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In these pages you will find tutorials designed to explain, in an accessible way, some of the principles behind the techniques used in our lab. They range from the very general (How does a microscope work?) to very specific approaches used in Three-Dimensional Electron Microscopy (3D-EM). This wiki is, by definition, a "work in progress"; we will periodically add new material and update what's already here. Blue links are for tutorials already available while red ones are for those we are still developing.
In the Leschziner Lab, we use electron microscopy to study structures of macromolecules. In particular, we look at single particles and use the techniques described in the sections below to generate three-dimensional structures of the proteins or protein complexes of interest. In addition to the study of protein structure, our lab is interested in improving techniques for 3D-EM.
We hope you find the material useful and welcome feedback on how to improve it. To give us feedback, please see the discussion tab at the top of the page, or e-mail us.
Contents
Introduction
The compound optical microscope, first recognized in 1590, is perhaps the most familiar of all laboratory instruments. The much younger electron microscope, for many years simply an exotic rarity, has also become a standard tool in biological research. As the convenience and accessibility of each has grown over the years, however, the understanding of relevant theory or even how the microscope itself functions has been lost among many users. While a firm understanding of the fundamental principles of optics and imaging is not often necessary for general function, eventually this knowledge becomes necessary for the proper use, interpretation of images, and choices of specific application. This is especially relevant for those who wish to innovate, or even effectively trouble-shoot, in the field.
This wiki is intended to provide both a brief overview of the fundamental concepts, as well as a more in-depth theoretical view for those readers who would like to examine more of the mathematics and physics behind imaging (such as with image formation, contrast, resolution limits). In particular, Fourier imaging will be discussed as it applies to the interpretation of data.
Since our focus is not directed toward crystalline data sets but rather what can be referred to as asymmetric structures, the image processing and data interpretation sections will deal with techniques particular to the latter.
General imaging principles
These conventions and basic physics concepts are fundamental tools for understanding optical and electron imaging.
Electron imaging
There are many caveats to imaging with electrons as opposed to photons.
- What is an Electron Microscope?
- History of the Electron Microscope
- Types of Signals
- Electron optics
- The Weak-Phase Object Approximation
- Contrast
- Ewald Sphere
- Aberrations
- Special Imaging Techniques
Electron Microscopy of Macromolecules
(Pipeline Picture)
Two-Dimensional Averaging
Once the data from imaging the macromolecules of interest are collected, several steps are taken to sift through the images and create a three-dimensional model.
Three-dimensional reconstruction by EM
A "30 Second Overview" of three-dimensional EM reconstruction.
Basics
Existing Reconstruction Methods
- Angular Reconstitution (AR)
- Random Conical Tilt (RCT)
- Orthogonal Tilt Reconstruction (OTR)
Software Available
An overview of the currently available software, links, and general descriptions.
Resolution
The concept of resolution in electron microscopy has brought out several debates in the past.
Hardware Innovations to the Electron Microscope
Some new, some old, there are many aspects of the microscope that may be changed, ostensibly altering the physics of image formation for the better.
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