1.1.1 Light Microscopes

• Many biological structures are too small to be seen by the naked eye
• Optical microscopes are an invaluable tool for scientists as they allow for tissues, cells and organelles to be seen and studied
• Light is directed through a thin layer of biological material (containing the tissue(s), cell(s) or organelle(s) to be observed) that is supported on a glass slide
• This light is focused through several lenses so that an image is visible through the eyepiece

Apparatus

• The key components of an optical microscope you will need to use are:
  • The eyepiece lens
  • The objective lenses
  • The stage
  • The light source
  • The coarse and fine focus
• Other apparatus used:
  • Forceps
  • Scissors
  • Scalpel
  • Coverslip
  • Slides
  • Pipette

Preparing specimens & samples

• Specimens must be prepared on a microscope slide to be observed under a light microscope
• This must be done carefully to avoid damaging the biological specimen and the structures within it
• The most common specimens to observe under a light microscope are cheek cells (animal cells) and onion cells (plant cells)
• Preparing a slide using a liquid specimen:
  • Add a few drops of the sample to the slide using a pipette
  • Cover the liquid/smear with a coverslip and gently press down to remove air bubbles
  • Wear gloves to ensure there is no cross-contamination of foreign cells
• Preparing a slide using a solid specimen:
  • Use scissors to cut a small sample of the tissue
  • Peel away or cut a very thin layer of cells from the tissue sample to be placed on the slide (using a scalpel or forceps)
  • Some tissue samples need to be treated with chemicals to kill/make the tissue rigid
  • Gently place a coverslip on top and press down to remove any air bubbles
  • A stain may be required to make the structures visible depending on the type of tissue being examined.
    • Commonly used stains include methylene blue to stain cheek cells and iodine to stain onion cells
  • Take care when using sharp objects and wear gloves to prevent the stain from dying your skin
• Preventing the dehydration of tissue:
  • The thin layers of material placed on slides can dry up rapidly
  • Adding a drop of water to the specimen (beneath the coverslip) can prevent the cells from being damaged by dehydration

Viewing the specimen

• When using an optical microscope always start with the low power objective lens:
  • It is easier to find what you are looking for in the field of view
  • This helps to prevent damage to the lens or coverslip in case the stage has been raised too high
• Unclear or blurry images:
  • Switch to the lower power objective lens and try using the coarse focus to get a clearer image
  • Consider whether the specimen sample is thin enough for light to pass through to see the structures clearly
  • There could be cross-contamination with foreign cells or bodies

A calibrated graticule must be used to take measurements of cells

• A graticule is a small disc that has an engraved scale. It can be placed into the eyepiece of a microscope to act as a ruler in the field of view
• As a graticule has no fixed units it must be calibrated for the objective lens that is in use. This is done by using a scale engraved on a microscope slide (a stage micrometer)
• By using the two scales together the number of micrometers each graticule unit is worth can be worked out
• After this is known the graticule can be used as a ruler in the field of view

Limitations of microscopy

• The size of cells or structures of tissues may appear inconsistent in different specimen slides
  • Cell structures are 3D and the different tissue samples will have been cut at different planes resulting in inconsistencies when viewed on a 2D slide
• Optical microscopes do not have the same magnification power as other types of microscopes and so there are some structures that cannot be seen
• The treatment of specimens when preparing slides could alter the structure of cells

Using units in microscopy

• You may be given a question in your Biology exam where the measurements for a magnification calculation have different units
• You need to ensure that you convert them both into the same unit before proceeding with the calculation (usually to calculate the magnification)
• Remember the following to help you convert between mm (millimetres), µm (micrometres) and nm (nanometres):

• If you are given a question with two different units in it, make sure you make a conversion so that both measurements have the same unit before doing your calculation

Higher Tier Only

• Magnification is calculated using the following equation:

Magnification = Drawing size ÷ Actual size

• One way to remember the equation is using an equation triangle:

An equation triangle for calculating magnification

• Rearranging the equation to find things other than the magnification becomes easy when you remember the triangle – whatever you are trying to find, place your finger over it and whatever is left is what you do, so:
  • Magnification = image size ÷ actual size
  • Actual size = image size ÷ magnification
  • Image size = actual size × magnification
• Remember magnification does not have any units and is just written as ‘X 10’ or ‘X 5000’

• You may also be asked to calculate the total magnification of a light microscope if given the magnification of the eyepiece lens and the magnification of the objective lens
• As these are two separate parts of a light microscope, each with its own magnifying power, you can simply multiply the two values to calculate the total magnification:

Magnification of light microscope = Magnification of eyepiece lens × Magnification of objective lens

Standard form

• When doing calculations and unit conversions, it is common to come across very big or very small numbers
• Standard form can be useful when working with these numbers
• Standard form is a way of writing very big and very small numbers using powers of 10

How to use standard form

• Using standard form, numbers are always written as follows: a × 10ⁿ
• The rules:
  • 1 ≤ a < 10 (the number 'a' must always be between 1 and 10)
  • n > 0 for LARGE numbers ('n' = how many times 'a' is multiplied by 10)
  • n < 0 for SMALL numbers ('n' = how many times 'a' is divided by 10)

Using standard form to convert between units

• For example, you can write 1 metre in millimetres using standard form:
  • 1 m = 1000 mm
  • So, 1 m = 1 mm × 1000
  • So, 1 m = 1 mm × 10 × 10 × 10
  • So, as we had to multiply 1 mm by 10 three times to get 1 m, we write this as:
  • 1 m = 1 × 10³ mm

• Writing 1 millimetre in metres using standard form is also possible and is just the opposite:
  • 1 mm = 0.001 m
  • So, 1 mm = 1 m ÷ 1000
  • So, 1 mm = 1 m ÷ 10 ÷ 10 ÷ 10
  • So, as we had to divide 1 m by 10 three times to get 1 mm, we write this as:
  • 1 mm = 1 × 10^-3 m

• Exactly the same process can be used if you needed to convert micrometres into millimetres. For example:
  • 1 µm = 0.001 mm
  • So, 1 µm = 1 mm ÷ 1000
  • So, 1 µm = 1 mm ÷ 10 ÷ 10 ÷ 10
  • So, as we had to divide 1 mm by 10 three times to get 1 µm, we write this as:
  • 1 µm = 1 × 10^-3 mm

Examples of using standard form in conversion calculations

• You could be asked to state 45 centimetres in millimetres using standard form:
  • 1 cm = 10 mm
  • So, 45 cm = 450 mm
  • So, 45 cm = 4.5 mm × 10 × 10
  • So, as we had to multiply 4.5 mm by 10 two times to get 45 cm, we write this as:
  • 45 cm = 4.5 × 10² mm

• You could also be asked to state 250 micrometres in millimetres using standard form:
  • 1 µm = 0.001 mm
  • So, 250 µm = 0.25 mm
  • So, 25 µm = 2.5 mm ÷ 10
  • So, as we had to divide 2.5 mm by 10 just once to get 250 µm, we write this as:
  • 250 µm = 2.5 × 10^-1 mm

Step One: Convert units

Step Two: Calculate the thickness of the leaf in mm

Step Three: Put these values into the equation for calculating magnification