MAGNIFICATION EQUATION
Magnification = image length / actual length
LIGHT MICROSCOPY
AKA Optical Microscopes use light and several lenses to magnify a sample.
Light from the Condenser Lens, and then through the Specimen where certain wavelengths are filtered to produce an image. The light then passes through the Objective Lens, which focuses it and can be changed in order to alter the magnification. Finally, the light passes through the Eyepiece Lens, which can also be changed to alter the magnification, and into the eye. The maximum magnification of light microscopes is usually ×1500, and their maximum resolution is 200nm, due to the wavelength of light.
ADVANTAGES
• Cheap to purchase and operate
• Small and portable
• Natural color of the specimen can be observed
• Living as well as dead material can be viewed
• Preparation is relatively quick and simple, requiring little expertise
• Unaffected by magnetic fields
DISADVANTAGES
• Magnifies objects up to 1500x
• Preparation may distort specimen
• The depth of the field is restricted
• Has a resolving power for biological specimens of around 1nm
There are two types of light microscope.
COMPOUND MICROSCOPES
contain several lenses and magnify a sample several hundred times. DISSECTING MICROSCOPES have a low final magnification but are useful when a large working distance between the objectives and the stage is required (e.g. during dissection). They have two eyepieces to produce a 3D stereoscopic view.
ELECTRON MICROSCOPE
Light microscope’s relatively low magnification and resolution are not satisfactory for viewing small/smaller things e.g. organelles within cells An Electron microscope is used instead Electrons have a much lower wavelength than light: so, they can be used to produce an image with a better (higher) resolution
ADVANTAGES
• Magnifies over 500000x
• Can provide 3D images
DISADVANTAGES
• Expensive to buy
• Expensive to produce electron beam
• Large + requires special rooms
• Lengthy + complex sample preparation
• Preparation distorts material
• Vacuum is required
• All images in black + white
There are 2 types of electron microscopes:
1. A Transmission Electron Microscope (TEM) – produces a 2D image
2. A Scanning Electron Microscope (SEM) – produces a 3D image
STAINING
Many specimens require preparation before being viewed by a light microscope, as some may not be colored or might distort when cut. Samples are Stained with colored stains that bind to certain chemicals or cell structures.
Staining in electron microscopy: using heavy metals to scatter electrons and produce contrast
MAGNIFICATION AND RESOLUTION
Magnification is the factor by which an image appears to be enlarged. It will be a whole number greater than 1 and is usually followed by an “x”, as in 10x magnification. When you look through microscope eyepieces, you are seeing a virtual image because in reality, what you are looking at is not as large as it appears through the eyepieces, and because there can be some distortion of the image.
Resolution is the shortest distance between two points that can still be visually distinguished as separate. The resolution of a typical unaided human eye is about 200 µm. Using a microscope decreases the resolution to distances as short as 0.2 µm. Resolution is a property of the eye. Resolving power is the ability of a lens to show two adjacent objects as discrete. Resolving power is a property of a lens.