1. Microscopy History Compound Light Microscopes Types of Microscopy
Measurement Systems
Measuring in a Microscope
Staining Procedures

2. Microscopy Early History of Devices that Alter Light:
Claudius Ptolemy (2nd Century B.C.)
Described refraction water
Seneca (1st Century A.D.)
Described magnification by a globe of water
Alhazen ( A.D.)
Described optical principles & anatomy of eye
Roger Bacon (1267 A.D.)
Described simple magnification

3. Microscopy Lenses first derived were used to improve eyesight
Pliny the Elder wrote of Nero’s use of emeralds to watch gladiators
Reinvention of spectacles occurred around 1280 to 1285 in Florence, Italy
Dutch spectacle maker Zaccharias Jansen was probably first to combine two lenses into compound microscope (1595)

4. Microscopy Robert Hooke (1665) Contemporary of Robert Boyle
Described cork with “cells” – first use of “cell” to describe structure of living organism
Used compound microscope

5. Microscopy Antoni van Leeuwenhoek Simple single lens microscopes
First described bacteria, blood, protozoa & sperm
Sent letters to Royal Society

6. Microscopy

7. Microscopy Problems with early microscopes Chromatic aberration
Spherical aberration

8. Microscopy Chromatic aberration
Occurs when different wavelengths of light are refracted through the lenses at different angles
Corrected using glass of different types

9. Microscopy Spherical aberration
Distortion because light hitting edge of lens does not have same focal length as middle
Correct using small apertures or diaphragms
Solved by Joseph Jackson Lister in 1830
Why reducing diaphragm improves contrast

10. Microscopy Microscope parts Ocular Objectives Stage Diaphragm
Condenser
Light Source
Course adjustment
Fine adjustment

11. Microscopy Modern compound microscope Diaphragm Condenser
Oil Immersion

12. Microscopy Compound Microscope Total Magnification Refractive Index
Ocular X Objective equals Total
Refractive Index
A measure of the relative velocity of light passing through a substance
Oil immersion
prevents light scattering between slide and objective – has same refractive index as slide

13. Microscopy Compound Microscope – Resolution
The ability of a lens to distinguish between two points as separate objects
Depends on wavelength of light – usually maximum resolution is wavelength / 2
Maximum for light microscope is about 0.2 microns or about 2000x

14. Microscopy Types of Modern Microscopy Bright field Dark field
Phase Contrast
Electron Microscopy
Scanning Electron Microscopy
Fluorescent (UV)

17. Microscopy Measurement Distance (Metric System) Meter (m) 100
Centimeter (cm) 10-2
Millimeter (mm) 10-3
Micrometer (µm) 10-6
Nanometer (nm) 10-9

18. Microscopy Measurement Volume (Metric System) Liter (L) 100
Cubic centimeter (cc) l cc = 1 mL
Milliliter (mL) 10-3
Microliter (µL) 10-6

19. Microscopy Unit Conversions Need a conversion factor
Example: µm = 1 mm
If not given, make it
Make a fraction; desired unit in numerator and original unit in denominator
Add appropriate numerical values from conversion factor and multiply

21. Microscopy Relation of Magnification to Field Diameter
Measuring in the Microscope Using Field Diameter

22. Microscopy Staining techniques – Preparation Smear Heat Fixation
Stain/counter stain
Negative stain

23. Microscopy Staining Techniques – Simple stains Mordant Crystal violet
Saffron
Methylene blue
Mordant
Intensifies stain
Iodine used in Gram stain

24. Microscopy Staining Techniques – Differential Stains
Stain one group of organisms/cells different than another
Gram stain
Acid Fast Stain

26. Microscopy Staining Techniques – Special Stains Capsule Endospore
Flagella