光学透明技术在植物多尺度成像中的应用

光学透明技术是一种通过各种化学试剂,将原本不透明的生物样本实现透明化,并在光学显微镜下深度成像的技术。结合多种光学显微成像新技术,光学透明技术可对整个组织进行成像和三维重建,深度剖析生物体内部空间特征与形成机制。近年来,多种植物光学透明技术和多尺度成像技术被陆续研发,并取得了丰硕的研究成果。该文综述了生物体光学透明技术的基本原理和一些新技术,重点介绍基于光学透明技术开发的新型成像方法及其在植物成像与细胞生物学中的应用,为后续植物整体、组织或器官的透明、成像与三维重构及功能研究提供理论依据和技术支持。     

Chemical clearing and dehydration of GFP expressing mouse brains

Generally, chemical tissue clearing is performed by a solution consisting of two parts benzyl benzoate and one part benzyl alcohol. However, prolonged exposure to this mixture markedly reduces the fluorescence of GFP expressing specimens, so that one has to compromise between clearing quality and fluorescence preservation. This can be a severe drawback when working with specimens exhibiting low GFP expression rates. Thus, we screened for a substitute and found that dibenzyl ether (phenylmethoxymethylbenzene, CAS 103-50-4) can be applied as a more GFP-friendly clearing medium. Clearing with dibenzyl ether provides improved tissue transparency and strikingly improved fluorescence intensity in GFP expressing mouse brains and other samples as mouse spinal cords, or embryos. Chemical clearing, staining, and embedding of biological samples mostly requires careful foregoing tissue dehydration. The commonly applied tissue dehydration medium is ethanol, which also can markedly impair GFP fluorescence. Screening for a substitute also for ethanol we found that tetrahydrofuran (CAS 109-99-9) is a more GFP-friendly dehydration medium than ethanol, providing better tissue transparency obtained by successive clearing. Combined, tetrahydrofuran and dibenzyl ether allow dehydration and chemical clearing of even delicate samples for UM, confocal microscopy, and other microscopy techniques.     

Rapid chemical clearing of white matter in the post-mortem human brain by 1,2-hexanediol delipidation

Three-dimensional (3D) imaging based on chemical tissue clearing in the post-mortem human brain is a promising approach for stereoscopic understanding of central nervous system diseases. Especially, delipidation of lipid-rich white matter (WM) is a rate-determining step in human brain clearing by hydrophilic reagents. In this study, we described the rapid delipidation of WM by a 1,2-hexanediol (HxD)-based aqueous solution. HxD delipidation enabled rapid clearing of a formalin-fixed human brain specimen including the WM. Although harsh HxD delipidation was applied to the brain tissue, conventional pathological staining patterns and various types of antigenicity were sufficiently preserved. Furthermore, HxD delipidation was compatible with 3D imaging of fluorescently-labeled tissue samples. HxD delipidation could be useful in future 3D neuropathological diagnosis.     

Role of extracellular reactive sulfur metabolites on microbial Se(0) dissolution

The dissolution of elemental selenium [Se(0)] during chemical weathering is an important step in the global selenium cycle. While microorganisms have been shown to play a key role in selenium dissolution in soils, the mechanisms of microbial selenium solubilization are poorly understood. In this study, we isolated a Bacillus species, designated as strain JG17, that exhibited the ability to dissolve Se(0) under oxic conditions and neutral pH. Growth of JG17 in a defined medium resulted in the production and accumulation of extracellular compounds that mediated Se(0) dissolution. Analysis of the spent medium revealed the presence of extracellular sulfite, sulfide, and thiosulfate. Abiotic Se(0) dissolution experiments with concentrations of sulfite, sulfide, and thiosulfate relevant to our system showed similar extents of selenium solubilization as the spent medium. Together, these results indicate that the solubilization of Se(0) by JG17 occurs via the release of extracellular inorganic sulfur compounds followed by chemical dissolution of Se(0) by the reactive sulfur metabolites. Our findings suggest that the production of reactive sulfur metabolites by soil microorganisms and the formation of soluble selenosulfur complexes can promote selenium mobilization during chemical weathering.     

