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College Physics for AP® Courses

27.9 *Extended Topic* Microscopy Enhanced by the Wave Characteristics of Light

College Physics for AP® Courses27.9 *Extended Topic* Microscopy Enhanced by the Wave Characteristics of Light
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  1. Preface
  2. 1 Introduction: The Nature of Science and Physics
    1. Connection for AP® Courses
    2. 1.1 Physics: An Introduction
    3. 1.2 Physical Quantities and Units
    4. 1.3 Accuracy, Precision, and Significant Figures
    5. 1.4 Approximation
    6. Glossary
    7. Section Summary
    8. Conceptual Questions
    9. Problems & Exercises
  3. 2 Kinematics
    1. Connection for AP® Courses
    2. 2.1 Displacement
    3. 2.2 Vectors, Scalars, and Coordinate Systems
    4. 2.3 Time, Velocity, and Speed
    5. 2.4 Acceleration
    6. 2.5 Motion Equations for Constant Acceleration in One Dimension
    7. 2.6 Problem-Solving Basics for One Dimensional Kinematics
    8. 2.7 Falling Objects
    9. 2.8 Graphical Analysis of One Dimensional Motion
    10. Glossary
    11. Section Summary
    12. Conceptual Questions
    13. Problems & Exercises
    14. Test Prep for AP® Courses
  4. 3 Two-Dimensional Kinematics
    1. Connection for AP® Courses
    2. 3.1 Kinematics in Two Dimensions: An Introduction
    3. 3.2 Vector Addition and Subtraction: Graphical Methods
    4. 3.3 Vector Addition and Subtraction: Analytical Methods
    5. 3.4 Projectile Motion
    6. 3.5 Addition of Velocities
    7. Glossary
    8. Section Summary
    9. Conceptual Questions
    10. Problems & Exercises
    11. Test Prep for AP® Courses
  5. 4 Dynamics: Force and Newton's Laws of Motion
    1. Connection for AP® Courses
    2. 4.1 Development of Force Concept
    3. 4.2 Newton's First Law of Motion: Inertia
    4. 4.3 Newton's Second Law of Motion: Concept of a System
    5. 4.4 Newton's Third Law of Motion: Symmetry in Forces
    6. 4.5 Normal, Tension, and Other Examples of Force
    7. 4.6 Problem-Solving Strategies
    8. 4.7 Further Applications of Newton's Laws of Motion
    9. 4.8 Extended Topic: The Four Basic Forces—An Introduction
    10. Glossary
    11. Section Summary
    12. Conceptual Questions
    13. Problems & Exercises
    14. Test Prep for AP® Courses
  6. 5 Further Applications of Newton's Laws: Friction, Drag, and Elasticity
    1. Connection for AP® Courses
    2. 5.1 Friction
    3. 5.2 Drag Forces
    4. 5.3 Elasticity: Stress and Strain
    5. Glossary
    6. Section Summary
    7. Conceptual Questions
    8. Problems & Exercises
    9. Test Prep for AP® Courses
  7. 6 Gravitation and Uniform Circular Motion
    1. Connection for AP® Courses
    2. 6.1 Rotation Angle and Angular Velocity
    3. 6.2 Centripetal Acceleration
    4. 6.3 Centripetal Force
    5. 6.4 Fictitious Forces and Non-inertial Frames: The Coriolis Force
    6. 6.5 Newton's Universal Law of Gravitation
    7. 6.6 Satellites and Kepler's Laws: An Argument for Simplicity
    8. Glossary
    9. Section Summary
    10. Conceptual Questions
    11. Problems & Exercises
    12. Test Prep for AP® Courses
  8. 7 Work, Energy, and Energy Resources
    1. Connection for AP® Courses
    2. 7.1 Work: The Scientific Definition
    3. 7.2 Kinetic Energy and the Work-Energy Theorem
    4. 7.3 Gravitational Potential Energy
    5. 7.4 Conservative Forces and Potential Energy
    6. 7.5 Nonconservative Forces
    7. 7.6 Conservation of Energy
    8. 7.7 Power
    9. 7.8 Work, Energy, and Power in Humans
    10. 7.9 World Energy Use
    11. Glossary
    12. Section Summary
    13. Conceptual Questions
    14. Problems & Exercises
    15. Test Prep for AP® Courses
  9. 8 Linear Momentum and Collisions
    1. Connection for AP® courses
