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Front Matter
Abstract
This front matter contains an introduction to the series, preface to the Field Guide, table of contents, and a glossary of symbols.

Library of Congress Cataloging-in-Publication Data

Tkaczyk, Tomasz S.

Field guide to microscopy / Tomasz S. Tkaczyk.

p. cm. -- (The field guide series)

Includes bibliographical references and index.

ISBN 978-0-8194-7246-5

1. Microscopy--Handbooks, manuals, etc. I. Title.

QH205.2.T53 2009

502.8′2--dc22

2009049648

Published by

SPIE

P.O. Box 10

Bellingham, Washington 98227-0010 USA

Phone: +1 360.676.3290

Fax: +1 360.647.1445

Email: spie@spie.org

Web: http://spie.org

The content of this book reflects the work and thought of the author. Every effort has been made to publish reliable and accurate information herein, but the publisher is not responsible for the validity of the information or for any outcomes resulting from reliance thereon.

Printed in the United States of America.

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Introduction to the Series

Welcome to the SPIE Field Guides—a series of publications written directly for the practicing engineer or scientist. Many textbooks and professional reference books cover optical principles and techniques in depth. The aim of the SPIE Field Guides is to distill this information, providing readers with a handy desk or briefcase reference that provides basic, essential information about optical principles, techniques, or phenomena, including definitions and descriptions, key equations, illustrations, application examples, design considerations, and additional resources. A significant effort will be made to provide a consistent notation and style between volumes in the series.

Each SPIE Field Guide addresses a major field of optical science and technology. The concept of these Field Guides is a format-intensive presentation based on figures and equations supplemented by concise explanations. In most cases, this modular approach places a single topic on a page, and provides full coverage of that topic on that page. Highlights, insights, and rules of thumb are displayed in sidebars to the main text. The appendices at the end of each Field Guide provide additional information such as related material outside the main scope of the volume, key mathematical relationships, and alternative methods. While complete in their coverage, the concise presentation may not be appropriate for those new to the field.

The SPIE Field Guides are intended to be living documents. The modular page-based presentation format allows them to be easily updated and expanded. We are interested in your suggestions for new Field Guide topics as well as what material should be added to an individual volume to make these Field Guides more useful to you. Please contact us at fieldguides@SPIE.org.

John E. Greivenkamp, Series Editor

College of Optical Sciences

The University of Arizona

The Field Guide Series

Keep information at your fingertips with all of the titles in the Field Guide series:

Field Guide to Geometrical Optics, John E. Greivenkamp (FG01)

Field Guide to Atmospheric Optics, Larry C. Andrews (FG02)

Field Guide to Adaptive Optics, Robert K. Tyson & Benjamin W. Frazier (FG03)

Field Guide to Visual and Ophthalmic Optics, Jim Schwiegerling (FG04)

Field Guide to Polarization, Edward Collett (FG05)

Field Guide to Optical Lithography, Chris A. Mack (FG06)

Field Guide to Optical Thin Films, Ronald R. Willey (FG07)

Field Guide to Spectroscopy, David W. Ball (FG08)

Field Guide to Infrared Systems, Arnold Daniels (FG09)

Field Guide to Interferometric Optical Testing, Eric P. Goodwin & James C. Wyant (FG10)

Field Guide to Illumination, Angelo V. Arecchi; Tahar Messadi; R. John Koshel (FG11)

Field Guide to Lasers, Rüdiger Paschotta (FG12)

Field Guide to Microscopy, Tomasz Tkaczyk (FG13)

Field Guide to Laser Pulse Generation, Rüdiger Paschotta (FG14)

Field Guide to Infrared Systems, Detectors, and FPAs, Second Edition, Arnold Daniels (FG15)

Field Guide to Optical Fiber Technology, Rüdiger Paschotta (FG16)

Preface to the Field Guide to Microscopy

In the 17th century Robert Hooke developed a compound microscope, launching a wonderful journey. The impact of his invention was immediate; in the same century microscopy gave name to “cells” and imaged living bacteria. Since then microscopy has been the witness and subject of numerous scientific discoveries, serving as a constant companion in humans’ quest to understand life and the world at the small end of the universe’s scale.

Microscopy is one of the most exciting fields in optics, as its variety applies principles of interference, diffraction, and polarization. It persists in pushing the boundaries of imaging limits. For example, life sciences in need of nanometer resolution recently broke the diffraction limit. These new super-resolution techniques helped name microscopy the method of the year by Nature Methods in 2008.

Microscopy will critically change over the next few decades. Historically, microscopy was designed for visual imaging; however, enormous recent progress (in detectors, light sources, actuators, etc.) allows the easing of visual constrains, providing new opportunities. I am excited to witness microscopy’s path toward both integrated, digital systems and nanoscopy.

This Field Guide has three major aims: (1) to give a brief overview of concepts used in microscopy; (2) to present major microscopy principles and implementations; and (3) to point to some recent microscopy trends. While many presented topics deserve a much broader description, the hope is that this Field Guide will be a useful reference in everyday microscopy work and a starting point for further study.

