High Data Output and Automated 3D Correlative Light–Electron Microscopy Method

Vicidomini, Giuseppe; Gagliani, Maria Cristina; Canfora, Michela; Cortese, Katia; Frosi, Fabio; Santangelo, Clara; Fiore, Pier Paolo Di; Boccacci, Patrizia; Diaspro, Alberto; Tacchetti, Carlo

doi:10.1111/j.1600-0854.2008.00815.x

Cited by 48 publications

(44 citation statements)

References 34 publications

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“…Furthermore, data fusion and LM-EM image matching are challenging issues and correspond to a large variety of scales, image contents and noises. As shown in [132], LM images are typically blurred when compared to EM images.…”

Section: Perspectives For Image Processing and Analysis Methodsmentioning

confidence: 99%

A Guided Tour of Selected Image Processing and Analysis Methods for Fluorescence and Electron Microscopy

Kervrann

Sorzano

Acton

et al. 2016

IEEE J. Sel. Top. Signal Process.

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Abstract-Microscopy imaging, including fluorescence microscopy and electron microscopy, has taken a prominent role in life science research and medicine due to its ability to investigate the 3D interior of live cells and organisms. A long-term research in bio-imaging at the sub-cellular and cellular scales consists then in inferring the relationships between the dynamics of macromolecules and their functions. In this area, image processing and analysis methods are now essential to understand the dynamic organization of groups of interacting molecules inside molecular machineries and to address issues in fundamental biology driven by advances in molecular biology, optics and technology. In this paper, we present recent advances in fluorescence and electron microscopy and we focus on dedicated image processing and analysis methods required to quantify phenotypes for a limited number but typical studies in cell imaging.

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Section: Perspectives For Image Processing and Analysis Methodsmentioning

confidence: 99%

A Guided Tour of Selected Image Processing and Analysis Methods for Fluorescence and Electron Microscopy

Kervrann

Sorzano

Acton

et al. 2016

IEEE J. Sel. Top. Signal Process.

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“…Hence, although new preparation techniques are emerging, we think that the Tokuyasu method stands out beyond other procedures for its simplicity, efficiency, and rapidity of observable results. The potential of immuno-EM is illustrated below in two CLEM protocols that have been recently released: the software-based high-data-output CLEM (HDO-CLEM) and the live-cell CLEM (van Rijnsoever et al 2008;Vicidomini et al 2008). …”

Section: Clem and Tokuyasu Cryosections: Exploiting The Advantages Ofmentioning

confidence: 99%

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

Cortese

Diaspro

Tacchetti

2009

J Histochem Cytochem.

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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.

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“…Cryo-fixation 29 Freeze-substitution 30 Tokuyasu cryo-sectioning 31 Immuno-(gold) labelling 32 3 3 a b s t r a c t 34 In recent years correlative microscopy, combining the power and advantages of different imaging system, 35 e.g., light, electrons, X-ray, NMR, etc., has become an important tool for biomedical research. Among all 36 the possible combinations of techniques, light and electron microscopy, have made an especially big step 37 forward and are being implemented in more and more research labs.…”

mentioning

confidence: 99%

Correlative microscopy

Loussert

Humbel

2015

Archives of Biochemistry and Biophysics

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In recent years correlative microscopy, combining the power and advantages of different imaging system, e.g., light, electrons, X-ray, NMR, etc., has become an important tool for biomedical research. Among all the possible combinations of techniques, light and electron microscopy, have made an especially big step forward and are being implemented in more and more research labs. Electron microscopy profits from the high spatial resolution, the direct recognition of the cellular ultrastructure and identification of the organelles. It, however, has two severe limitations: the restricted field of view and the fact that no live imaging can be done. On the other hand light microscopy has the advantage of live imaging, following a fluorescently tagged molecule in real time and at lower magnifications the large field of view facilitates the identification and location of sparse individual cells in a large context, e.g., tissue. The combination of these two imaging techniques appears to be a valuable approach to dissect biological events at a submicrometer level. Light microscopy can be used to follow a labelled protein of interest, or a visible organelle such as mitochondria, in time, then the sample is fixed and the exactly same region is investigated by electron microscopy. The time resolution is dependent on the speed of penetration and fixation when chemical fixatives are used and on the reaction time of the operator for cryo-fixation. Light microscopy can also be used to identify cells of interest, e.g., a special cell type in tissue or cells that have been modified by either transfections or RNAi, in a large population of non-modified cells. A further application is to find fluorescence labels in cells on a large section to reduce searching time in the electron microscope. Multiple fluorescence labelling of a series of sections can be correlated with the ultrastructure of the individual sections to get 3D information of the distribution of the marked proteins: array tomography. More and more efforts are put in either converting a fluorescence label into an electron dense product or preserving the fluorescence throughout preparation for the electron microscopy. Here, we will review successful protocols and where possible try to extract common features to better understand the importance of the individual steps in the preparation. Further the new instruments and software, intended to ease correlative light and electron microscopy, are discussed. Last but not least we will detail the approach we have chosen for correlative microscopy.

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High Data Output and Automated 3D Correlative Light–Electron Microscopy Method

Cited by 48 publications

References 34 publications

A Guided Tour of Selected Image Processing and Analysis Methods for Fluorescence and Electron Microscopy

A Guided Tour of Selected Image Processing and Analysis Methods for Fluorescence and Electron Microscopy

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

Correlative microscopy

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