    2. 8.1 Linear Momentum and Force
    3. 8.2 Impulse
    4. 8.3 Conservation of Momentum
    5. 8.4 Elastic Collisions in One Dimension
    6. 8.5 Inelastic Collisions in One Dimension
    7. 8.6 Collisions of Point Masses in Two Dimensions
    8. 8.7 Introduction to Rocket Propulsion
    9. Glossary
    10. Section Summary
    11. Conceptual Questions
    12. Problems & Exercises
    13. Test Prep for AP® Courses
  10. 9 Statics and Torque
    1. Connection for AP® Courses
    2. 9.1 The First Condition for Equilibrium
    3. 9.2 The Second Condition for Equilibrium
    4. 9.3 Stability
    5. 9.4 Applications of Statics, Including Problem-Solving Strategies
    6. 9.5 Simple Machines
    7. 9.6 Forces and Torques in Muscles and Joints
    8. Glossary
    9. Section Summary
    10. Conceptual Questions
    11. Problems & Exercises
    12. Test Prep for AP® Courses
  11. 10 Rotational Motion and Angular Momentum
    1. Connection for AP® Courses
    2. 10.1 Angular Acceleration
    3. 10.2 Kinematics of Rotational Motion
    4. 10.3 Dynamics of Rotational Motion: Rotational Inertia
    5. 10.4 Rotational Kinetic Energy: Work and Energy Revisited
    6. 10.5 Angular Momentum and Its Conservation
    7. 10.6 Collisions of Extended Bodies in Two Dimensions
    8. 10.7 Gyroscopic Effects: Vector Aspects of Angular Momentum
    9. Glossary
    10. Section Summary
    11. Conceptual Questions
    12. Problems & Exercises
    13. Test Prep for AP® Courses
  12. 11 Fluid Statics
    1. Connection for AP® Courses
    2. 11.1 What Is a Fluid?
    3. 11.2 Density
    4. 11.3 Pressure
    5. 11.4 Variation of Pressure with Depth in a Fluid
    6. 11.5 Pascal’s Principle
    7. 11.6 Gauge Pressure, Absolute Pressure, and Pressure Measurement
    8. 11.7 Archimedes’ Principle
    9. 11.8 Cohesion and Adhesion in Liquids: Surface Tension and Capillary Action
    10. 11.9 Pressures in the Body
    11. Glossary
    12. Section Summary
    13. Conceptual Questions
    14. Problems & Exercises
    15. Test Prep for AP® Courses
  13. 12 Fluid Dynamics and Its Biological and Medical Applications
    1. Connection for AP® Courses
    2. 12.1 Flow Rate and Its Relation to Velocity
    3. 12.2 Bernoulli’s Equation
    4. 12.3 The Most General Applications of Bernoulli’s Equation
    5. 12.4 Viscosity and Laminar Flow; Poiseuille’s Law
    6. 12.5 The Onset of Turbulence
    7. 12.6 Motion of an Object in a Viscous Fluid
    8. 12.7 Molecular Transport Phenomena: Diffusion, Osmosis, and Related Processes
    9. Glossary
    10. Section Summary
    11. Conceptual Questions
    12. Problems & Exercises
    13. Test Prep for AP® Courses
  14. 13 Temperature, Kinetic Theory, and the Gas Laws
    1. Connection for AP® Courses
    2. 13.1 Temperature
    3. 13.2 Thermal Expansion of Solids and Liquids
    4. 13.3 The Ideal Gas Law
    5. 13.4 Kinetic Theory: Atomic and Molecular Explanation of Pressure and Temperature
    6. 13.5 Phase Changes
    7. 13.6 Humidity, Evaporation, and Boiling
    8. Glossary
    9. Section Summary
    10. Conceptual Questions
    11. Problems & Exercises
    12. Test Prep for AP® Courses
  15. 14 Heat and Heat Transfer Methods
    1. Connection for AP® Courses
    2. 14.1 Heat
    3. 14.2 Temperature Change and Heat Capacity
    4. 14.3 Phase Change and Latent Heat
    5. 14.4 Heat Transfer Methods
    6. 14.5 Conduction
    7. 14.6 Convection
    8. 14.7 Radiation
    9. Glossary
    10. Section Summary
    11. Conceptual Questions
    12. Problems & Exercises
    13. Test Prep for AP® Courses
  16. 15 Thermodynamics
    1. Connection for AP® Courses
    2. 15.1 The First Law of Thermodynamics
    3. 15.2 The First Law of Thermodynamics and Some Simple Processes
    4. 15.3 Introduction to the Second Law of Thermodynamics: Heat Engines and Their Efficiency
