This document discusses four types of light microscopy and compares light microscopes to electron microscopes. [1] Light microscopes use visible light and have lower maximum resolving power and magnification than electron microscopes. [2] Electron microscopes use beams of electrons which allow for greater resolving power and magnification, ranging from 10,000x to 100,000x. [3] They are able to provide detailed views of bacteria, viruses and intracellular structures that light microscopes cannot see.
This document discusses four types of light microscopy and compares light microscopes to electron microscopes. [1] Light microscopes use visible light and have lower maximum resolving power and magnification than electron microscopes. [2] Electron microscopes use beams of electrons which allow for greater resolving power and magnification, ranging from 10,000x to 100,000x. [3] They are able to provide detailed views of bacteria, viruses and intracellular structures that light microscopes cannot see.
This document discusses four types of light microscopy and compares light microscopes to electron microscopes. [1] Light microscopes use visible light and have lower maximum resolving power and magnification than electron microscopes. [2] Electron microscopes use beams of electrons which allow for greater resolving power and magnification, ranging from 10,000x to 100,000x. [3] They are able to provide detailed views of bacteria, viruses and intracellular structures that light microscopes cannot see.
This document discusses four types of light microscopy and compares light microscopes to electron microscopes. [1] Light microscopes use visible light and have lower maximum resolving power and magnification than electron microscopes. [2] Electron microscopes use beams of electrons which allow for greater resolving power and magnification, ranging from 10,000x to 100,000x. [3] They are able to provide detailed views of bacteria, viruses and intracellular structures that light microscopes cannot see.
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Four kinds of light microscopy
Pearson education Microscopy ELECTRONS
beams of electrons are used to produce images
Electron Microscopy THE LIGHT MICROSCOPE v THE ELECTRON MICROSCOPE FEATURE LIGHT MICROSCOPE ELECTRON MICROSCOPE
Electromagnetic Visible light Electrons
spectrum used 760nm (red) – 390nm app. 4nm Colours visible Monochrome Maximum app. 200nm 0.2nm resolving power Fine detail Maximum magnification x1000 – x1500 x500 000 Radiation Tungsten or quartz High voltage (50kV) source halogen lamp tungsten lamp Lenses Glass Magnets Interior Air-filled Vacuum Focussing Human eye (retina), fluorescent (TV) screen, screen photographic film photographic film Microscopy • Electron Microscopy – Light microscopes cannot resolve structures closer than 200 nm – Electron microscopes have greater resolving power and magnification – Magnifies objects 10,000X to 100,000X – Detailed views of bacteria, viruses, internal cellular structures, molecules, and large atoms – Two types • Transmission electron microscopes • Scanning electron microscopes • (‘Its not how much they magnify that is key - but how well they resolve…’) Interaction with matter Backscattered electrons Topography and composition
Secondary electrons Topography
Transmitted Electrons Structure and composition
X-rays Composition The Transmission Electron Microscope
• electrons scatter when they pass through
thin sections of a specimen • transmitted electrons (those that do not scatter) are used to produce image • denser regions in specimen, scatter more electrons and appear darker A transmission electron microscope (TEM) The Scanning Electron Microscope
• uses electrons reflected from the surface of
a specimen to create image • produces a 3-dimensional image of specimen’s surface features Scanning electron microscope (SEM) THE LIGHT MICROSCOPE v THE ELECTRON MICROSCOPE
ELECTRON FEATURE LIGHT MICROSCOPE MICROSCOPE Preparation of Temporary mounts Tissues must be specimens living or dead dehydrated = dead Fixation Alcohol OsO4 or KMnO4 Embedding Wax Resin Sectioning Hand or microtome Microtome only. slices 20 000nm Slices 50nm Whole cells visible Parts of cells visible Stains Water soluble dyes Heavy metals
Support Glass slide Copper grid
Sample Preparation for Electron Microscopy
Preparing samples for electron microscopy is time consuming. It takes
about a week to prepare a sample for the TEM.
compound microscope: mount specimen in water on slide, put on cover slip
SEM: fixative, dehydrate, coat with gold
TEM: fixative, dehydrate, embed in resin block, cut block into pyramid shape, cut slice with microtome, mount on grid, stain Details in light microscope samples can be enhanced by stains that absorb light;
Similarly TEM samples of biological tissues can utilize high
atomic number stains to enhance contrast.
The stain absorbs electrons or scatters part of the electron beam
which otherwise is projected onto the imaging system.
Compounds of heavy metals such as osmium, lead, uranium or
gold (in immunogold labelling) may be used prior to TEM observation to selectively deposit electron dense atoms in or on the sample in desired cellular or protein regions, requiring an understanding of how heavy metals bind to biological tissues. Preparation methods • shadowing – coating specimen with a thin film of a heavy metal
• freeze-etching – freeze specimen then fracture along lines of greatest weakness (e.g., membranes) Dr. Gilla Kaplan/Rockfeller University, New York City Ebola
