Correlative Light Electron Microscopy (CLEM)
CLEM (Correlative Light Electron Microscopy) combines the capabilities of two typically separate microscopy platforms: light (or fluorescent) microscopy (LM) and electron microscopy (EM). The advantage of LM is that it can provide wide field images of whole, often living, cells, but its resolution is limited. The advantage of EM is that it can provide much higher resolution images, up to molecular dimensions, but only over specific regions of a cell at a time and not in living cells. CLEM combines the advantages of both techniques, allowing scientists to spot cellular structures and processes of interest in whole cell images with LM and then zoom in for a closer look with EM. This dual examination provides valuable complementary and often unique information.
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Correlative Cryo-Fluorescence and Cryo-Scanning Electron Microscopy as a Straightforward Tool to Study Host-Pathogen Interactions
Correlative light and electron microscopy is an imaging technique that enables identification and targeting of fluorescently tagged structures with subsequent imaging at near-to-nanometer resolution. We established a novel correlative cryo-fluorescence microscopy and cryo-scanning electron microscopy workflow, which enables imaging of the studied object of interest very close to its natural state, devoid of artifacts caused for instance by slow chemical fixation. This system was tested by investigating the interaction of the zoonotic bacterium Borrelia burgdorferi with two mammalian cell lines of neural origin in order to broaden our knowledge about the cell-association mechanisms that precedes the entry of the bacteria into the cell.Read article -
Imaging of Host Cell-bacteria Interactions using Correlative Microscopy under Cryo-conditions
Pathogenic bacteria have developed intriguing strategies to establish and promote infections in their respective hosts. Most bacterial pathogens initiate infectious diseases by adhering to host cells surface. Knowledge of the interplay between the pathogenic organism and the host cells can provide fundamental new insight into the underlying mechanisms of the infectious process and therefore of the disease. Various microscopy techniques have proven to be critical tools to study these events.Read article -
Correlative In-Resin Super-Resolution and Electron Microscopy Using Standard Fluorescent Proteins
We introduce a method for correlative in-resin super-resolution fluorescence and electron microscopy (EM) of biological structures in mammalian culture cells. Cryo-fixed resin embedded samples offer superior structural preservation, performing in-resin super-resolution, however, remains a challenge.Read article -
Correlative Light and Electron Microscopy – Get Your Free CLEM e-Book for Download
The urge to go deeper into the microscopic world has led researchers to combine the versatility of the light microscopy (LM) with the resolution power of the electron microscope (EM) to produce Correlative Light and Electron Microscopy (CLEM). CLEM’s most significant and powerful characteristic for cell biology research is an ability to study the same cell using two different microscopy platforms. The Essential Knowledge Briefing describes the basics of CLEM and also reviews potential pitfalls and problems as well as tricks for solving them.Read article -
Correlating Intravital Multi-Photon Microscopy to 3D Electron Microscopy of Invading Tumor Cells Using Anatomical Reference Points
Cancer research unsing multiphoton microscopy and 3D electron microscopy. Correlative microscopy combines the advantages of both light and electron microscopy to enable imaging of rare and transient events at high resolution. Performing correlative microscopy in complex and bulky samples such as an entire living organism is a time-consuming and error-prone task.Read article -
Customized Patterned Substrates for Highly Versatile Correlative Light-Scanning Electron Microscopy
Correlative light electron microscopy (CLEM) combines the advantages of light and electron microscopy, thus making it possible to follow dynamic events in living cells at nanometre resolution. Various CLEM approaches and devices have been developed, each of which has its own advantages and technical challenges. We here describe our customized patterned glass substrates, which improve the feasibility of correlative fluorescence/confocal and scanning electron microscopy.Read article -
New Insights into Cilia and Flagella by Cryo-EM
Cilia and flagella were the first organelles to be discovered and have been studied for centuries. However, their essential role in humans and how ciliary defects cause diseases are still not well understood. Cryo-EM has recently shed new light on their inner workings and solved some long-standing mysteries, only to raise new questions on how cilia and flagella function.Read article -
ICln: A New Regulator of Non-Erythroid 4.1R Localisation and Function
To optimise the efficiency of cell machinery, cells can use the same protein (often called a hub protein) to participate in different cell functions by simply changing its target molecules. There are large data sets describing protein-protein interactions ("interactome") but they frequently fail to consider the functional significance of the interactions themselves.Read article -
A Precise and Rapid Mapping Protocol for Correlative Light and Electron Microscopy of small invertebrate organisms
CLEM (correlative live cell and electronmicroscopy) seeks to bridge the data acquired with different imaging strategies, typically between light microscopy and electron microscopy. It has been successfully applied in cell cultures, although its use in multicellular systems is hampered by difficulties in locating the ROI (region of interest).Read article -
Brief Introduction to High-Pressure Freezing
Water is the most abundant cellular constituent and therefore important for preserving cellular ultra-structure. Currently the only way to fix cellular constituents without introducing significant structural alterations is by cryo-fixation. There are currently two common methods employed; plunge freezing and high pressure freezing.Read article -
Postlipolytic Insulin-dependent Remodeling of Micro Lipid Droplets in Adipocytes
Despite the lipolysis–lipogenesis cycle being a fundamental process in adipocyte biology, very little is known about the morphological changes that occur during this process. The remodeling of lipid droplets to form micro lipid droplets (mLDs) is a striking feature of lipolysis in adipocytes, but once lipolysis ceases, the cell must regain its basal morphology.Read article -
A Genetically Encoded Tag for Correlated Light and Electron Microscopy of Intact Cells, Tissues, and Organisms
Electron microscopy (EM) achieves the highest spatial resolution in protein localization, but specific protein EM labeling has lacked generally applicable genetically encoded tags for in situ visualization in cells and tissues. Here we introduce ‘"miniSOG"’ (for mini Singlet Oxygen Generator), a fluorescent flavoprotein engineered from Arabidopsis phototropin 2.Read article -
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.Read article -
High Data Output and Automated 3D Correlative Light–Electron Microscopy Method
Correlative light/electron microscopy (CLEM) allows the simultaneous observation of a given subcellular structure by fluorescence light microscopy (FLM) and electron microscopy. The use of this approach is becoming increasingly frequent in cell biology. In this study, we report on a new high data output CLEM method based on the use of cryosections.Read article -
CLEM: Combining the Strengths of Light and Electron Microscopy
In recent years light microscopy studies have been dominated by live cell imaging while electron microscopy has been used for high-resolution studies. Latterly, there has been increasing interest in combining these techniques. This combination is called Correlative Light Electron Microscopy (CLEM). Due to the high resolution made possible by electron microscopy, artefacts induced during preparation of a sample can, however, also be clearly seen.Read article