Cell Theory: Skills (DP IB Biology)

Skills in Microscopy

• Many biological structures are too small to be seen by the naked eye

• Optical (light) microscopes are an invaluable tool for scientists as they allow for tissues, cells and organelles to be seen and studied

• For example, the movement of chromosomes during mitosis can be observed using a microscope

How optical (light) microscopes work

• Light is directed through the thin layer of biological material that is supported on a glass slide

• This light is focused through several lenses so that an image is visible through the eyepiece

• The magnifying power of the microscope can be increased by rotating the higher power objective lens into place

Apparatus

The key components of an optical (light) microscope are:

• The eyepiece lens

• The objective lenses

• The stage

• The light source

• The coarse and fine focus

• Other tools that may be used:

  • Forceps

  • Scissors

  • Scalpel

  • Coverslip

  • Slides

  • Pipette

A diagram of an optical microscope

Image showing all the components of an optical (light) microscope

Method

• Preparing a temporary mount 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 temporary mount 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 be treated with chemicals to kill / make the tissue rigid

  • A stain may be required to make the structures visible depending on the type of tissue being examined

  • Gently place a coverslip on top and press down to remove any air bubbles

  • Take care when using sharp objects and wear gloves to prevent the stain from dying your skin

• Place the microscope slide on the stage, fix in place using the stage clips (ensure the microscope is plugged in and on)

• 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 incase the stage has been raised too high

• Whilst looking through the eyepiece lens move the coarse focusing knob until the specimen comes into focus. The fine focusing knob should be used to sharpen the focus on particular parts (and at higher objective lens only)

• To examine the whole slide, move it carefully with your hands (or if using a binocular microscope use the stage adjusting knobs)

• Once you have focused on the object/structure then carefully move to a higher objective lens (10X and 40X). If resistance is felt do not continue to move the turret

  • At the higher objective powers only use the fine focusing knob

  • Do not move the stage down when moving to higher objective lens

• 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

• Use a calibrated graticule 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 measurements made using these microscope apparatus are a form of quantitative observations

Diagram of an eyepiece graticule and stage micrometer

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

Magnification calculations

• Magnification is how many times bigger the image of a specimen observed is in comparison to the actual (real-life) size of the specimen

• The magnification (M) of an object can be calculated if both the size of the image (I), and the actual size of the specimen (A), is known

The magnification equation triangle

An equation triangle for calculating magnification

Using the appropriate units

• The size of cells is typically measured using the micrometer (μm) scale, with cellular structures measured in either micrometers (μm) or nanometers (nm)

• When doing calculations all measurements must be in the same units. It is best to use the smallest unit of measurement shown in the question

• To convert units, multiply or divide depending if the units are increasing or decreasing

• Magnification does not have units

Diagram to show conversion of units

• There are 1000 nanometers (nm) in a micrometre (µm)

• There are 1000 micrometres (µm) in a millimetre (mm)

• There are 1000 millimetres (mm) in a metre (m)

Producing a scale bar

• A scale bar is a straight line on the drawing or micrograph that represents the actual size before the image was enlarged

• It can be used to calculate magnification from biological drawings and images

• To add a scale bar to a biological drawing of a microscope specimen:

  1. Use the eyepiece graticule and stage micrometer to calculate the distance between two markings on the eyepiece graticule; this is the graticule unit

  2. Remove the stage micrometer and add the specimen to the microscope stage

  3. Measure the length of the specimen using the eyepiece graticule which will be in graticule units

  4. Determine the length of the specimen in micrometers by multiplying the number of graticule units by the length of each unit (calculated in step 1)

• Your scale bar should represent 20% of the actual length of your specimen. If you specimen is 300µm then your scale bar would represent 60µm

  1. Draw your specimen as directed and measure the length of your drawing in mm; your scale bar should be 20% of the length of your specimen drawing; if your drawing is 150mm then your scale bar should be 30mm long

  2. Add the actual length your scale bar represents underneath your scale bar e.g. 60µm

Using a scale bar

• If the calculation required includes a scale bar on the micrograph or drawing then follow these steps:

  1. Use a ruler to measure the length of the scale bar in millimetres (mm)

  2. Convert this measurement into the same units as the number on the scale bar

  3. Insert these numbers into the magnification formula above (note: the size of the image is the measured length of the scale bar and the actual size is the number on the scale bar)

NOS: Measurement using instruments is a form of quantitative observation

• Microscopy can give us accurate quantitative observations about cells

  • Quantitative observations are a collection of data which are focused on numbers and values such as measurements of length, height, volume, or values of quantity and frequency

• Using instruments such as eyepiece graticules and stage micrometers allow us to take measurements on a small scale such as in micrometers (µm) and nanometers (nm) (using electron microscopes)

  • Data can be collected about cell and organelle sizes

• Qualitative data is non-numerical data such as colour and presence of structures which can also be determined using microscopes

• Making observations and taking measurements form the basis for developing new hypotheses in Biology