    5. 15.4 Carnot’s Perfect Heat Engine: The Second Law of Thermodynamics Restated
    6. 15.5 Applications of Thermodynamics: Heat Pumps and Refrigerators
    7. 15.6 Entropy and the Second Law of Thermodynamics: Disorder and the Unavailability of Energy
    8. 15.7 Statistical Interpretation of Entropy and the Second Law of Thermodynamics: The Underlying Explanation
    9. Glossary
    10. Section Summary
    11. Conceptual Questions
    12. Problems & Exercises
    13. Test Prep for AP® Courses
  17. 16 Oscillatory Motion and Waves
    1. Connection for AP® Courses
    2. 16.1 Hooke’s Law: Stress and Strain Revisited
    3. 16.2 Period and Frequency in Oscillations
    4. 16.3 Simple Harmonic Motion: A Special Periodic Motion
    5. 16.4 The Simple Pendulum
    6. 16.5 Energy and the Simple Harmonic Oscillator
    7. 16.6 Uniform Circular Motion and Simple Harmonic Motion
    8. 16.7 Damped Harmonic Motion
    9. 16.8 Forced Oscillations and Resonance
    10. 16.9 Waves
    11. 16.10 Superposition and Interference
    12. 16.11 Energy in Waves: Intensity
    13. Glossary
    14. Section Summary
    15. Conceptual Questions
    16. Problems & Exercises
    17. Test Prep for AP® Courses
  18. 17 Physics of Hearing
    1. Connection for AP® Courses
    2. 17.1 Sound
    3. 17.2 Speed of Sound, Frequency, and Wavelength
    4. 17.3 Sound Intensity and Sound Level
    5. 17.4 Doppler Effect and Sonic Booms
    6. 17.5 Sound Interference and Resonance: Standing Waves in Air Columns
    7. 17.6 Hearing
    8. 17.7 Ultrasound
    9. Glossary
    10. Section Summary
    11. Conceptual Questions
    12. Problems & Exercises
    13. Test Prep for AP® Courses
  19. 18 Electric Charge and Electric Field
    1. Connection for AP® Courses
    2. 18.1 Static Electricity and Charge: Conservation of Charge
    3. 18.2 Conductors and Insulators
    4. 18.3 Conductors and Electric Fields in Static Equilibrium
    5. 18.4 Coulomb’s Law
    6. 18.5 Electric Field: Concept of a Field Revisited
    7. 18.6 Electric Field Lines: Multiple Charges
    8. 18.7 Electric Forces in Biology
    9. 18.8 Applications of Electrostatics
    10. Glossary
    11. Section Summary
    12. Conceptual Questions
    13. Problems & Exercises
    14. Test Prep for AP® Courses
  20. 19 Electric Potential and Electric Field
    1. Connection for AP® Courses
    2. 19.1 Electric Potential Energy: Potential Difference
    3. 19.2 Electric Potential in a Uniform Electric Field
    4. 19.3 Electrical Potential Due to a Point Charge
    5. 19.4 Equipotential Lines
    6. 19.5 Capacitors and Dielectrics
    7. 19.6 Capacitors in Series and Parallel
    8. 19.7 Energy Stored in Capacitors
    9. Glossary
    10. Section Summary
    11. Conceptual Questions
    12. Problems & Exercises
    13. Test Prep for AP® Courses
  21. 20 Electric Current, Resistance, and Ohm's Law
    1. Connection for AP® Courses
    2. 20.1 Current
    3. 20.2 Ohm’s Law: Resistance and Simple Circuits
    4. 20.3 Resistance and Resistivity
    5. 20.4 Electric Power and Energy
    6. 20.5 Alternating Current versus Direct Current
    7. 20.6 Electric Hazards and the Human Body
    8. 20.7 Nerve Conduction–Electrocardiograms
    9. Glossary
    10. Section Summary
    11. Conceptual Questions
    12. Problems & Exercises
    13. Test Prep for AP® Courses
  22. 21 Circuits, Bioelectricity, and DC Instruments
    1. Connection for AP® Courses
    2. 21.1 Resistors in Series and Parallel
    3. 21.2 Electromotive Force: Terminal Voltage
    4. 21.3 Kirchhoff’s Rules
    5. 21.4 DC Voltmeters and Ammeters
    6. 21.5 Null Measurements
    7. 21.6 DC Circuits Containing Resistors and Capacitors
    8. Glossary
    9. Section Summary
    10. Conceptual Questions
    11. Problems & Exercises
