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Measurement & magnification in microscopes

"Magnification" can often be meaningless because of the electronic manipulation, resizing etc. of  images on computers and also different computer screen sizes. Magnification needs to be specifically measured. Often, it is more meaningful to give an absolute measurement of something seen through a microscope.

Measurements in microscopes are accomplished by calibrating the system using two items:

  1. a stage micrometer - essentially a small ruler viewed through the microscope, it is an etched scale with divisions. This will appear larger as magnification is increased.

  2. an eyepiece graticule - an etched scale or framework that is located in the ocular i.e. eyepiece of the microscope. This does not appear larger as magnification is increased and is therefore a fixed reference.


 

Wild M5 low-power stereo microscope:

Stage micrometer - scale for measurement on a "low power" dissecting/stereo microscope
This is a 10 mm scale divided into 100 divisions, where
each small division = 0.1 mm = 100 micrometers = 100 mm.

 

Eyepiece measuring graticule in the Wild M5 stereo microscope
The divisions are arbitrary and need calibrating against a known scale.


Calibration simply means recording what the eyepiece graticule/scale divisions cover on the stage micrometer at each magnification. For practical purposes, it is more accurate to take many eyepiece divisions, measure them against a length on the stage micrometer and then calculate what one eyepiece division measures.

Stereo microscope stage micrometer (large figures) and eyepeiece micrometer (small figures)
photographed at x50. This combines the scales seen in the two photos above.
100 eyepiece divisions on the small scale cover 2 mm on the large scale,
where "20" = 2,000 micrometers.
Therefore, 1 eyepiece division = 20 micrometers at x50.

 

 

Leitz Orthoplan "high-power"/compound research microscope
- with Nomarski (DIC - Differential Interference Contrast) and dark-field optics:
 

Stage micrometer - scale for measurement on a high-power microscope
This is a 1 mm scale divided into 100 divisions.
Each small division = 1/100th mm = 0.01 mm = 10 micrometers or 10 mm

 

Eyepiece graticule in the Leitz Orthoplan microscope
(this was originally part of a 35 mm automatic film camera, now obsolete)
The corner lines and ring spacings can be used for measurement.


Again - calibration simply means recording what an eyepiece feature covers on the stage micrometer at each magnification. For accuracy, it is more accurate to take several eyepiece divisions, measure them against a long length on the stage micrometer and then calculate what one eyepiece division/feature measures.
 

 

Both Leitz high-power microscope graticules photographed together at x400.
The whole photo-field covers 215 micrometers left-to-right, 0.215 mm or approx. 1/5th mm.
The outer rings cover 35 micrometers
The inner rings cover 20 micrometers
The gap between the 2 rings covers 9 micrometers at x 400.

The magnification of this image?
"0 to 20" = 1/5th mm = 0.2 mm = 200 micrometers covering 142 mm on a 800x 600 display.
Therefore: 142,000 / 200 = x710
NB - the microscope was set at x400

 

Both high-power microscope graticules photographed together at x1000.
The repeat period of the vertical black bars on the stage micrometer is 10 micrometers.
This measures approx. 65 mm on an 800x600 display.
The double outer rings are approx 6.5 mm wide, covering 1 micrometer.
The central dark zone of the outer rings measures approx. 2 mm,
covering 10 x (2/65) = 0.3 micrometers
(this is close to the resolution limit of the best light microscopes. being about 0.2 micometers).

The magnification on this image?
- on a 800x 600 display
The repeat period of the vertical black bars, representing 10 micrometers, is 65 mm.
65 mm = 65,000 micrometers, therefore divide by 10 to get 1 micrometer
65,000 / 10 = x6,500 magnification.

 

Another way of measuring things is to photograph a specimen and then photograph a scale under the same conditions and compare/superimpose the two without digitally changing sizes.

A further method is to photograph a specimen and scale at identical magnifications - these can then be manipulated for measurement in a photo program using separate image layers.

 


Wild M5 low-power microscope (left)
Leitz Orthoplan microscope (right)

 


First nymph examined for gut contents, from Ringmoor Down.
The 'head' and first pair of legs are removed.
The extruded contents are to the left.

 

 

 

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