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

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 (962-1038 A.D.) Described optical principles & anatomy of eye Roger Bacon (1267 A.D.) Described simple magnification

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)

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

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

Microscopy

Microscopy Problems with early microscopes Chromatic aberration Spherical aberration

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

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

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

Microscopy Modern compound microscope Diaphragm Condenser Oil Immersion

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

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

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

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

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

Microscopy Unit Conversions Need a conversion factor Example: 1000 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

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

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

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

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

Microscopy Staining Techniques – Special Stains Capsule Endospore Flagella