    12. Test Prep for AP® Courses
  23. 22 Magnetism
    1. Connection for AP® Courses
    2. 22.1 Magnets
    3. 22.2 Ferromagnets and Electromagnets
    4. 22.3 Magnetic Fields and Magnetic Field Lines
    5. 22.4 Magnetic Field Strength: Force on a Moving Charge in a Magnetic Field
    6. 22.5 Force on a Moving Charge in a Magnetic Field: Examples and Applications
    7. 22.6 The Hall Effect
    8. 22.7 Magnetic Force on a Current-Carrying Conductor
    9. 22.8 Torque on a Current Loop: Motors and Meters
    10. 22.9 Magnetic Fields Produced by Currents: Ampere’s Law
    11. 22.10 Magnetic Force between Two Parallel Conductors
    12. 22.11 More Applications of Magnetism
    13. Glossary
    14. Section Summary
    15. Conceptual Questions
    16. Problems & Exercises
    17. Test Prep for AP® Courses
  24. 23 Electromagnetic Induction, AC Circuits, and Electrical Technologies
    1. Connection for AP® Courses
    2. 23.1 Induced Emf and Magnetic Flux
    3. 23.2 Faraday’s Law of Induction: Lenz’s Law
    4. 23.3 Motional Emf
    5. 23.4 Eddy Currents and Magnetic Damping
    6. 23.5 Electric Generators
    7. 23.6 Back Emf
    8. 23.7 Transformers
    9. 23.8 Electrical Safety: Systems and Devices
    10. 23.9 Inductance
    11. 23.10 RL Circuits
    12. 23.11 Reactance, Inductive and Capacitive
    13. 23.12 RLC Series AC Circuits
    14. Glossary
    15. Section Summary
    16. Conceptual Questions
    17. Problems & Exercises
    18. Test Prep for AP® Courses
  25. 24 Electromagnetic Waves
    1. Connection for AP® Courses
    2. 24.1 Maxwell’s Equations: Electromagnetic Waves Predicted and Observed
    3. 24.2 Production of Electromagnetic Waves
    4. 24.3 The Electromagnetic Spectrum
    5. 24.4 Energy in Electromagnetic Waves
    6. Glossary
    7. Section Summary
    8. Conceptual Questions
    9. Problems & Exercises
    10. Test Prep for AP® Courses
  26. 25 Geometric Optics
    1. Connection for AP® Courses
    2. 25.1 The Ray Aspect of Light
    3. 25.2 The Law of Reflection
    4. 25.3 The Law of Refraction
    5. 25.4 Total Internal Reflection
    6. 25.5 Dispersion: The Rainbow and Prisms
    7. 25.6 Image Formation by Lenses
    8. 25.7 Image Formation by Mirrors
    9. Glossary
    10. Section Summary
    11. Conceptual Questions
    12. Problems & Exercises
    13. Test Prep for AP® Courses
  27. 26 Vision and Optical Instruments
    1. Connection for AP® Courses
    2. 26.1 Physics of the Eye
    3. 26.2 Vision Correction
    4. 26.3 Color and Color Vision
    5. 26.4 Microscopes
    6. 26.5 Telescopes
    7. 26.6 Aberrations
    8. Glossary
    9. Section Summary
    10. Conceptual Questions
    11. Problems & Exercises
    12. Test Prep for AP® Courses
  28. 27 Wave Optics
    1. Connection for AP® Courses
    2. 27.1 The Wave Aspect of Light: Interference
    3. 27.2 Huygens's Principle: Diffraction
    4. 27.3 Young’s Double Slit Experiment
    5. 27.4 Multiple Slit Diffraction
    6. 27.5 Single Slit Diffraction
    7. 27.6 Limits of Resolution: The Rayleigh Criterion
    8. 27.7 Thin Film Interference
    9. 27.8 Polarization
    10. 27.9 *Extended Topic* Microscopy Enhanced by the Wave Characteristics of Light
    11. Glossary
    12. Section Summary
    13. Conceptual Questions
    14. Problems & Exercises
    15. Test Prep for AP® Courses
  29. 28 Special Relativity
    1. Connection for AP® Courses
    2. 28.1 Einstein’s Postulates
    3. 28.2 Simultaneity And Time Dilation
    4. 28.3 Length Contraction
    5. 28.4 Relativistic Addition of Velocities
    6. 28.5 Relativistic Momentum
    7. 28.6 Relativistic Energy
    8. Glossary
    9. Section Summary
    10. Conceptual Questions
    11. Problems & Exercises
    12. Test Prep for AP® Courses
  30. 29 Introduction to Quantum Physics
    1. Connection for AP® Courses
