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
The reliability of analytical
data generated from chemical
and physical analyses is
critically dependent on three
factors:
1. Validity of the analytical
methods used
2. Reliability of the instruments
used for the experiments
3. Proper training of the analysts

The light microscope also called compound
microscope because it consists of two lens systems,
the objective lens, which forms a real image of the
specimen, and the eyepiece, which forms an image
at infinity that can be viewed by the operator
 The overall magnification is the product of the
magnification of these two groups of lenses

Microscope performance (resolving power)

This is the capability of the microscope to discriminate between
two points separated by a minute distance (distance two objects
must be apart and still be seen as separate and distinct).

Human eye can resolve two points as close as 100 µm apart.

The maximum resolution of the light microscope 200nm. Objects
closer together than 0.2 µm will not be distinctly seen. Increasing
the magnification will not make the objects more distinct, just
bigger.
Ocular
Nosepiece͢
Objectives
Arm
Stage Adjustment
Knob
Iris Diaphragm
Base
Coarse Adjustment
Knob
Fine Adjustment
Knob
Light Source

Because the size of objects in the field of view is
different at each magnification , you have to calculate
the diameters of the fields of view at each
magnification . This process is called “ Calibration of
microscope “
The field of view is the area that is visible to you when you look
through the eyepiece, when you increase the magnification
you focus on a smaller area
By knowing the size of the field of view ( diameter), you can
measure the size of objects in the microscope

To determine field diameter using a stage micrometer, one places
the stage micrometer on the microscope stage.

Next , looking through the eye piece (using the lowest
magnification) , one uses the mechanical stage controls to line up
divisions of the micrometer with edges of field of view.

Once a microscope is calibrated at one magnification it should
not be necessary to repeat calibration for other objective lenses.
The scale is inversely proportional to the magnification itself.

Field of view 1 X magnification 1 = Field of view 2 X
magnification 2

If field diameter at 40X is measured to be 5.8 mm ,then
the diameter at 400X must be 0.58mm
5.8 x 40X = ? x 400X
HPD = 5.8 x 40X
400X

Some stage micrometers are finely divided only at one
end. One of the larger divisions is positioned at one edge
of the field of view, so that the fine part of the scale
overlaps the opposite side. The field diameter ca then
be determined to the maximum available precision
› An ocular micrometer
scale, or reticule, is a scale
etched on a glass disk and
placed within an eyepiece,
the scale is over imposed
over any image seen in the
microscope, allowing the
user to measure any object
in the field of view, such
measurement requires the
reticule to be calibrated

T o calibrate and / or use an eyepiece reticule, start by
focusing the eyepiece itself on the reticule. Line the
ocular reticule with a stage micrometer , then measure
the distance over which the eyepiece reticule extends
and divide by the number of divisions to determine
distance per division