Chemical and biological leaching of enargite

Enargite (Cu 3 AsS 4 ) was leached faster by bacteria in sulfuric acid medium (pH 1.6) with added ferric sulfate than by chemical leaching at the same or higher iron concentration. During chemical leaching with ferric iron, the copper dissolution rate decreased from an initial value of 0.03% per hour to a value of 0.002% per hour. Enargite is oxidized to elemental sulfur and dissolved arsenic (As 3+ and As 5+ ). Less than 10% of sulfur is oxidized to sulfate. The arsenic and copper dissolutions observed in bacterial leaching experiments suggest the existence of a specific bacterial action on the leaching of enargite, demonstrated by the ability of bacteria to oxidize enargite at very low concentration of dissolved iron and by the higher dissolution rate obtained in bacterial leaching compared to chemical ferric leaching.     

Determining the impacts of fermentative bacteria on wollastonite dissolution kinetics

Silicate minerals can be a source of calcium and alkalinity, enabling CO₂ sequestration in the form of carbonates. For this to occur, the mineral needs to be first dissolved in an acidifying process such as the biological process of anaerobic fermentation. In the present study, the main factors which govern the dissolution process of an alkaline silicate mineral (wollastonite, CaSiO₃) in an anaerobic fermentation process were determined. Wollastonite dissolution kinetics was measured in a series of chemical batch experiments in order to be able to estimate the required amount of alkaline silicate that can neutralize the acidifying fermentation process. An anaerobic fermentation of glucose with wollastonite as the neutralizing agent was consequently performed in a fed-batch reactor. Results of this experiment were compared with an abiotic (control) fed-batch reactor in which the fermentation products (i.e. organic acids and alcohols) were externally supplied to the system at comparable rates and proportions, in order to provide chemical conditions similar to those during the biotic (fermentation) experiment. This procedure enabled us to determine whether dissolution of wollastonite was solely enhanced by production of organic acids or whether there were other impacts that fermentative bacteria could have on the mineral dissolution rate. The established pH profiles, which were the direct indicator of the dissolution rate, were comparable in both experiments suggesting that the mineral dissolution rate was mostly influenced by the quantity of the organic acids produced.     

Chemical Dehydration for Rapid Paraffin Embedding

We describe chemical dehydration with 2,2-dimethoxypropane (DMP) for rapid paraffin embedding using a mixture of DMP and mineral oil followed by mineral oil as clearing intermediates. This method is useful for classical histological techniques as well as for histochemistry and immunocytochemistry.     

恢复东湖水生生态系统途径初探

借鉴国内外其他湖泊的治理经验 ,讨论了如何恢复东湖水生生态系统的一些途径 ,其中探讨了①物理方法 :截污 ,清淤与引水冲污 ;②化学方法 :化学除磷与抑藻或杀藻 ;③生物方法 :用生物量输出氮磷与用生物方法对小型浮游藻类进行控制。     

Effects of Submerged Vegetation on Water Clarity Across Climates

A positive feedback between submerged vegetation and water clarity forms the backbone of the alternative state theory in shallow lakes. The water clearing effect of aquatic vegetation may be caused by different physical, chemical, and biological mechanisms and has been studied mainly in temperate lakes. Recent work suggests differences in biotic interactions between (sub)tropical and cooler lakes might result in a less pronounced clearing effect in the (sub)tropics. To assess whether the effect of submerged vegetation changes with climate, we sampled 83 lakes over a gradient ranging from the tundra to the tropics in South America. Judged from a comparison of water clarity inside and outside vegetation beds, the vegetation appeared to have a similar positive effect on the water clarity across all climatic regions studied. However, the local clearing effect of vegetation decreased steeply with the contribution of humic substances to the underwater light attenuation. Looking at turbidity on a whole-lake scale, results were more difficult to interpret. Although lakes with abundant vegetation (>30%) were generally clear, sparsely vegetated lakes differed widely in clarity. Overall, the effect of vegetation on water clarity in our lakes appears to be smaller than that found in various Northern hemisphere studies. This might be explained by differences in fish communities and their relation to vegetation. For instance, unlike in Northern hemisphere studies, we find no clear relation between vegetation coverage and fish abundance or their diet preference. High densities of omnivorous fish and coinciding low grazing pressures on phytoplankton in the (sub)tropics may, furthermore, weaken the effect of vegetation on water clarity.     

