Micrometry

Microscopes: window to the world of the cell

Why use Light/ electron microscopes?

OBJ: (a) Explain and distinguish between resolution and magnification

Resolution- The ability to distinguish between two points. up to 200nm for LM; 0.5 nm for EM Magnification- the number of times larger an observed object is to its actual size

Total magnification= objective lens X eyepiece lens Total magnification= observed size of object/ actual size of object

Contrasts in microscopes and microscopy techniques

Light Microscopy
Able to view live samples

Scanning Electron microscopy

Transmission Electron Microscopy

Samples dead
Samples need to be sectioned

Sample preparation Samples need to be coated simpler

Lower Magnification & resolution (200 nm)

Samples viewed in colour

Higher degree of magnification and resolution (0.5nm)

Samples black and white or grayscale

Stage Micrometer
A glass slide with a scale etched onto it. However its unit is not always SI unit, but an arbitrary unit. So we need to calibrate the stage micrometer, i.e. to convert the arbitrary unit to SI unit. How? Use your ruler

Measurement of microscope samples

A

stage micrometer is a glass slide with etchings of a known or measurable length. Think of it as a tiny ruler

 graticules are microscope eyepieces with a scale etched (usually with 100 divisions) into it for the routine measuring of samples. Graticules need to be calibrated with the micrometer before it can be effectively used. (Etchings in non-S.I units)

Calibrating Stage Micrometer

Stage Micrometer
Entire scale =100 divisions Therefore 1 division =

Ruler measurement
10mm 10/100 =0.1mm =100mm

Try to keep all measurements to 2 decimal places and utilize unit prefixes

Micrometry
Place the micrometer on the microscope stage and superimpose or align the graticule scale onto it.
Place your sample under the same magnification and line it against the graticule
20 20 divisions of graticule

7 divisions 0 0
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 Each micrometer division= mm

0.05 mm

0.65 mm

20 divisions of the graticule= 0.65 mm 1 division = 0.65mm /20

=0.0325 mm or 32.85m Length of cell=32.5m X 7 graticule =227.5m divisions

WARNING!!!!
Be sure to be mindful of the no. of divisions on the graticule it may not always be 100 divisions The same goes for your micrometerit may not always be 10 mm. Make sure when you do the graticule calibration you know which scale belongs to which. Be sure units are correct and unit conversions are correct.

Eyepiece graticule vs. plastic graticule

Eyepiece graticule
1. Calibration tool 2. Location
Glass slide on stage Stage micrometer

Plastic graticule
Clear

Ruler Accurate Inaccurate

3. Clarity of scale
Eyepiece

4. Accuracy of measurement

Not clear

Eyepiece graticule vs. plastic graticule

Eyepiece graticule
1. Calibration tool 2. Location
Stage micrometer

Plastic graticule
Ruler
slide on stage

Eyepiece
Clear Accurate Not clear Inaccurate

3. Clarity of scale
4. Accuracy of measurement