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Microscopy

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 (2 nd Century B.C.) Described refraction water Seneca (1 st Century A.D.)

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Microscopy

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  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 (962-1038 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 • 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)

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

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

  17. Microscopy • Magnification • Relation of Magnification to Field Diameter • Measuring in the Microscope

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

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

  20. Microscopy • Staining Techniques – Special Stains • Capsule • Endospore • Flagella

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