Managing invasions at the cost of native habitat? An experimental test of the impact of fire on the invasion of <Emphasis Type="Italic">Chromolaena odorata</Emphasis> in a South African savanna

Successfully managing invasive plants in natural systems is extremely difficult. Recently however, progress has been made with an approach focused on changing ecosystem processes through the disturbance regime. We performed a large-scale (3 ha) full-factorial field experiment in densely invaded woodland in Hluhluwe-iMfolozi Park, a savanna reserve in South Africa, to study the effect of fire on the control of the pan-tropical invasive exotic shrub Chromolaena odorata in combination with the conventional method, i.e. manual clearing and herbicide application. We show how fire interacted with the conventional clearing of C. odorata and induced an intense canopy fire that caused a shift from woodland to grassland. After 2.5 years of monitoring, grasses were still dominant and re-invasion minimal. It is important to note that fire without prior clearing did not have the same effect and was not successful in reducing densities of C. odorata. An integrated control practice targeting the species with mechanical and chemical methods, while simultaneously targeting its habitat through fire, effectively controlled dense C. odorata thickets during the course of the experiment. However, this approach transformed regular surface fires into high-intensity canopy fires that are rare in savannas. We discuss how this altered fire regime may threaten native habitats, including fire-sensitive forest patches and riverine woodlands within the savanna mozaic. This is an important dilemma for managers that should not be overlooked and asks for long-term data on the impact of control programs on the native vegetation.     

Goethite Dissolution in the Presence of Phytosiderophores: Rates, Mechanisms, and the Synergistic Effect of Oxalate

The purpose of this study was the elucidation of the chemical mechanism of an important process in iron acquisition by graminaceous plants: the dissolution of iron oxides in the presence of phytosiderophores. We were particularly interested in the effects of diurnal root exudation of phytosiderophores and of the presence of other organic ligands in the rhizosphere of graminaceous plants on the dissolution mechanism. Phytosiderophores of the type 2′-deoxymugineic acid (DMA) were purified from the root exudates of wheat plants (Triticum aestivum L. cv. Tamaro). DMA-promoted dissolution of goethite under steady-state and non-steady-state conditions and its dependence on pH, adsorbed DMA concentration, and the presence of the organic ligand oxalate were studied. We show that dissolution of goethite by phytosiderophores follows a surface controlled ligand promoted dissolution mechanism. We also found that oxalate, an organic ligand commonly found in rhizosphere soils, has a synergistic effect on the steady-state dissolution of goethite by DMA. Under non-steady-state addition of the phytosiderophore, mimicking the diurnal exudation pattern of phytosiderophore release, a fast dissolution of iron is triggered in the presence of oxalate. To investigate the efficiency of these mechanisms in plant iron acquisition, wheat plants were grown on a substrate amended with goethite as only iron source. The chlorophyll status of these plants was similar to iron-fertilized plants and significantly higher than in plants grown in iron free nutrient solutions. This demonstrates that wheat can efficiently mobilize iron, even from well crystalline goethite that is usually considered unavailable for plant nutrition.     

Using wintergreen oil for mounting mosquito larvae: a safer alternative to xylene

Permanent mounting of fourth instar mosquito larvae is essential for identifying Aedes spp. This procedure requires extensive exposure to xylene, a clearing agent in the mounting process. We investigated wintergreen oil as a substitute for xylene. Five hundred larvae were mounted on slides to evaluate shrinkage or expansion of specimens after clearing using xylene or wintergreen oil. We examined the ventral brush and siphonal hair tufts for species identification and for preservation of morphological characteristics after clearing specimens in xylene or wintergreen oil. Shrinkage of the length of whole larvae and width of the head, thorax and abdomen after mounting was significantly greater after clearing with xylene than with wintergreen oil. The length of the comb scale nearest the ventral brush was similar for both clearing agents. The clarity of the specimens after mounting was improved by clearing with wintergreen oil, but the integrity of the ventral brush and siphonal hair tufts were similar for both clearing agents.     

Offsets for land clearing: No net loss or the tail wagging the dog?

