Advanced Correlative Light/Electron Microscopy: Current Methods and New Developments Using Tokuyasu Cryosections

Microscopy is an essential tool for analysis of cellular structures and function. With the advent of new fluorescent probes and super-resolution light microscopy techniques, the study of dynamic processes in living cells has been greatly facilitated. Fluorescence light microscopy provides analytical, quantitative, and three-dimensional (3D) data with emphasis on analysis of live cells using fluorescent markers. Sample preparation is easy and relatively inexpensive, and the use of appropriate tags provides the ability to track specific proteins of interest. Of course, only electron microscopy (EM) achieves the highest definition in terms of ultrastructure and protein labeling. To fill the gap between light microscopy and EM, correlative light and electron microscopy (CLEM) strategies have been developed. In particular, hybrid techniques based upon immuno-EM provide sensitive protein detection combined with high-resolution information on cell structures and protein localization. By adding the third dimension to EM with electron tomography (ET) combined with rapid freezing, CLEM techniques now provide additional tools for quantitative 3D analysis. Here, we overview the major methods applied and highlight the latest advances in the field of CLEM. We then focus on two selected techniques that use cryosections as substrate for combined biomolecular imaging. Finally, we provide a perspective of future developments in the field. (J Histochem Cytochem 57:1103–1112, 2009)     

辽宁省辽河水生态系统健康评价

2009年6-8月,在对辽宁省辽河铁岭段、沈阳段、盘锦段20个断面水文、水质、着生藻类、栖息地状况实地调查的基础上,采用主成分分析方法,进行指标的筛选与指标权重的确定,构建了该河流水生态系统健康评价指标体系和健康评价标准体系,并用改进的灰色关联度法对辽河6个断面的水生态系统健康状况进行了评价.结果表明：辽宁省辽河3个评价断面的水生态系统健康程度一般,2个评价断面分别为较差和极差,仅有1个评价断面达到了亚健康程度,说明辽河河流水生态系统生态退化严重,需要进一步加强辽河水系的生态恢复及水环境污染综合治理.     

Immuno‐ and Correlative Light Microscopy‐Electron Tomography Methods for 3D Protein Localization in Yeast

Compartmentalization of eukaryotic cells is created and maintained through membrane rearrangements that include membrane transport and organelle biogenesis. Three‐dimensional reconstructions with nanoscale resolution in combination with protein localization are essential for an accurate molecular dissection of these processes. The yeast Saccharomyces cerevisiae is a key model system for identifying genes and characterizing pathways essential for the organization of cellular ultrastructures. Electron microscopy studies of yeast, however, have been hampered by the presence of a cell wall that obstructs penetration of resins and cryoprotectants, and by the protein dense cytoplasm, which obscures the membrane details. Here we present an immuno‐electron tomography (IET) method, which allows the determination of protein distribution patterns on reconstructed organelles from yeast. In addition, we extend this IET approach into a correlative light microscopy‐electron tomography procedure where structures positive for a specific protein localized through a fluorescent signal are resolved in 3D. These new investigative tools for yeast will help to advance our understanding of the endomembrane system organization in eukaryotic cells.      

Correlative Microscopy of Vitreous Sections Provides Insights into BAR-Domain Organization In Situ

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Hierarchy establishment among potentially similar buds

A plant has a great excess of buds each with the potential of developing an entire shoot system. The general question tackled was to what extent shoot size and time of bud development are important for bud hierarchy. Pea seedlings with two shoots, which were either equal or unequal in size, were obtained by the early removal of the seminal shoot. When these two shoots were also removed, one of the two cotyledon buds next to the bases of the shoots developed into the new shoot system. The determination of which of the buds became dominant was studied as a function of the relative sizes of the two primary cotyledonary shoots, of differences in the timing of the removal of these shoots and of the size of the buds. The bud that became dominant was not necessarily the larger one, nor did it always emerge from the axil of the larger shoot. Instead, it was usually the bud that was inhibited for a shorter period by the shoot next to it. It is suggested that the fate of a bud is predominantly determined by developmental parameters, for example lime of release, which are correlated with its developmental status and not necessarily with its physical size or with the past development of its shoot.     

Higher‐order assemblies of oligomeric cargo receptor complexes form the membrane scaffold of the Cvt vesicle

Selective autophagy is the mechanism by which large cargos are specifically sequestered for degradation. The structural details of cargo and receptor assembly giving rise to autophagic vesicles remain to be elucidated. We utilize the yeast cytoplasm‐to‐vacuole targeting (Cvt) pathway, a prototype of selective autophagy, together with a multi‐scale analysis approach to study the molecular structure of Cvt vesicles. We report the oligomeric nature of the major Cvt cargo Ape1 with a combined 2.8 Å X‐ray and negative stain EM structure, as well as the secondary cargo Ams1 with a 6.3 Å cryo‐EM structure. We show that the major dodecameric cargo prApe1 exhibits a tendency to form higher‐order chain structures that are broken upon interaction with the receptor Atg19 in vitro. The stoichiometry of these cargo–receptor complexes is key to maintaining the size of the Cvt aggregate in vivo. Using correlative light and electron microscopy, we further visualize key stages of Cvt vesicle biogenesis. Our findings suggest that Atg19 interaction limits Ape1 aggregate size while serving as a vehicle for vacuolar delivery of tetrameric Ams1.     

A comparison of absolute performance of different correlative and mechanistic species distribution models in an independent area

To investigate the comparative abilities of six different bioclimatic models in an independent area, utilizing the distribution of eight different species available at a global scale and in Australia. Global scale and Australia. We tested a variety of bioclimatic models for eight different plant species employing five discriminatory correlative species distribution models (SDMs) including Generalized Linear Model (GLM), MaxEnt, Random Forest (RF), Boosted Regression Tree (BRT), Bioclim, together with CLIMEX (CL) as a mechanistic niche model. These models were fitted using a training dataset of available global data, but with the exclusion of Australian locations. The capabilities of these techniques in projecting suitable climate, based on independent records for these species in Australia, were compared. Thus, Australia is not used to calibrate the models and therefore it is as an independent area regarding geographic locations. To assess and compare performance, we utilized the area under the receiver operating characteristic (ROC) curves (AUC), true skill statistic (TSS), and fractional predicted areas for all SDMs. In addition, we assessed satisfactory agreements between the outputs of the six different bioclimatic models, for all eight species in Australia. The modeling method impacted on potential distribution predictions under current climate. However, the utilization of sensitivity and the fractional predicted areas showed that GLM, MaxEnt, Bioclim, and CL had the highest sensitivity for Australian climate conditions. Bioclim calculated the highest fractional predicted area of an independent area, while RF and BRT were poor. For many applications, it is difficult to decide which bioclimatic model to use. This research shows that variable results are obtained using different SDMs in an independent area. This research also shows that the SDMs produce different results for different species; for example, Bioclim may not be good for one species but works better for other species. Also, when projecting a “large” number of species into novel environments or in an independent area, the selection of the “best” model/technique is often less reliable than an ensemble modeling approach. In addition, it is vital to understand the accuracy of SDMs' predictions. Further, while TSS, together with fractional predicted areas, are appropriate tools for the measurement of accuracy between model results, particularly when undertaking projections on an independent area, AUC has been proved not to be. Our study highlights that each one of these models (CL, Bioclim, GLM, MaxEnt, BRT, and RF) provides slightly different results on projections and that it may be safer to use an ensemble of models.     

Cone-shaped HIV-1 capsids are transported through intact nuclear pores

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