Light Microscopes (OCR GCSE Combined Science A (Gateway))

Microscope Equipment

• 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

The components of an optical microscope

Calibration, Sample Prep & Setup

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

Care must be taken to avoid smudging the glass slide or trapping air bubbles under the coverslip

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

The stage micrometer scale is used to find out how many micrometers each graticule unit represents

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):

Converting 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

Calculation of Magnification

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