Digital microscopy

Cover

Contents

Contributors

Preface

Chapter 1: Microscope Basics

I. Introduction

II. How Microscopes Work

A. The Finite Tube-Length Microscope

B. Infinity Optics Microscopes

III. Objective Basics

A. Types of Objectives

B. "Mixing and Matching" Objectives

C. Coverslip Selection

IV. Mounting Video Cameras on the Microscope

A. Basic Considerations

B. Empty Magnification

C. Camera Pixel Number and Resolution

Reference

Chapter 2: The Optics of Microscope Image Formation

I. Introduction

II. Physical Optics: The Superposition of Waves

III. Huygens' Principle

IV. Young's Experiment: Two-Slit Interference

V. Diffraction from a Single Slit

VI. The Airy Disk and the Issue of Microscope Resolution

VII. Fourier or Reciprocal Space: The Concept of Spatial Frequencies

VIII. Resolution of the Microscope

IX. Resolution and Contrast

X. Conclusions

XI. Appendix I

A. Fourier Series

XII. Appendix II. A. Kirchoff's Scalar Theory of Diffraction: Recasting Huygen's Principle in an Electrodynamic Context

B. Generalizing the Problem

C. Scalar Spherical Waves

D. Green's Theorem

E. Solution for a Plane

F. Huygens' Principle

XIII. Appendix III

A. Diffraction by a Circular Aperture from Which the Airy Disk Comes

Acknowledgments

References

Chapter 3: Proper Alignment of the Microscope

I. Key Components of Every Light Microscope

A. Light Source

B. Lamp Collector

C. Diffusers and Filters

D. Field Diaphragm

E. Condensers

F. Aperture Diaphragm

G. Condenser Carrier or Substage

H. The Specimen Stage

I. The Objective

J. Revolving Nosepiece

K. Infinity Space

L. Tube, Eyepiece, and Video Adapters

II. Koehler Illumination

A. Aligning the Microscope for Koehler Illumination

B. What Are the Benefits of Koehler Illumination?

C. Resolution and Contrast

Chapter 4: Mating Cameras to Microscopes

I. Introduction. II. Optical Considerations

Chapter 5: Fundamentals of Fluorescence and Fluorescence Microscopy

I. Introduction

II. Light Absorption and Beer's Law

III. Atomic Fluorescence

IV. Organic Molecular Fluorescence

V. Excited State Lifetime and Fluorescence Quantum Efficiency

VI. Excited State Saturation

VII. Nonradiative Decay Mechanisms

VIII. Fluorescence Resonance Energy

IX. Fluorescence Depolarization

X. Measuring Fluorescence in the Steady State

XI. Construction of a Monochromator

XII. Construction of a Photomultiplier Tube

XIII. Measuring Fluorescence in the Time-Domain

A. Boxcar-Gated Detection Method

B. Streak Camera Method

C. Photon Correlation Method

D. Note on the Process of "Deconvolution"

E. Phase Modulation Method

XIV. Filters for the Selection of Wavelength

XV. The Fluorescence Microscope

XVI. The Power of