Light v Electron Microscope Table
Staining Specimens
Specimens to be viewed under a microscope sometimes need to be stained, as the
cytoplasm and other cell structures may be transparent or difficult to distinguish
o Note that most of the colours seen in images taken using a light microscope
are the result of added stains
Chloroplasts are the exception to this; they show up green, which is
their natural colour
The type of stain used is dependent on what type of specimen is being prepared and
what the researcher wants to observe within the specimen
o Different molecules absorb different dyes depending on their chemical
nature
Specimens or sections are sometimes stained with multiple dyes to ensure that
several different tissues within the specimen show up; this is known as differential
staining
Some common stains include
o Haemotoxylin
Stains plant and animal cell nuclei purple, brown or blue
o Methylene blue
Stains animal cell nuclei blue
o Acetocarmine
Stains chromosomes in dividing nuclei of plant and animal cells
o Iodine
Stains starch-containing material in plant cells blue-black
o Toluidine blue
Stains tissues that contain DNA and RNA blue
o Phloroglucinol
Stains a chemical called lignin found in some plant cells red/pink
Toluidine blue and phloroglucinol have been used to stain this tissue specimen taken from
a leaf
Core Practical: Light Microscopy
Microscope Images
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
o Light is directed through a thin layer of biological material that is
supported on a glass slide
o This light is focused through several lenses so that an image is
visible through the eyepiece
o 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
o The eyepiece lens
o The objective lenses
o The stage
o The light source
o The coarse and fine focus
Other tools that may be used
o Forceps
o Scissors
o Scalpel
o Coverslip
o Slides
o Pipette
o Staining solution
The components of an optical microscope
Method
Preparing a slide using a liquid specimen
o Add a few drops of the sample to the slide using a pipette
o Cover the liquid / smear with a coverslip and gently press down
to remove air bubbles
o Wear gloves to ensure there is no cross-contamination of foreign
cells
Methods of preparing a microscope slide using a solid specimen
o Take care when using sharp objects and wear gloves to prevent the
stain from dying your skin
o Use scissors or a scalpel to cut a small sample of the tissue
o 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
o Apply a stain to make cells more visible
o 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
o 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
o 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
o It is easier to find what you are looking for in the field of view
o This helps to prevent damage to the lens or coverslip in case the
stage has been raised too high
Preventing the dehydration of tissue
o The thin layers of material placed on slides can dry up rapidly
o Adding a drop of water to the specimen beneath the coverslip can
prevent the cells from being damaged by dehydration
Unclear or blurry images
o Switch to the lower power objective lens and try using the coarse
focus to get a clearer image
o Consider whether the specimen sample is thin enough for light to
pass through to see the structures clearly
o 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
o 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
o 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
o The drawing must have a title
o The magnification under which the observations shown by the
drawing are made must be recorded
o A sharp pencil should be used
o Drawings should be on plain white paper
o Lines should be clear, single lines with no sketching
o No shading
o The drawing should take up as much of the space on the page as
possible
o Well-defined structures should be drawn
o The drawing should be made with proper proportions
o Label lines should not cross or have arrowheads and
should connect directly to the part of the drawing being labelled
o 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
o 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
o 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.
Measurements of Microscopic Images
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:
o An eyepiece lens, which often has a magnification of x10
o A series of (usually 3) objective lenses, each with a different
magnification
o 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
o 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.
Worked example
An image of an animal cell is 30 mm in diameter and it has been magnified by
a factor of x3000.
What is the actual diameter of the cell?
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
o 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
o 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
Worked example
Calculate the size of the units of the eyepiece graticule in the image below.
Note that the large divisions in the top half of the image show the stage
micrometer and that each stage micrometer division is 1 mm across.
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 = 1 mm (1000 µm)
1 graticule unit = 1000 ÷ 40 = 25 µm
o An object that spanned five eyepiece graticule units could therefore
be measured as follows
5 x 25 µm = 125 µm
Exam Tip
The biggest pitfall with these kinds of calculations is forgetting to convert the
units so that they match before embarking on a calculation. E.g. if image size is
measured in mm but the actual size of an object is given in µm then both need
to be converted into µm before using the equation triangle above.
To convert a measurement from mm into µm the measurement must be
multiplied by 1000 (there are 1000 µm in 1 mm).
