Micrometry

Microscopes: window to the world of the cell

Why use Light/ electron

microscopes?

• Total magnification= objective lens X eyepiece lens

• Total magnification= observed size of object/ actual size of object

OBJ:(a) Explain and distinguish between resolution and magnificationResolution- 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

Contrasts in microscopes and microscopy techniques

Light Microscopy

Scanning Electron microscopy

Transmission Electron Microscopy

Samples dead

Able to Able to view live view live samplessamplesSample preparation simpler

Samples need to be coated

Samples need to be sectioned

Lower Magnification & resolution (200 nm)

Higher degree of magnification and resolution (0.5nm)

Samples viewed in colour

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

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

Measurement of microscope samples

• 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)

Stage Micrometer Ruler measurement

Entire scale = divisions

Therefore 1 division =

Calibrating Stage Micrometer

100 10mm

10/100 =0.1mm=100m

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

MicrometryPlace the micrometer on the microscope stage and

superimpose or align the graticule scale onto it.

Each micrometer division= 0.05 mm

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 mm

0 20

0 0.65 mm20 divisions of the graticule= 0.65 mm

1 division = 0.65mm /20 =0.0325 mm or 32.85µmLength of cell=32.5µm X 7 graticule divisions =227.5µm

Place your sample under the same magnification and line it against the graticule

20 divisions of graticule

7 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

Plastic graticule

1. Calibration tool

2. Location

3. Clarity of scale

4. Accuracy of measurement

Stage micrometer

Eyepiece

Clear

Accurate

Ruler

Glass slide on stage

Not clear Inaccurate

Eyepiece graticule vs. plastic graticule

Eyepiece graticule

Plastic graticule

1. Calibration tool

2. Location

3. Clarity of scale

4. Accuracy of measurement

Stage micrometer

Eyepiece

Clear

Accurate

Ruler

slide on stage

Not clear

Inaccurate