    2. 29.1 Quantization of Energy
    3. 29.2 The Photoelectric Effect
    4. 29.3 Photon Energies and the Electromagnetic Spectrum
    5. 29.4 Photon Momentum
    6. 29.5 The Particle-Wave Duality
    7. 29.6 The Wave Nature of Matter
    8. 29.7 Probability: The Heisenberg Uncertainty Principle
    9. 29.8 The Particle-Wave Duality Reviewed
    10. Glossary
    11. Section Summary
    12. Conceptual Questions
    13. Problems & Exercises
    14. Test Prep for AP® Courses
  31. 30 Atomic Physics
    1. Connection for AP® Courses
    2. 30.1 Discovery of the Atom
    3. 30.2 Discovery of the Parts of the Atom: Electrons and Nuclei
    4. 30.3 Bohr’s Theory of the Hydrogen Atom
    5. 30.4 X Rays: Atomic Origins and Applications
    6. 30.5 Applications of Atomic Excitations and De-Excitations
    7. 30.6 The Wave Nature of Matter Causes Quantization
    8. 30.7 Patterns in Spectra Reveal More Quantization
    9. 30.8 Quantum Numbers and Rules
    10. 30.9 The Pauli Exclusion Principle
    11. Glossary
    12. Section Summary
    13. Conceptual Questions
    14. Problems & Exercises
    15. Test Prep for AP® Courses
  32. 31 Radioactivity and Nuclear Physics
    1. Connection for AP® Courses
    2. 31.1 Nuclear Radioactivity
    3. 31.2 Radiation Detection and Detectors
    4. 31.3 Substructure of the Nucleus
    5. 31.4 Nuclear Decay and Conservation Laws
    6. 31.5 Half-Life and Activity
    7. 31.6 Binding Energy
    8. 31.7 Tunneling
    9. Glossary
    10. Section Summary
    11. Conceptual Questions
    12. Problems & Exercises
    13. Test Prep for AP® Courses
  33. 32 Medical Applications of Nuclear Physics
    1. Connection for AP® Courses
    2. 32.1 Medical Imaging and Diagnostics
    3. 32.2 Biological Effects of Ionizing Radiation
    4. 32.3 Therapeutic Uses of Ionizing Radiation
    5. 32.4 Food Irradiation
    6. 32.5 Fusion
    7. 32.6 Fission
    8. 32.7 Nuclear Weapons
    9. Glossary
    10. Section Summary
    11. Conceptual Questions
    12. Problems & Exercises
    13. Test Prep for AP® Courses
  34. 33 Particle Physics
    1. Connection for AP® Courses
    2. 33.1 The Yukawa Particle and the Heisenberg Uncertainty Principle Revisited
    3. 33.2 The Four Basic Forces
    4. 33.3 Accelerators Create Matter from Energy
    5. 33.4 Particles, Patterns, and Conservation Laws
    6. 33.5 Quarks: Is That All There Is?
    7. 33.6 GUTs: The Unification of Forces
    8. Glossary
    9. Section Summary
    10. Conceptual Questions
    11. Problems & Exercises
    12. Test Prep for AP® Courses
  35. 34 Frontiers of Physics
    1. Connection for AP® Courses
    2. 34.1 Cosmology and Particle Physics
    3. 34.2 General Relativity and Quantum Gravity
    4. 34.3 Superstrings
    5. 34.4 Dark Matter and Closure
    6. 34.5 Complexity and Chaos
    7. 34.6 High-Temperature Superconductors
    8. 34.7 Some Questions We Know to Ask
    9. Glossary
    10. Section Summary
    11. Conceptual Questions
    12. Problems & Exercises
  36. A | Atomic Masses
  37. B | Selected Radioactive Isotopes
  38. C | Useful Information
  39. D | Glossary of Key Symbols and Notation
  40. Answer Key
    1. Chapter 1
    2. Chapter 2
    3. Chapter 3
    4. Chapter 4
    5. Chapter 5
    6. Chapter 6
    7. Chapter 7
    8. Chapter 8
    9. Chapter 9
    10. Chapter 10
    11. Chapter 11
    12. Chapter 12
    13. Chapter 13
    14. Chapter 14
    15. Chapter 15
    16. Chapter 16
    17. Chapter 17
    18. Chapter 18
    19. Chapter 19
    20. Chapter 20
    21. Chapter 21
    22. Chapter 22
    23. Chapter 23
    24. Chapter 24
    25. Chapter 25
    26. Chapter 26
    27. Chapter 27
    28. Chapter 28
    29. Chapter 29
    30. Chapter 30
    31. Chapter 31
    32. Chapter 32
    33. Chapter 33
    34. Chapter 34
  41. Index

