2.1.1 microscopes

microscope definition

an instrument employing lenses to produce a magnified image and fine detail of objects too small to see with the naked eye

unit for measurement in cells

micrometre (um)

unit for measurement in biological molecules

nanometre (nm)

magnification definition

how many time the size of an image is larger than the object itself

resolution definition

the degree to which it is possible to distinguish between two separate objects which are close to each other

2 categories of microscope and their waves

optical (employ light waves)

electron (employ electron waves)

how an optical microscope works

shines light through the specimen to be viewed. contains two lenses: objective and eyepiece => so object is magnified twice

how electron microscope works

same as light but with electron beams:

shine electron beam through specimen

lenses in the 2 microscopes

optical: glass

electron: magnets

resolution, light or electron?

resolution much higher in electron microscope than optical microscope

2 types of electron microscope

transmission electron microscope (TEM), scanning electron microscope (SEM)

how does a TEM work

tissue/cell is infiltrated with plastic resin (=> very hard) and is cut into very thin slices using a diamond knife

a beam of electrons is passed through exceedingly thin slices of material and produces an image on the screen

allows interior of cells to be seen

dimensional appearance, TEM vs SEM?

TEM: 2D

SEM: 3D

how does SEM work

specimen in shadowed with a layer of heavy metal and covered with carbon

allows surface of specimen to be seen

it is scanned by a fine electron beam which is scattered from surface of specimen and transmitted to a detector

how does freeze-fracture work?

SEM only:

freeze substance

crack open randomly

see inside of cells

resolution, TEM vs SEM

TEM is higher

magnification, TEM vs SEM

TEM is higher

colour, TEM vs SEM

both black and white

light microscopes advantages

• small and portable

• unaffected by magnetic fields

• natural colour of material can be observed

• preparation of material is relatively quick and simple, requires little expertise

• cheap to operate

• living material may be viewed

• cheap to purchase (£100-500)

• material rarely distorted by preparation

electron microscopes disadvantages

• very large and must be operated in special rooms

• affected by magnetic fields

• all images are in black and white

• preparation of material is lengthy, requires considerable expertise and complex equipment

• expensive: requires up to 100,000 V to produce the electron beam

• a high vacuum is required => living material can’t be viewed

• expensive to buy (£1,000,000)

• preparation of material may distort it

light microscope disadvantages

• magnifies object up to x1500

• can resolve objects up to 0.2um apart

• depth of field is restricted

electron microscopes advantages

• magnifies object up to x500,000

• can resolve objects 0.001um apart

• possible to see a greater depth of field (SEM 3D imagery)

light microscope labelled

observing a slide procedure

1. use coarse focus knob to give maximum distance between stage and low power objective lens

2. place a slide on the stage the right way up and centrally over the hole. Switch on power, adjust brightness until dim light is shining through the hole in the stage. Don’t look through eyepiece yet

3. Use coarse focus to move the slide as close to the objective lens as possible without the lens touching the slide

4. Look through the eyepiece

5. adjust light if needed and use coarse focus to increase distance between lens and stage

6. use fine focus once you can see something

7. move slide around to look at different parts

types of temporary slides

a dry mount, a wet mount, smear slides, squash slide

dry mount:

place specimen on slide, place cover slip to cover it and flatten it

wet mount:

put water and/or stain on specimen on the slide, lower coverslip at an angle to minimise trapping bubbles, mop up excess stain

irrigation technique for a simple stain:

if a specimen is already mounted, a drop of stain can be placed at one end of the coverslip and drawn through using filter paper slightly pushed under the edge of the coverslip

smear slides:

place a drop of sample e.g. blood onto slide. push second slide at angle backwards into the drop, drag second slide back away from drop at an angle to smear the blood

squash slide:

squash specimen onto slide so is flat

preparing permanant slides:

1. fixation: preserving the material in a life-like condition with minimum distortion

2. dehydration: removing water from the fixed specimen with alcohols to remove fixative and water from the tissue and replace them with dehydrating fluid

3. clearing: removing the dehydrating alcohols to leave specimen transparent

4. embedding: placing in a mould with wax or resin to form a block

5. sectioning: using a microtome to produce thin slices from the block

6. staining: treating the thin slices with chemical agents to “dye” different structures different colours

7. mounting: securing the stained slice to a slide under a coverslip

why is staining necessary?

the interior of cells is often transparent

staining provides…

• contrast between components of cells e.g. organelles to be identified

• contrast between a structure e.g. a cell and its background

lipid stain

Sudan red- turns red

Sudan red

lipid stain- turns red

starch stain

iodine- blue/black

iodine

stain for starch- blue/black

methylene blue

stain for DNA/chromosomes- blue

DNA/chromosomes stains

methylene blue (toluidine blue, heomatoxylin)- all turn blue (acetic orsein-red)

Eosin

stain for (the proteins in) cytoplasm- turns pink/red

(proteins in) cytoplasm stain

eosin- pink/red

differential staining definition

staining that uses more than one chemical stain- makes differences between cells or between different structures within cells more visible

microscope calibration method

1. use a stage micrometre- slide with a scale on it measuring 10mm. each small division is 0.1mm

2. line up graticule in eyepiece lens with the scale on the stage micrometre

3. count number of divisions on eyepiece graticule equivalent to each division on stage micrometre and hence calculate the length that one eyepiece division is equivalent to

4. repeat with other objective lenses

graticule length formula

length of 1 graticule unit= distance between two divisions on the stage micrometre (0.1mm/100um)/ number of eyepiece units in that distance

biological drawing rules

1. drawing must fill >50% of the area given

2. single clear lines drawn with a sharp and hard pencil. No sketching or discontinuous/broken lines

3. complete outline of structures only, no single lines to represent a feature

4. NO shading or colour

5. correct proportions of structures: draw what you see NOT a textbook illustration

6. labelling:

   a) label lines must be in pencil and drawn with a ruler

   b) NO arrows at the end of label lines

   c) label lines must touch the feature labelled

   d) label lines must not cross each other

7. informative title- include which lens has been used

8. scale or magnification must be included

9. annotation must be included under the label- anything that cannot be:

   a) drawn e.g. colour, texture, fine detail

   b) whose outline cannot be completed e.g. creases, hairs, granulation