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Light microscopy
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Simplified Light Microscope
Light Microscopes
Light microscopes allow us to observe prokaryotic cells and identify
large structures e.g. mitochondria in eukaryotic cells.
X-ray diffraction
Electron microscope
Light microscope
Bacteria,
Blue-green algae
Water molecule (1.7 Å)
Amino acids
proteins
Human eye
Eukaryotic cells
viruses
10-7
10-6
10-5
10-4
Scale in mm (millimetres, 10-3 metres
10-3
0.01
0.1
1.0
10-4
1.0
10
100
1000
10-3
0.01
0.1
Scale in mm (millimetres, 10-6 metres, also called “microns”.
The limit of resolution
•
The light microscope can be used to observe whole cells plus large
internal structures e.g. nuclei, chloroplasts and mitochondria.
•
But the limit of resolution is about 0.2 mm.
Temporary mounts
•
Temporary mounts are discarded
after viewing. Specimens are
transferred to a glass slide possibly
in a droplet of water.
•
The specimen may need arrangement on the slide if tissues / cells
overlap.
•
A glass cover-slip is placed over the specimen.
•
Excess water and
water droplets are
removed.
Dry Tissue
Staining
•
Most biological specimens in thin section are
transparent making them all but invisible, so
stains are used.
•
A selective/ differential stain may bind to
molecules in e.g. only the nucleus. The outline of
the nucleus is made clearer against the different
colour of the background.
•
Selective staining works because of local
differences e.g. pH variations, the presence of
localized biomolecules.
Artefacts
• Specimens are prepared for microscopy with physical and chemical
procedures e.g. tissue mashing that introduce changes – artefacts.
Summary
• A magnified image of an object is
seen with the light microscope.
• Specimens must be thin for light to
pass through.
• Differential staining improves
visibility against a transparent,
non-staining background.
• Nucleus, nucleolus, chromosomes
and chloroplasts are observable in
the intact cell.
• Prokaryotic cells can be observed
after staining.
Measuring and estimating magification
Question:
If the eye piece magnification is x10
and the objective Lens magnification is x40
What is the total image magnification ?
40 X 10 = 400x
In the exam you may be shown an image e.g. an
amoeba and told that the real length of the amoeba is
25mm
The diagram on paper is 40 mm
Q: what is the magnification ?
Image size on paper
magnification
=
Organism’s real size
=
=
40 mm
25 mm
40,000 mm
25 mm
Make units
same! (x mm by
1000)
= X 1600
Calculating object size
Typical image in a text book:
A stained cheek cell
showing the
nucleus, X200
The actual object (real) size can be
easily calculated.
Step 1 Measure cell in mm.
30mm
Step 2 Convert to mm for convenience (‘mm are smaller than mm, expect more
of them so X 1000) = 30,000mm. To convert mm into mm, divide by 1000.
•
The image size, 30,000mm is 200X too large.
•
The actual length of the cell is:
30,000mm
200
= 150mm
EPU calibration
•
The objective lens is calibrated using a special microscope slide
called a stage micrometer.
•
The stage micrometer is simply a ruler.
•
Unlike the normal ruler measuring in mm the stage micrometer
measures in micrometres (µm).
Stage Micrometer scale
1mm
0 10 20 30 40 50 60 70 80 90 100
•
The ruler on the stage micrometer is exactly 1mm
•
The 1mm line is divided into 100 equal divisions
Estimating size under the microscope
1. Remove the eyepiece and unscrew the top lens.
2. Insert the eyepiece graticule (transparent disc with accurately spaced
marks) in the eyepiece. Screw the top lens back in place and replace
the eyepiece.
eyepiece graticule
Estmating size under the microscope
3. Place a graduated slide (the stage micrometer) on the stage and with the 10X
objective in place, centre the marks of the slide in the field of view and focus.
Graduated slide
Accurate scale markings
in central ring of
graduated slide
Estmating size under the microscope
4. Use the graduated slide to calibrate the graticule. The slide is marked in parts of
a millimetre. By aligning the two scales as shown below you can work out how
the divisions of the graticule correspond to micrometres at this magnification.
Aligned eyepiece graticule
scale with stage micrometer
5. By noting the length of an unknown structure in graticule divisions you can then
convert this into absolute units of length, e.g. µm.
6. Each objective lens needs to be calibrated in the same way.
Once calibrated objects can be measured in EPUs.
EPUs are converted into absolute measurement using the figures obtained by
the calibration.
Summary
• Magnification is the ratio of image to object
size.
• Magnification is achieved by eye piece and
objective lens.
• Magnification is image size divided by object
size.
• Object size is image size divided by
magnification.
• Resolution is the ability to discriminate two
individual points.
• In light microscopy resolution is determined
by the quality of the lens and ultimately the
wavelength of light.