3.8 Core Practical 5: Light Microscopy

• Many biological structures are too small to be seen by the naked eye
• Optical, or light, 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 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

Preparation of microscope slides

• The key components of an optical 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
  • Staining solution

The components of an optical microscope

Method

• 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
• Methods of preparing a microscope slide using a solid specimen
  • Take care when using sharp objects and wear gloves to prevent the stain from dying your skin
  • Use scissors or a scalpel to cut a small sample of the tissue
  • Use forceps to peel away or cut a very thin layer of cells from the tissue sample to be placed on the slide 
    • The tissue needs to be thin so that the light from the microscope can pass through
  • Apply a stain to make cells more visible
  • Gently place a coverslip on top and press down to remove any air bubbles

• Some tissue samples need to be treated with chemicals to kill cells or make the tissue rigid
  • This involves fixing the specimen using the preservative formaldehyde, dehydrating it using a series of ethanol solutions, impregnating it with paraffin or resin for support and then cutting thin slices from the specimen
  • The paraffin is removed from the slices and a stain is applied before the specimen is mounted and a coverslip is applied

Slide Preparation Table

Using a microscope

• 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
• 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
• 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

Limitations

• 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 this 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

Drawing cells

• To record the observations seen under the microscope, or from photomicrographs taken, a labelled biological drawing is often made
  • Biological drawings are line drawings that show specific features that have been observed when the specimen was viewed
• There are a number of rules or conventions that are followed when making a biological drawing
  • The drawing must have a title
  • The magnification under which the observations shown by the drawing are made must be recorded
  • A sharp pencil should be used 
  • Drawings should be on plain white paper
  • Lines should be clear, single lines with no sketching
  • No shading
  • The drawing should take up as much of the space on the page as possible
  • Well-defined structures should be drawn
  • The drawing should be made with proper proportions
  • Label lines should not cross or have arrowheads and should connect directly to the part of the drawing being labelled
  • Label lines should ideally be kept to one side of the drawing in parallel to the top of the page, and should be drawn with a ruler
  • Only visible structures should be drawn; not structures that the viewer thinks they should be able to see!
• Drawings of cells are typically made when visualizing cells at a higher magnification power

An example of a cellular drawing taken from a high-power image of phloem tissue

• Plan drawings that show the arrangement of cells within a tissue or organ are typically made using samples viewed under lower magnifications
  • Individual cells are never drawn in a plan diagram

An example of a tissue plan diagram drawn from a low-power image of a transverse section of a root. Note that there is no cell detail present.

• Magnification is how many times bigger the image of a specimen observed is in comparison to the actual, real-life size of the specimen
• A light microscope has two types of lens:
  • An eyepiece lens, which often has a magnification of x10
  • A series of (usually 3) objective lenses, each with a different magnification
  • To calculate the total magnification, the magnification of the eyepiece lens and the objective lens are multiplied together:

total magnification = eyepiece lens magnification x objective lens magnification

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

magnification =  image size  actual size

• • Remember to ensure that the image size (I) and the actual size (A) of the specimen have the same units before doing the calculation

The equation for calculating magnification can be rearranged to calculate either actual size, image size, or magnification.

Using an eyepiece graticule & stage micrometer

• A graticule is a small disc that has an engraved ruler
• It can be placed into the eyepiece of a microscope to act as a ruler in the field of view, so is sometimes known as an eyepiece graticule
• As an eyepiece graticule has no fixed units it must be calibrated for the objective lens that is in use
  • The graticule in the eyepiece remains the same size when the magnification of the microscope is altered, so recalibration is needed at each viewing magnification
• Calibration of the eyepiece graticule is done a microscope slide with an engraved scale known as a stage micrometer
• By using the eyepiece graticule and the stage micrometer together, the size of each graticule unit can be calculated
  • After this is known the graticule can be used as a ruler to measure objects in the field of view

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

Step 1: Observe the number of eyepiece unit divisions per micrometer unit

In the image, the stage micrometer has three lines

Each micrometer division has 40 eyepiece graticule divisions within it

Step 2: Calculate the size of each eyepiece graticule unit

40 graticule divisions = 10 µm

1 graticule unit = 10 ÷ 40 = 0.25 µm

• • An object that spanned five eyepiece graticule units could therefore be measured as follows

5 x 0.25 µm = 1.25 µm