Microscope measurements & calculations

What is used for measuring cells?

• Eyepiece graticule - light microscope - glass disc placed in eyepiece of a microscope with a scale etched into glass disc

• 10 mm long & divided into 100 subdivisions

  

Why must the eyepiece graticule be calibrated for a particular lens?

• Scale cannot be used directly to measure size of objects under a microscope’s objective lens because each objective lens will magnify to a different degree

• Once calibrated, graticule can remain in position for future use, provided same objective lens is used

• Sensible to record results of calibration for a particular objective lens & leave this attached to microscope

What is used for calibrating eyepiece graticule?

• Stage micrometer

• Scale etched - 2mm long & smallest subdivisions are 0.01 mm

Calculating length of divisions on eyepiece graticule

• Find ratio between eyepiece graticule scale & stage micrometer scale

• Divide stage micrometre scale with eyepiece graticule scale

Calculating scale for different objective lenses

• Divide differences in magnification

• Divide this by calibration unit

Artefact

A visible structural detail produced by the

processing of the specimen.

‣ Not a part of the living tissue.

‣ e.g. an air bubble.

Explain how to measure part of a specimen when observed through an optical microscope

• Use an eyepiece graticule.

‣ Calibrate the eyepiece graticule using a stage micrometer.

‣ Measure the required part of the specimen (e.g. the diameter of a cell).

‣ Take repeat measurements and calculate a mean.

‣ Use the calibrated eyepiece graticule units to calculate the size of the required part.

How to prepare a dry mount

• Use a scalpel/microtome to cut a thin slice of the specimen.

‣ Use tweezers to place the specimen in the middle of a clean slide.

‣ Place a cover slip over the top.

How to prepare a wet mount

• Use a pipette to place a drop of water onto a clean slide.

‣ Use a scalpel/microtome to cut a thin slice of the specimen (so light can pass through).

‣ Use tweezers to place specimen on top of water.

‣ Lower cover slip at an angle over specimen, to avoid trapping air bubbles.

‣ If needed, add stain to make structures more visible.

Principles for scientific drawings

• Drawing must take up at least 50% of the space provided.

‣ Continuous lines - no sketching.

‣ No shading/hatching/colouring.

‣ Ruled label lines in pencil with no arrowheads.

‣ Label lines should not cross.

‣ State magnification factor / include a scale bar.

‣ Correct proportions.

‣ Include a title.

Principles for scientific drawings: low-power tissue plan

• Don’t draw individual cells, show only the distribution of tissues.

‣ Draw all tissues completely enclosed by pencil lines (there should be no gaps or crossed lines).

‣ Draw an accurate interpretation of the distribution of tissues (e.g. thickness/size of each tissue must be similar to microscope image).

‣ Label the different tissues present.

How did the scientific community distinguish between

artefacts & organelles?

• Prepared specimens in different ways.

‣ If an object could be seen with one preparation technique but not another, it was likely to be an artefact.