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

Arm

Coarse Adjustment KnobFine AdjustmentKnobLight Source

Nosepiece� Objectives

Stage Adjustment Knob Iris Diaphragm

Base

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