Summary   Offsets (also known as mitigation banks, compensatory habitat, set-asides) is a policy instrument recently introduced in several States in Australia to permit some land clearing while striving for no net loss in the extent and condition of native vegetation overall. Offsetting is criticized with respect to the amount of gain required to compensate for losses from clearing, the equivalence of losses and gains, the time lag between losses and gains and a poor record of compliance. Despite these criticisms, we conclude that offsetting is a useful policy instrument while governments continue to permit some clearing of native vegetation. However, offsets will only contribute to no net loss if (i) clearing is restricted to vegetation that is simplified enough so that its functions can be restored elsewhere with confidence or clearing is restricted to vegetation that is unlikely to persist and is not practicable to restore irrespective of clearing; (ii) any temporary loss in habitat between clearing and the maturation of an offset, or differences between the habitat lost from clearing and gained through an offset, does not represent significant risk to a species, population or ecosystem process; (iii) there will be gains of sufficient magnitude on the offset site to compensate for losses from clearing; (iv) best practice adaptive management is applied to offsets; (v) offsets are in place for at least the same duration as the impacts from clearing; and (vi) there is adequate compliance. Land clearing with offsets outside these parameters is inconsistent with 'no net loss'.     

Microwave-assisted technology for the clearing and staining of arbuscular mycorrhizal fungi in roots

The use of microwave irradiation as a source of energy to clear and stain intra-radical arbuscular mycorrhizal fungi propagules has been tested on a variety of indigenous and cultivated herbaceous plants. The aim of the study was to evaluate the efficiency of microwave irradiation on root softening, fungi tissue staining, and preservation of DNA integrity for subsequent molecular analyses. The proposed methodology has been adapted from the standard procedures used to detect and quantify mycorrhizal root colonization levels. Using a domestic microwave oven, tissue clearing and staining required together between 30 s and 1.5 min of microwave treatment to be completed, depending the diameter size of the roots. The well-performing chemical stains tested were acid fuchsin, trypan blue, and aniline blue. The acid fuchsin clearing and staining processes, as performed, were also demonstrated to preserve DNA integrity for further molecular analyses. Irradiation by microwaves has been used with success in our laboratory within the frame of several studies. It offers considerable time saving over traditional method, reducing processing times from several hours to a few minutes while decreasing considerably the amount of chemicals and energy required to perform analyses.     

Formulation and Stability Testing of Itraconazole Crystalline Nanoparticles

Itraconazole (ITZ) crystalline nanoparticles were prepared using relatively simple, low-cost sonoprecipitation technique, in which both the solvent and antisolvent were organic in nature. The effect of stabilizer type (hydroxypropyl methylcellulose, hydroxypropyl cellulose, Inutec SP1®, and pluronic F127), drying method (oven and freeze drying) and matrix former used (Avicel PH101, and Aerosil®200) on the dissolution performance as a key characteristic of nanocrystals was evaluated. In 10 min, all of the prepared nanocrystals showed 3.77−8.59 times improvement in percent drug dissolved compared to pure ITZ. Concerning the effect of stabilizer type, the following rank order can be given: pluronic F127 ≥ hydroxypropyl cellulose ≥ hydroxypropyl methylcellulose (HPMC) > inutec SP1. Freeze-dried ITZ nanocrystals containing Avicel PH 101 showed better dissolution rate compared to other nanocrystals. The chemical structure of itraconazole nanocrystals was not changed as revealed by Fourier transform infrared. Stability study of selected nanocrystals (F5, F7, and F8) revealed physical and chemical stability of F7 and F8, while a decrease in dissolution rate of F5 was observed (although being chemically stable) when stored under high relative humidity conditions. Although inutec is less potent than pluronic F127 and HPMC regarding their effect on dissolution rate enhancement, it is equipotent to pluronic F127 in preserving the rapid drug dissolution.Key words: itraconazole, nanocrystals, nanoparticles, stability study     

Size-exclusion chromatography of enzymatically treated cellulose and related polysaccharides: a review

Size-exclusion chromatography (SEC) of wood pulp polysaccharides (cellulose and hemicelluloses) has become widely used for the analysis of these polymers, including monitoring of transformations occurring under various chemical and biological processes. The present review provides information on the different procedures and methodologies developed for the characterization of molecular mass distribution (MMD) and chemical composition of cellulose and hemicelluloses by SEC. This paper also includes the significant results obtained in the characterization of enzymatically treated wood polysaccharides. Conventional SEC with double detection (refractometric and UV), "hyphenated" with a fractional dissolution, was demonstrated to be an efficient method for simultaneous determination of MMD and chemical heterogeneity.     