Learning Objectives

By the end of this section, you will be able to:

  • Discuss the different types of microscopes.

Physics research underpins the advancement of developments in microscopy. As we gain knowledge of the wave nature of electromagnetic waves and methods to analyze and interpret signals, new microscopes that enable us to “see” more are being developed. It is the evolution and newer generation of microscopes that are described in this section.

The use of microscopes (microscopy) to observe small details is limited by the wave nature of light. Owing to the fact that light diffracts significantly around small objects, it becomes impossible to observe details significantly smaller than the wavelength of light. One rule of thumb has it that all details smaller than about λλ size 12{λ} {} are difficult to observe. Radar, for example, can detect the size of an aircraft, but not its individual rivets, since the wavelength of most radar is several centimeters or greater. Similarly, visible light cannot detect individual atoms, since atoms are about 0.1 nm in size and visible wavelengths range from 380 to 760 nm. Ironically, special techniques used to obtain the best possible resolution with microscopes take advantage of the same wave characteristics of light that ultimately limit the detail.

Making Connections: Waves

All attempts to observe the size and shape of objects are limited by the wavelength of the probe. Sonar and medical ultrasound are limited by the wavelength of sound they employ. We shall see that this is also true in electron microscopy, since electrons have a wavelength. Heisenberg’s uncertainty principle asserts that this limit is fundamental and inescapable, as we shall see in quantum mechanics.

