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Automated Microscopy

The traditional microscope that you know and love is operated manually. Picture the scene: the microscopist chooses the light source, gently places the sample the moveable stage, selects the objective lens, and scans to select the field of view. This process is perfect for processing and analyzing a small number of samples per day. But…

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Data Analysis for Three-dimensional Volume Scanning Electron Microscopy

In recent years, three-dimensional (3D) scanning electron microscopy techniques have gained recognition in the biological sciences. In particular, array tomography, serial block face scanning electron microscopy (SBFSEM) and focused ion beam scanning electron microscopy (FIBSEM) (described in Three-Dimensional Scanning Electron Microscopy for Biology) have shown an increase in biological applications, elucidating ultrastructural details of cells…

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Three-Dimensional Scanning Electron Microscopy for Biology

Scanning electron microscopy (SEM) is a powerful technique, traditionally used for imaging the surface of cells, tissues and whole multicellular organisms (see An Introduction to Electron Microscopy for Biologists)(Fig. 1). While the resultant images appear to be three dimensional (3D), they actually contain no depth information. However, there are several SEM techniques that can obtain…

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How to Troubleshoot Problems with Fluorescently Tagged Proteins

Using fluorescent proteins as imaging probes is a widespread and versatile technique in microscopy. You can use them in a wide range of living systems, from single cultured cells to complete organisms and animals. Fluorescently tagged proteins can be used to track and examine real-time localization, interactions and translocation of your protein of interest, as…

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Live-Cell Imaging: Choosing the Right Technique

If you want to see in real time what is going on inside your cell then you should be performing live-cell imaging. Live-cell imaging techniques allow real-time examination of almost every aspect of cellular function under normal and experimental conditions. With all live-cell imaging experiments, the main challenges are to keep your cells alive and healthy…

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Beginners Guide to PALM Sample Preparation

In the first part of this article series we went through how Photoactivated Localization Microscopy (PALM) works. Now that you have a good understanding of the technique, it is time to start thinking about how to prepare a good sample for PALM. In this article we will cover how to choose which fluorescent proteins to…

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An introduction to Photoactivated Localization Microscopy (PALM)

A Nobel Prize-winning breakthrough Superresolution microscopy has stepped into the lime light earlier this year when Eric Betzig, Stefan Hell and William Moerner were awarded the Nobel Prize in Chemistry for their work on breaking through the diffraction barrier of fluorescence microscopy. The part Eric Betzig played in this ground-breaking achievement was the invention of…

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A Beginners Guide to The Point Spread Function

Suppose that you have a tiny fluorescent object, such as a 10nm-diameter fluorescent bead or even a single fluorescent molecule, and you try to observe it under a fluorescence microscope.  Provided that the object is bright enough, even though it is well below the resolution limit of your microscope you can still see the object;…

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