as well as continued maceration of a specimen to a degree that the specimen virtually disappears. Confusingly, decay does not always appear equally within a collection of slides mounted at the same time in the same medium. The reasons for the deteriorative processes have been discussed but are controversial especially for gum-chloral media. Comparing data from conservation sciences, chemical handbooks, and documented ingredients, we discuss here how far chemical and physical deterioration probably are inherent to many media and are caused by the chemical and physical properties of their components and by chemicals dragged along from previous preparation steps like fixation, chemical maceration, and physical clearing. Some recipes even contain a macerating agent, which proceeds with its destructive work. We provide permeability data for oxygen and water vapor of several polymers contained in mounting media and coverslip seals. Calculation of the penetration rate of moisture in one example reveals that water molecules reach a specimen within a few days up to about a month; this lays to rest extensive discussions about the permanent protection of a mounted specimen by a mounting medium and a coverslip seal. Based on the ever growing evidence of the unsuitable composition and application of many, and possibly almost all, mounting media, we strongly encourage changing the perspective on microscope slides from immediate usability and convenience of preparation towards durability and reversibility, concepts taken from conservation sciences. Such a change has already been suggested by Upton (1993) more than 20 years ago for gum-chloral media, but these media are still encouraged nowadays by scientists. Without a new perspective, taxonomic biology will certainly lose a large amount of its specimen basis for its research within the next few decades. Modern non-invasive techniques like Raman spectroscopy may help to identify mounting media and coverslip seals on a given slide as well as to understand ageing of the media. An outlook is given on potential future studies. In order to improve the situation of existing collections of microscope slides, we transfer concepts as per the Smithsonian Collections Standards and Profiling System, developed for insect collections more than 25 years ago, to collections of slides. We describe historical and current properties and usage of glass slides, coverslips, labels, and adhesives under conservational aspects. In addition, we summarize and argue from published and our own experimental information about restorative procedures, including re-hydration of dried-up specimens previously mounted in a fluid medium. Alternatives to microscope slides are considered. We also extract practical suggestions from the literature concerning microscope equipment, cleaning of optical surfaces, health risks of immersion oil, and recent improvements of temporary observation media especially in connection with new developments in digital software.

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Section: Microscope Equipmentmentioning

confidence: 99%

Collection management and study of microscope slides: Storage, profiling, deterioration, restoration procedures, and general recommendations

Neuhaus

Schmid

Riedel

2017

Zootaxa

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“…The CLSM images of these parasite eggs revealed detailed morphological features that were helpful in identification efforts. Many of these subtle anatomical features are less visible during LM analysis of archaeoparasitological specimens, thus using advanced microscopy techniques, such as CLSM, can be useful for determining the taxonomic designations of parasite eggs recovered from archaeological contexts [11,13]. At the time of imaging, Physaloptera sp.…”

Section: Discussionmentioning

confidence: 99%

Application of Autofluorescence for Confocal Microscopy to Aid in Archaeoparasitological Analyses

Morrow

Elowsky

2019

Korean J Parasitol

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Confocal laser scanning microscopy (CLSM) was used to examine archaeoparasitological specimens from coprolites associated with La Cueva de los Muertos Chiquitos (CMC) located near present-day Durango, Mexico. The eggs for 4 different types of parasites recovered from CMC coprolites were imaged using CLSM to assist with identification efforts. While some of the parasite eggs recovered from CMC coprolites were readily identified using standard light microscopy (LM), CLSM provided useful data for more challenging identifications by highlighting subtle morphological features and enhancing visualization of parasite egg anatomy. While other advanced microscopy techniques, such as scanning electron microscopy (SEM), may also detect cryptic identifying characters, CLSM is less destructive to the specimens. Utilizing CLSM allows for subsequent examinations, such as molecular analyses, that cannot be performed following SEM sample preparation and imaging. Furthermore, CLSM detects intrinsic autofluorescence molecules, making improved identification independent of resource and time-intensive protocols. These aspects of CLSM make it an excellent method for assisting in taxonomic identification and for acquiring more detailed images of archaeoparasitological specimens.

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“…An Olympus BX60 microscope was used for analysis of the structure of the cyst and cuticular capsules using brightfield (BF) and differential interference contrast (DIC). Moreover, filters for detecting UV autofluorescence according to Perry et al [25] were used in the analysis. Permanent slides were prepared using Hoyer's mounting medium (prepared according to Morek et al [26] and closed with a coverslip.…”

Section: Methodsmentioning

confidence: 99%

Analysis of Encystment, Excystment, and Cyst Structure in Freshwater Eutardigrade Thulinius ruffoi (Tardigrada, Isohypsibioidea: Doryphoribiidae)

Janelt

Poprawa

2020

Diversity

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Encystment in tardigrades is relatively poorly understood. It is seen as an adaptive strategy evolved to withstand unfavorable environmental conditions. This process is an example of the epigenetic, phenotypic plasticity which is closely linked to the molting process. Thulinius ruffoi is a freshwater eutardigrade and a representative of one of the biggest eutardigrade orders. This species is able to form cysts. The ovoid-shaped cysts of this species are known from nature, but cysts may also be obtained under laboratory conditions. During encystment, the animals undergo profound morphological changes that result in cyst formation. The animals surround their bodies with cuticles that isolate them from the environment. These cuticles form a cuticular capsule (cyst wall) which is composed of three cuticles. Each cuticle is morphologically distinct. The cuticles that form the cuticular capsule are increasingly simplified. During encystment, only one, unmodified and possibly functional buccal-pharyngeal apparatus was found to be formed. Apart from the feeding apparatus, the encysted specimens also possess a set of claws, and their body is covered with its own cuticle. As a consequence, the encysted animals are fully adapted to the active life after leaving the cyst capsule.

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Looking at tardigrades in a new light: using epifluorescence to interpret structure

Cited by 19 publications

References 21 publications

Collection management and study of microscope slides: Storage, profiling, deterioration, restoration procedures, and general recommendations

Collection management and study of microscope slides: Storage, profiling, deterioration, restoration procedures, and general recommendations

Application of Autofluorescence for Confocal Microscopy to Aid in Archaeoparasitological Analyses

Analysis of Encystment, Excystment, and Cyst Structure in Freshwater Eutardigrade Thulinius ruffoi (Tardigrada, Isohypsibioidea: Doryphoribiidae)

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