The most obvious method of obtaining better detail is to utilize shorter wavelengths. Ultraviolet (UV) microscopes have been constructed with special lenses that transmit UV rays and utilize photographic or electronic techniques to record images. The shorter UV wavelengths allow somewhat greater detail to be observed, but drawbacks, such as the hazard of UV to living tissue and the need for special detection devices and lenses (which tend to be dispersive in the UV), severely limit the use of UV microscopes. Elsewhere, we will explore practical uses of very short wavelength EM waves, such as x rays, and other short-wavelength probes, such as electrons in electron microscopes, to detect small details.

Another difficulty in microscopy is the fact that many microscopic objects do not absorb much of the light passing through them. The lack of contrast makes image interpretation very difficult. Contrast is the difference in intensity between objects and the background on which they are observed. Stains (such as dyes, fluorophores, etc.) are commonly employed to enhance contrast, but these tend to be application specific. More general wave interference techniques can be used to produce contrast. Figure 27.51 shows the passage of light through a sample. Since the indices of refraction differ, the number of wavelengths in the paths differs. Light emerging from the object is thus out of phase with light from the background and will interfere differently, producing enhanced contrast, especially if the light is coherent and monochromatic—as in laser light.

The schematic shows a rectangular object on a background material. Two oscillatory waves go from left to right across the image. At the left of the image, the waves have the same wavelength and are in phase. The lower wave goes through the object, where its wavelength becomes significantly shorter. To the right of the object the waves again have the same wavelength but they are now almost completely out of phase.
Figure 27.51 Light rays passing through a sample under a microscope will emerge with different phases depending on their paths. The object shown has a greater index of refraction than the background, and so the wavelength decreases as the ray passes through it. Superimposing these rays produces interference that varies with path, enhancing contrast between the object and background.

Interference microscopes enhance contrast between objects and background by superimposing a reference beam of light upon the light emerging from the sample. Since light from the background and objects differ in phase, there will be different amounts of constructive and destructive interference, producing the desired contrast in final intensity. Figure 27.52 shows schematically how this is done. Parallel rays of light from a source are split into two beams by a half-silvered mirror. These beams are called the object and reference beams. Each beam passes through identical optical elements, except that the object beam passes through the object we wish to observe microscopically. The light beams are recombined by another half-silvered mirror and interfere. Since the light rays passing through different parts of the object have different phases, interference will be significantly different and, hence, have greater contrast between them.

The schematic shows an optical setup for an interference microscope. A light source produces a beam of light that is split into two beams by a beam splitter, which is a half silvered mirror. The beams are steered around the opposite side of a square and recombine at the corner diagonally opposite the beam splitter. The object being analyzed is placed in one arm so that the beam in that arm goes through the object.
Figure 27.52 An interference microscope utilizes interference between the reference and object beam to enhance contrast. The two beams are split by a half-silvered mirror; the object beam is sent through the object, and the reference beam is sent through otherwise identical optical elements. The beams are recombined by another half-silvered mirror, and the interference depends on the various phases emerging from different parts of the object, enhancing contrast.

Another type of microscope utilizing wave interference and differences in phases to enhance contrast is called the phase-contrast microscope. While its principle is the same as the interference microscope, the phase-contrast microscope is simpler to use and construct. Its impact (and the principle upon which it is based) was so important that its developer, the Dutch physicist Frits Zernike (1888–1966), was awarded the Nobel Prize in 1953. Figure 27.53 shows the basic construction of a phase-contrast microscope. Phase differences between light passing through the object and background are produced by passing the rays through different parts of a phase plate (so called because it shifts the phase of the light passing through it). These two light rays are superimposed in the image plane, producing contrast due to their interference.

The schematic shows two beams of light going up from the bottom of the image and crossing at a point labeled object. After passing through the object, the beams diverge and then are focused by a convex lens. The light passes through a plate called the phase plate, and the beams then focus on the image plane. The background light diverges after passing through the phase plate so that it spreads away from the primary light beam on the image plane.
Figure 27.53 Simplified construction of a phase-contrast microscope. Phase differences between light passing through the object and background are produced by passing the rays through different parts of a phase plate. The light rays are superimposed in the image plane, producing contrast due to their interference.