I would like to express my special thanks to my colleague here at Rice University, Mark Pierce, for his crucial advice throughout the writing process and his tremendous help in acquiring microscopy images.

This Field Guide is dedicated to my family: my wife, Dorota, and my daughters, Antonina and Karolina.

Tomasz Tkaczyk

Rice University

Table of Contents

Glossary of Symbols xi

Basics Concepts 1

Nature of Light 1

The Spectrum of Microscopy 2

Wave Equations 3

Wavefront Propagation 4

Optical Path Length (OPL) 5

Laws of Reflection and Refraction 6

Total Internal Reflection 7

Evanescent Wave in Total Internal Reflection 8

Propagation of Light in Anisotropic Media 9

Polarization of Light and Polarization States 10

Coherence and Monochromatic Light 11

Interference 12

Contrast vs Spatial and Temporal Coherence 13

Contrast of Fringes (Polarization and Amplitude Ratio) 15

Multiple Wave Interference 16

Interferometers 17

Diffraction 18

Diffraction Grating 19

Useful Definitions from Geometrical Optics 21

Image Formation 22

Magnification 23

Stops and Rays in an Optical System 24

Aberrations 25

Chromatic Aberrations 26

Spherical Aberration and Coma 27

Astigmatism, Field Curvature, and Distortion 28

Performance Metrics 29

Microscope Construction 31

The Compound Microscope 31

The Eye 32

Upright and Inverted Microscopes 33

The Finite Tube Length Microscope 34

Infinity-Corrected Systems 35

Telecentricity of a Microscope 36

Magnification of a Microscope 37

Numerical Aperture 38

Resolution Limit 39

Useful Magnification 40

Depth of Field and Depth of Focus 41

Magnification and Frequency vs Depth of Field 42

Köhler Illumination 43

Alignment of Köhler Illumination 45

Critical Illumination 46

Stereo Microscopes 47

Eyepieces 48

Nomenclature and Marking of Objectives 50

Objective Designs 51

Special Objectives and Features 53

Special Lens Components 55

Cover Glass and Immersion 56

Common Light Sources for Microscopy 58

LED Light Sources 59

Filters 60

Polarizers and Polarization Prisms 61

Specialized Techniques 63

Amplitude and Phase Objects 63

The Selection of a Microscopy Technique 64

Image Comparison 65

Phase Contrast 66

Visibility in Phase Contrast 69

The Phase Contrast Microscope 70

Characteristic Features of Phase Contrast 71

Amplitude Contrast 72

Oblique Illumination 73

Modulation Contrast 74

Hoffman Contrast 75

Dark Field Microscopy 76

Optical Staining: Rheinberg Illumination 77

Optical Staining: Dispersion Staining 78

Shearing Interferometry: The Basis for DIC 79

DIC Microscope Design 80

Appearance of DIC Images 81

Reflectance DIC 82

Polarization Microscopy 83

Images Obtained with Polarization Microscopes 84

Compensators 85

Confocal Microscopy 86

Scanning Approaches 87

Images from a Confocal Microscope 89

Fluorescence 90

Configuration of a Fluorescence Microscope 91

Images from Fluorescence Microscopy 93

Properties of Fluorophores 94

Single vs Multi-Photon Excitation 95

Light Sources for Scanning Microscopy 96

Practical Considerations in LSM 97

Interference Microscopy 98

Optical Coherence Tomography/Microscopy 99

Optical Profiling Techniques 100

Optical Profilometry: System Design 101

Phase-Shifting Algorithms 102

Resolution Enhancement Techniques 103

Structured Illumination: Axial Sectioning 103

Structured Illumination: Resolution Enhancement 104

TIRF Microscopy 105

Solid Immersion 106

Stimulated Emission Depletion 107

STORM 108

4Pi Microscopy 109

The Limits of Light Microscopy 110

Other Special Techniques 111

Raman and CARS Microscopy 111

SPIM 112

Array Microscopy 113

Digital Microscopy and CCD Detectors 114

Digital Microscopy 114

Principles of CCD Operation 115

CCD Architectures 116

CCD Noise 118

Signal-to-Noise Ratio and the Digitization of CCD 119

CCD Sampling 120

Equation Summary 122

Bibliography 128

Index 133

Glossary of Symbols

a(x, y), b(x, y)

Background and fringe amplitude

AO

Vector of light passing through amplitude Object

AR, AS

Amplitudes of reference and sample beams

b

Fringe period

c

Velocity of light

C

Contrast, visibility

Cac

Visibility of amplitude contrast

Cph

Visibility of phase contrast

Cph-min

Minimum detectable visibility of phase contrast

d

Airy disk dimension

d

Decay distance of evanescent wave

d

Diameter of the diffractive object

d

Grating constant

d

Resolution limit

D

Diopters

D

Number of pixels in the x and y directions (Nx and Ny, respectively)

