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IB Biology - The Microscope

Chapter 1.1 - Introduction to cells

April 25, 2020
  • Use of a light microscope to investigate the structure of cells and tissues, with drawing of cells. Calculation of the magnification of drawings and the actual size of structures and ultrastructures shown in drawings or micrographs.

  • Scale bars are useful as a way of indicating actual sizes in drawings and micrographs.

Resolution (Resolving Power)

  • The microscopy resolving power defined by its ability to distinguish the two points of the object 
  • Affected by the wavelength of the light, the shorter the wavelength, the better the resolving power 

From the above diagram, the resolving power decreases from left to right,

  • left one the shorter wavelength --> "hit" precisely on the two points --> give a distinctive and sharp image
  • right one the longer wavelength --> can't hit on the top points --> give an image which merging two points 

The Decrease the wavelength, the better the resolving power, the sharper the image

Light Microscope

  • magnification power x2000
  • maximum resolution is 200nm in size
    (size smaller then 200nm the resulting image will be like the right one from the above diagram) 
  • use light for illumination 
  • can view nucleus, mitochondria, cytoplasm, cell membrane, chloroplasts and cell wall these organelles 
  • placing the specimen on the glass slide
  • as most of the cell is transparent, using coloured dye to stain the cell to improve the visualisation
  • can view living organisms 
  • the first light microscope was invented in the early 1800s --> helping scientist to write the cell theory 

The light microscope possesses hardware including,

  • mirror: reflecting the light to illuminate the specimen 
  • stage: holding the specimen 
  • focusing knobs: adjust the image if it is out-focused 
  • eyepiece lens: when we view the image, containing a magnifying lens 
  • objective lenses: contain magnifying lenses, turning the disk to choose the magnifying lens 

the coloured image given by the light microscope

Two magnifying lenses: eyepiece lens and objective lens in light microscope.

Electron Microscope

  • using an electron beam as an illumination 
  • better resolution then the light microscope as the electron wavelength is much shorter than the light 
  • giving black and white image, artificially colourize the image afterwards 
  • specimen is mounted on metal gride, CANNOT view living organisms 
  • 2 types of electron microscope: transmission electron microscope, scanning electron microscope 
  • the first electron microscope was invented in the 1940s

the wavelength of electron beam is much smaller than light, so TEM, SEM have much greater resolution

Transmission electron microscope (TEM)

  • maximum magnification power x1 000 000
  • maximum resolution is 1nm in size
  • coating heavy metal on the object 
  • can view the section of the object ​

Scanning electron microscope (SEM)

  • maximum magnification power x200 000
  • maximum resolution is 1nm in size
  • coating carbon or gold on the object
  • can view the external appearance of the object 

you can see from the above image, the TEM (right) gives section image while SEM (left) gives the appearance of the pollen grain

Freeze-etching

  • one of the methods to prepare specimen for TEM and SEM
  • for viewing the 3D arrangement of lipids and proteins in the cell membrane 
  • freezing part: using liquid nitrogen to freeze and fracture the cell into pieces 
  • etching part: evaporating the ice, the surface is exposed 
  • the heavy metal coat on the surface under vacuum 

Magnification and Scale

How to calculate the actual size of the specimen from the microscopy image?

e.g. you use a light microscope with 40x magnifying lens to view a cell with 10mm diameter in the image. The actual size of the cell:

(10x40) = 10mm / x

x = 0.025mm

the actual size is 0.025mm

  • Be reminded that, the magnifying power is 10x40 because the light microscope has 2 magnifying lenses which are eyepieces lens (x10) and objective lens (x40 in this example)!

Magnifying power of light microscopy = eyepieces lens x objective lens

In a microscopy image, you usually see a scale, how to calculate the actual size with the use of scale?

e.g.:

  • the scale state in the image is 3.33μm 
  • the scale bar length measured by ruler (lets say) is 5mm --> 5 x 1000 = 5 000μm
  • the size of the image measured by ruler (lets say) is 60mm ---> 60 x 1000 = 60 000μm 
  • Hence, the (3.33/5000) = (x/60000), x=39.96μm 

be awarded of the unit, "μm" is 1000x smaller than "mm" (1mm = 1000μm)

you can also calculate the magnifying power from the scale,

x = size of image / size of object

x = 60000μm / 39.96μm

x= x1502

In this IB biology topic, you have to know:

  • what is resolution? why the electron microscope has greater resolution than the light microscope?
  • what cell organelles can be seen by light microscope 
  • the SEM and TEM use carbon and heavy metal for coating respectively 
  • how to calculate the magnification, actual size of the object with the scale bar 

That is the end of the topics~~

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