The document discusses two topics: 1. Measuring the size of objects under a microscope using an eyepiece graticule. An eyepiece graticule must be calibrated for each magnification by comparing it to a stage micrometer. 2. The process of cell fractionation which separates cell components by breaking open cells and centrifuging to separate organelles based on density.

1. We are learning to:
• Calibrate an eye piece graticule and use it to measure the size of an object
under microscope
• Describe the process of cell fractionation

2. What is an eyepiece graticule
We know that
So, size of real object = size of image /magnification
The size of an object under a microscope is measured using a scale fixed in
the eyepiece lens of a microscope. The scale is called ‘ EYEPIECE GRATICULE’.
The divisions on an eyepiece graticule (EPG) have no measurement units at their own.
The units are assigned to the EPG using a stage micrometer. This is called calibrating an EPG.
Since the same image appears of different sizes under different objective lenses on the same
microscope, the calibration has to be done separately for each magnification.

3. An Eyepiece graticule
The size of an image under a microscope can be measured using a fixed scale called eyepiece graticule.
After correct calibration the same EPG can be used for any measurements carried out under a microscope.
An EPG does not have any units on it, the units are assigned by calibrating the EPG against another scale called SM.
This is called calibration and is only need to be conducted once. It has to be done separately for each magnification.
Objective graticule/objective micrometer
It is a scale printed on a microscope. It can be used for direct measurements of an object as well as for calibration
of an eyepiece graticule.
NOTE: *A micrometre may be used by being directly mounted on the object being viewed.
However, this is expensive and impractical and thus not common.

4. Cell fractionation
In cell fractionation, cells are broken up and the different organelles are separated out.
Cell fractionation comprises of following stages:
• Freezing/cooling the tissue in a buffer solution with the same
water potential as the cell cytoplasm
• Homogenation
Breaking up the tissue in a blender releases organelles from cells
Any cells and large debris are filtered off from the homogenate
to get a mixture of organelles alone.
• Ultracentrifugation
Various organelles are from the the homogenate using a centrifuge machine
A centrifuge spins the homogenate starting with a a slow speed.
The heaviest organelles settles down while the rest of the mixture is transferred to
to another tube and spun at a speed higher than before.
The process is repeated until all required organelles are separated.
Figure: A centrifuge machine
Figure: Stages of cell fractionation

5. An Eyepiece graticule
The size of an image under a microscope can be measured using a fixed scale called eyepiece graticule.
After correct calibration the same EPG can be used for any measurements carried out under a microscope.
An EPG does not have any units on it, the units are assigned by calibrating the EPG against another scale called SM.
This is called calibration and is only need to be conducted once. It has to be done separately for each magnification.
Objective graticule/objective micrometer
It is a scale printed on a microscope. It can be used for direct measurements of an object as well as for calibration
of an eyepiece graticule.
NOTE: *A micrometre may be used by being directly mounted on the object being viewed.
However, this is expensive and impractical and thus not common.

6. The graticule calibration is done in the following steps
1. Set up the microscope to the required magnification to view the sample.
2. Place a stage graticule on the stage.
3. Line up the two scales (the stage and eyepiece graticules) similar to the diagram.
4. Count the number of divisions on the eyepiece graticule equivalent to each division on the stage micrometre
5. As the length equivalent to each division on the stage micrometre are known,
it is possible to calculate the length of one eyepiece division.

7. Example:
Assume 1 stage micrometre division = 0.01mm (its provided on the stage micrometre)
If 100 eyepiece divisions = 30 stage micrometre division
= ……………………(no. of stage divs.) x ……………………..( length of 1 division on micrometre)
= …………………..(a)
1 eyepiece graticule unit = ………………………(a) /………………..(no. of eyepiece graticule divs)
= ………………………
To calculate the size of an object count the divisions a specimen covers on the eyepiece graticule and multiply it by
the 1 eyepiece graticule unit calculated above.
EPG calibration Object size measurement Object size measurement

8. Types of microscopes
1. Simple convex lenses
2. The compound light-microscope
3. The electron microscope
a. The transmission electron microscope
b. The scanning electron microscope
Light Microscope Electron Microscope
Low purchase and operation cost High purchase and operation cost
Small and portable Large and requires special operation room
Simple and easy sample preparation Lengthy and complex sample preparation
The material under study is rarely damaged The material under study is damaged during sample
preparation
Vacuum is not required Requires vacuum
Natural colors of sample are maintained All images are in black-and-white
Resolving power = 0.2 µm Resolving power = 0.1 nm
Magnification is up to 2000 times Magnification over 500, 000
Both living and dead specimens can be studied Only dead specimens are observed as they need to be fixed in
plastic and viewed under vacuum
Specimen stained to improve visibility Specimen stained with electron dense material (heavy
metals like gold.

9. Types of microscopes
1. The electron microscope
In an electron microscope an electron beam is used instead of photons (light), which increases the resolution
of the microscope 20 (in SEM) to 2000 (in TEM) times.
a. The transmission electron microscope
b. The scanning electron microscope

10. Comparison of a transmission electron microscope (TEM) and scanning electron microscope (SEM)
Transmission Electron Microscope (TEM) Scanning Electron Microscope (SEM)
A beam of electrons passes through the specimen, which on
reflection is directed on a fluorescence screen to get a
photomicrograph.
The beam of electrons scans the surface of the specimen rather
than penetrating into the specimen. The scattered electrons
generates an image of the surface contours.
A flat 2D images 3D images
Resolution 2000 time better than the light microscope Resolution 20 time better than the light microscope
Requires a very thin specimen which is hard to prepare and
introduces artifacts in the end image
Thick sample can be tested
Image not in natural colours Coloured image
Limitation of the TEM
• The upper limit of the resolution is hard to achieve due to difficulties in preparing an adequately thin sample.
• The sample gets damaged due to vacuum and electrons energy/living sample cannot be observed.
• No coloured image