#3. Microscopy

Most cells are very small, and their structures can only be seen by using a microscope.









1. Light microscopes

• light rays pass through the specimen on a slide 

• focused by an objective lens and an eyepiece lens. 

• ---> magnified image of the specimen on the retina of your eye/screen/camera.

2. Electron microscopes

• uses beams of electrons 
• specimen very thin, placed in a vacuum to allow electrons to pass through it. 
• electrons are focused onto a screen/photographic film ---> magnified image of the specimen.

3. Magnification and Resolution

• Amount of magnification depends on the resolution of the microscope (ability to distinguish 2 objects as separate). 

• The smaller the objects that can be distinguished --> the higher the resolution. 

• wavelength: beam of electrons  <<<  light

         resolution:  electron microscope >>> light microscope

• with electron microscope, we can see much more fine detail of a cell.

Units: millimetre, micrometre, nanometre

4. Magnification calculations

Work out the real size of an object knowing the magnification:

a. This drawing of a mitochondrion has been magnified 100 000 times.

• Use ruler to measure its length in mm (50 mm).

• Convert this measurement to µm by multiplying by 1 000.
                    50 x 1 000 = 50 000 µg

• Substitute into the equation:

b. This is a the drawing of a chloroplast:

- The magnification for this drawing:

- The length of the chloroplast:

• Measure the length of the image in mm (80 mm) and convert to µm ---> 80 000 µm.

• Calculate its real length:

5. Measuring cells using a graticule

• Eyepiece graticule is a little scale bar placed in the eyepiece of light microscope. 
• The graticule is marked off in 'graticule units'.
• Turn the eyepiece so that the graticule scale lies over the object: the width of one cell is 23 graticule units.

Calibration: the conversion of graticule units into real units (mm, µm).

• use a special slide called a stage micrometer that is marked off in a tiny scale. The smallest markings are often 0.01 mm (10 µg) apart.

• Take the specimen off the stage or the microscope and replace it with the stage micrometer. Use the same objective lens. 

• Line up the micrometer scale and the eyepiece graticule scale (by turning the eyepiece and moving the micrometer on the stage). Make sure that 2 large markings on each scale are lined up.

• The 50 mark (stage micrometer) is lined up with the 1.0 mark (eyepiece graticule). 

• Work towards the right until you see another two lines lined up. 

• The 68 mark (stage micrometer) is lined up with the 9.0 mark (eyepiece graticule). So you can say that:  

                 80 small eyepiece graticule markings = 18 stage micrometer markings
                                                                      = 18 x 0.01 mm = 0.18 mm
                                                                      = 180 µm
                so 1 small eyepiece graticule marking = 180: 80 = 2.25 µm

• The plant cell was 23 eyepiece graticule units long --> its real width is: 23 x 2.25 = 51.75 µm

Syllabus 2015: The microscope in cell studies Candidates should be able to: (a) [PA] use an eyepiece graticule and stage micrometer scale to measure cells and be familiar with units (millimetre, micrometre, nanometre) used in cell studies; (b) explain and distinguish between resolution and magnification, with reference to light microscopy and electron microscopy; (g) [PA] calculate linear magnification of drawings and photographs; (h) [PA] calculate actual sizes of specimens from drawings and photographs;

Syllabus 2016-2018: The microscope in cell studies An understanding of the principles  of microscopy shows why light and electron microscopes have been essential in improving our knowledge of cells. a)   compare the structure of typical animal and plant cells by making temporary preparations of live material  and using photomicrographs b)   calculate the linear magnifications of drawings, photomicrographs and electron micrographs c)   use  an eyepiece graticule  and stage micrometer scale to measure cells and be familiar with units (millimetre, micrometre, nanometre) used in cell studies d)   explain and distinguish between resolution and magnification, with reference to light microscopy and electron microscopy e)   calculate actual sizes  of specimens from drawings, photomicrographs and electron micrographs