Microscopes

Magnification - Number of times greater an image is than the actual object.

Magnification= Image size vvghorggggg The length of the scale bar

gggghghghhghActual object size gghhghfThe length the scale bar represent

Actual object size = Image size

jjjgjgjgjhghjgjgjgjMagnification

Resolution - Ability to distinguish between two separate points; the degree of detail that can be seen in an image.

Micrometres and nanometres

• 1µm (micrometre) = 1/1000mm or 1 x 10⁻³ mm or 1 x 10⁻⁶ m

To get from mm to µm multiply by 1000

• 1nm (nanometre) = 1/1000µm or 1 x 10⁻⁶ mm or 1 x 10⁻⁹ m

To get from µm to nm multiply by 1000

Light Microscopes - Use glass lenses to refract light rays and produce a magnified image of an object

Eyepiece lens magnifies and focuses the image from the object onto the eye.

Objective lens collects light passing through the specimen and produces a magnifies image.

Condenser lens focuses the light onto the specimen held between the cover slip and slide.

Condenser Iris diaphragm is closed slightly to produce a narrow beam of light.

• Specimens need to be thin and stained to show up clearly.

Transmission Electron Microsopes (TEM) - pass beams of electrons through a specimen to produce images of an object.

Electron gun and anode - produce a beam of electrons

Condenser Electromagnetic lens - directs the electron beam onto the specimen

Grid - specimen is placed on it

Objective electromagnetic lens - produces an image

projector electromagnetic lenses - focus the magnified image onto the screen

Screen or photographic plate - shows the image of the specimen

• Electrons have shorter wavelengths than light rays so give a much higher resolution.

Scaning Elctron Microscopes (SEM) - Bounces electron beams off of the surface of an object giving a 3D image. Allows organelles to be seen.

• The original images produced by an electron microscope are in black, white and grey only but false colours are often added using a computer to help identify diffent structures.
• Specimen needs to be thin and stained.
• In electron microscopy stains are heavy metals e.g lead or osmium.
• The ions are large and positively charged. the negatively charged electrons do not pass through cells and therefore do not show up on the screen.
• Ions of the metals are taken up by some parts of the cell more than others. Structures which have taken up the stain appear darker than other areas

Type vhuygjhHighest Magnification ggjjjjgjkgBest Resolution

Light hghjghgjggx 2000 ghghgfhgfhfghfhgfhgghf300nm

TEM hhhhhghhx 500,000 + (2D) hghghghhhhh0.1nm

SEM hgjhhjhghgx 25,000 (3D) jjhjhjhjfgguggjhgj10nm

Calibrating a microscope

Why? To measure the size of a structure under the field of view with a specific magnification

How? using an eyepiece graticule (a scale of unknow lengthinserted into an eyepiece) and using a stage micrometre (a scale of known length 10mm) placed onto the stage and superimposed on top of the eyepiece graticule.

• To measure cells the graticule must first be calibrated using a micrometer
• A stage micrometer is a microscope slide with a scale very accuractely etched into 100µm divisions.
• The graticule has 100 "dinks" - find how many dinks on the micrometre fit into 100 on the gratucule. Then add µm onto the number as the unit of measurement