A polarization microscope also enhances contrast by utilizing a wave characteristic of light. Polarization microscopes are useful for objects that are optically active or birefringent, particularly if those characteristics vary from place to place in the object. Polarized light is sent through the object and then observed through a polarizing filter that is perpendicular to the original polarization direction. Nearly transparent objects can then appear with strong color and in high contrast. Many polarization effects are wavelength dependent, producing color in the processed image. Contrast results from the action of the polarizing filter in passing only components parallel to its axis.

Apart from the UV microscope, the variations of microscopy discussed so far in this section are available as attachments to fairly standard microscopes or as slight variations. The next level of sophistication is provided by commercial confocal microscopes, which use the extended focal region shown in Figure 27.31(b) to obtain three-dimensional images rather than two-dimensional images. Here, only a single plane or region of focus is identified; out-of-focus regions above and below this plane are subtracted out by a computer so the image quality is much better. This type of microscope makes use of fluorescence, where a laser provides the excitation light. Laser light passing through a tiny aperture called a pinhole forms an extended focal region within the specimen. The reflected light passes through the objective lens to a second pinhole and the photomultiplier detector, see Figure 27.54. The second pinhole is the key here and serves to block much of the light from points that are not at the focal point of the objective lens. The pinhole is conjugate (coupled) to the focal point of the lens. The second pinhole and detector are scanned, allowing reflected light from a small region or section of the extended focal region to be imaged at any one time. The out-of-focus light is excluded. Each image is stored in a computer, and a full scanned image is generated in a short time. Live cell processes can also be imaged at adequate scanning speeds allowing the imaging of three-dimensional microscopic movement. Confocal microscopy enhances images over conventional optical microscopy, especially for thicker specimens, and so has become quite popular.

The next level of sophistication is provided by microscopes attached to instruments that isolate and detect only a small wavelength band of light—monochromators and spectral analyzers. Here, the monochromatic light from a laser is scattered from the specimen. This scattered light shifts up or down as it excites particular energy levels in the sample. The uniqueness of the observed scattered light can give detailed information about the chemical composition of a given spot on the sample with high contrast—like molecular fingerprints. Applications are in materials science, nanotechnology, and the biomedical field. Fine details in biochemical processes over time can even be detected. The ultimate in microscopy is the electron microscope—to be discussed later. Research is being conducted into the development of new prototype microscopes that can become commercially available, providing better diagnostic and research capacities.

Schematic of a confocal microscope. There is a sample at the bottom, a pinhole at the top, and a pinhole at the right side. The sample is a horizontal rectangle that is rather thick in the vertical direction. A green laser beam coming from the right is focused through the right pinhole, then reflects downward off of a dichroic mirror. It is then collected by a horizontal objective lens and focused onto the sample. The focus is not a point, but an extended zone where the beam diameter is minimal. Two solid red rays leave the focal plane of the objective lens and diverge upward. This plane is inside the sample and is labeled object in focal plane. These rays pass through the objective lens and begin to converge. After passing through the dichroic mirror, they continue upward and are focused through the top pinhole. After passing through this pinhole, these rays enter a detector. Two dashed red rays leave the sample at a point slightly above the focal plane, which is labeled object not in focal plane. These rays follow similar paths as the solid red rays, but they do not focus on the pinhole and so are blocked and do not reach the detector.
Figure 27.54 A confocal microscope provides three-dimensional images using pinholes and the extended depth of focus as described by wave optics. The right pinhole illuminates a tiny region of the sample in the focal plane. In-focus light rays from this tiny region pass through the dichroic mirror and the second pinhole to a detector and a computer. Out-of-focus light rays are blocked. The pinhole is scanned sideways to form an image of the entire focal plane. The pinhole can then be scanned up and down to gather images from different focal planes. The result is a three-dimensional image of the specimen.
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