根尖整体透明技术改良

整体透明观察技术是植物形态发育研究的基础手段之一, 是无需制作切片直接观察植物体内部形态结构的有效方法。该技术采用高折射率介质降低光在样品中的散射, 提高光通量, 增加视野深度, 从而实现组织样品透明观察。然而透明剂能改变透明液的渗透势和pH值, 从而对细胞形态保持产生负面影响。目前, 针对植物叶片和胚珠已建立了相对成熟的整体透明观察体系, 但根尖由于细胞壁较薄, 现有的整体透明方法常导致细胞形态改变, 不确定性增加(如根尖整体形态改变和细胞发生严重的质壁分离)。该研究以拟南芥(Arabidopsis thaliana)幼苗为实验材料, 通过检测根尖形态、细胞质壁分离情况和细胞清晰度, 对常用的透明液组分、pH值和透明时间进行优化, 旨在建立一种适用于根尖等较脆弱组织材料的整体透明方法。     

Chemical weathering of soil minerals and the role of biological processes

Existing weathering models based on direct, abiotic factors, and biological factors through systemic feedbacks seem to describe the weathering rates observed in the field with good accuracy. The Swedish weathering rate model, PROFILE is the only existing model with this capacity. The PROFILE model is widely used and has been used successfully in sustainability assessments for forests and agriculture. The author has reviewed available experiments, as well as existing knowledge on chemical weathering kinetics and concludes that the physical and chemical conditions existing in forest soils do not allow any significant direct surface actions on minerals by microorganisms or tree roots. The reported tracks and holes in minerals have been mistakenly identified as being produced by roots, but this misunderstanding is caused by a lack of understanding of crystallography and chemical dissolution mechanisms. The holes and tracks have been produced by known chemical mechanisms.     

Optical clearing for improved contrast in second harmonic generation imaging of skeletal muscle

Using second harmonic generation (SHG) imaging microscopy, we have examined the effect of optical clearing with glycerol to achieve greater penetration into specimens of skeletal muscle tissue. We find that treatment with 50% glycerol results in a 2.5-fold increase in achievable SHG imaging depth. Signal processing analyses using fast Fourier transform and continuous wavelet transforms show quantitatively that the periodicity of the sarcomere structure is unaltered by the clearing process and that image quality deep in the tissue is improved with clearing. Comparison of the SHG angular polarization dependence also shows no change in the supramolecular organization of acto-myosin complexes. By contrast, identical treatment of mouse tendon (collagen based) resulted in a strong decrease in SHG response. We suggest that the primary mechanism of optical clearing in muscle with glycerol treatment results from the reduction of cytoplasmic protein concentration and concomitant decrease in the secondary inner filter effect on the SHG signal. The lack of glycerol concentration dependence on the imaging depth indicates that refractive index matching plays only a minor role in the optical clearing of muscle. SHG and optical clearing may provide an ideal mechanism to study physiology in highly scattering skeletal or cardiac muscle tissue with significantly improved depth of penetration and achievable imaging depth.     

A guidebook for DISCO tissue clearing

Histological analysis of biological tissues by mechanical sectioning is significantly time‐consuming and error‐prone due to loss of important information during sample slicing. In the recent years, the development of tissue clearing methods overcame several of these limitations and allowed exploring intact biological specimens by rendering tissues transparent and subsequently imaging them by laser scanning fluorescence microscopy. In this review, we provide a guide for scientists who would like to perform a clearing protocol from scratch without any prior knowledge, with an emphasis on DISCO clearing protocols, which have been widely used not only due to their robustness, but also owing to their relatively straightforward application. We discuss diverse tissue‐clearing options and propose solutions for several possible pitfalls. Moreover, after surveying more than 30 researchers that employ tissue clearing techniques in their laboratories, we compiled the most frequently encountered issues and propose solutions. Overall, this review offers an informative and detailed guide through the growing literature of tissue clearing and can help with finding the easiest way for hands‐on implementation.