DA

Vector of light diffracted at the amplitude object

DOF

Depth of focus

DP

Vector of light diffracted at phase object

Dpinhole

Pinhole diameter

dxy, dz

Spatial and axial resolution of confocal microscope

E

Electric field

E

Energy gap

Ex and Ey

Components of electric field

F

Coefficient of finesse

F

Fluorescent emission

f

Focal length

fc, fF

Focal length for lines C and F

fe

Effective focal length

FOV

Field of view

FWHM

Full width at half maximum

h

Planck’s constant

h, h′

Object and image height

H

Magnetic field

I

Intensity of light

i, j

Pixel coordinates

Io

Intensity of incident light

Imax, Imin

Maximum and minimum intensity in the image

I, It, Ir

Irradiances of incident, transmitted, and reflected light

I1, I2, I3

Intensity of successive images

I0, I2π/3, I4π/3

Intensities for the image point and three consecutive grid positions

k

Number of events

k

Wave number

L

Distance

lc

Coherence length

m

Diffraction order

M

Magnification

Mmin

Minimum microscope magnification

MP

Magnifying power

MTF

Modulation transfer function

Mu

Angular magnification

n

Refractive index of the dielectric media

n

Step number

N

Expected value

N

Intensity decrease coefficient

N

Total number of grating lines

na

Probability of two-photon excitation

NA

Numerical aperture

ne

Refractive index for propagation velocity of extraordinary wave

nm, nr

Refractive indices of the media surrounding the phase ring and the ring itself

no

Refractive index for propagation velocity of ordinary wave

n1

Refractive index of media 1

n2

Refractive index of media 2

o, e

Ordinary and extraordinary beams

OD

Optical density

OPD

Optical path difference

OPL

Optical path length

OTF

Optical transfer function

OTL

Optical tube length

P

Probability

Pavg

Average power

PO

Vector of light passing through phase object

PSF

Point spread function

Q

Fluorophore quantum yield

r

Radius

r

Reflection coefficients

r, m, and o

Relative, media, or vacuum

rAS

Radius of aperture stop

rPR

Radius of phase ring

rλ

Radius of filter for wavelength λ

s, s′

Shear between wavefront object and image space

S

Pinhole/slit separation

s

Lateral shift in TIR perpendicular component of electromagnetic vector

svl.jpg

Lateral shift in TIR for parallel component of electromagnetic vector

SFD

Factor depending on specific Fraunhofer approximation

SM

Vector of light passing through surrounding media

SNR

Signal-to-noise ratio

SR

Strehl ratio

t

Lens separation

t

Thickness

t

Time

t

Transmission coefficient

T

Time required to pass the distance between wave oscillations

T

Throughput of a confocal microscope

tc

Coherence time

u, u′

Object, image aperture angle

Vd, Ve

Abbe number as defined for lines d, F, C or e, F′, C′

Vm

Velocity of light in media

w

Width of the slit

x, y

Coordinates

z

Distance along the direction of propagation

z

Fraunhofer diffraction distance

z, z′

Object and image distances

zm, zo

Imaged sample depth, length of reference path

α

Angle between vectors of interfering waves

α

Birefringent prism angle

α

Grating incidence angle in plane perpendicular to grating plane

αs

Visual stereo resolving power

β

Grating diffraction angle in plane perpendicular to grating plane

γ

Angle of fringe localization plane

γ

Convergence angle of a stereo microscope

γ

Incidence / diffraction angle from the plane perpendicular to grating plane

Γ

Retardation

δ

Birefringence

δ

Excitation cross section of dye

δz

Depth perception

Δb

Axial phase delay

Δf

Variation in focal length

Δk

Phase mismatch

Δz, Δz′

Object and image separations

Δλ

Wavelength bandwidth

Δv

Frequency bandwidth

Δφ

Phase delay

Δφ

Phase difference

Δφ

Phase shift

Δφmin

Minimum perceived phase difference

ε

Angle between interfering beams

ε

Dielectric constant, i.e., medium permittivity

η

Quantum efficiency

θ′

Refraction angle

θcr

Critical angle

θi

Incidence angle

θr

Reflection angle

λ

Wavelength of light

λp

Peak wavelength for the mth interference order

μ

Magnetic permeability

ν

Frequency of light

ν

Repetition frequency

ξ

Propagation direction

ρ

Spatial frequency

σ

Molecular absorption cross section

σdark

Dark noise

σphoton

Photon noise

σread

Read noise

τ

Integration time

τ

Length of a pulse

τ

Trans mitt ance

φ

Incident photon flux

φ

Optical power

φ

Phase difference generated by a thin film

φo

Initial phase

φo

Phase delay through the object

φp

Phase delay in a phase plate

φTF

Phase difference generated by a thin film

ω

Angular frequency

ω

Bandgap frequency

⊥ and ║

Perpendicular and parallel components of the light vector

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