Understanding the Light Microscope: A Computer-Aided Introduction

Front Cover
Academic Press, Sep 17, 1999 - Computers - 192 pages
Understanding the light microscope consists of four original computer programs with an explanatory book. Author Dan Goldstein says using the programs can teach aspects of microscopy and diffraction not often found in medical or biological courses, adding, "... what one non-mathematician has created should not be beyond the understanding of others!"

The book aims to provide understanding at a level deeper than customary in existing texts and in a form accessible to microscope users, particularly biologists. It covers simple ray optics, the aberrations of "real" (thick) lenses, polarized light, and the influence of diffraction on imaging. The book can be read alone, but appreciation of its contents is greatly enhanced when used in conjunction with the programs.

The Zernike program allows simulation of the effects of aperture, spherical aberration and focus of the objective lens, as well as coherence of the illumination. There is a range of illumination options, including: bright field; oblique; phase-contrast, central, and peripheral dark field; Schlieren; modulation contrast; apodization; interference microscopy (both shearing and differential); fluorescence; and confocal scanning. The program can simulate Fraunhofer and Fresnel diffraction by slits and gratings, interference of light from two slits, and the formation of a bright region in the middle of the shadow of an opaque object.

The Kohler program is interactive, and is intended to teach the operation of bright field and phase- contrast microscopes. Microscope settings can be made random for adjustment practice.

The Nicol program simulates aspects of quantitative polarized-light microscopy. Object properties can be made random for practice in the analysis of unknown anisotropic specimens.

Snellius is a ray-tracing program showing the effect of aberrations that occur in simple and compound lenses. Users can define their own lens systems, and save the results to disc.

There is a help function for all four programs.

The programs, which have been in intermittent evolution since 1986, enable researchers using microscopy to back up practical experience with theory. Advanced medical, biological, and biomedical undergraduates, as well as optical physicists in industry and academia, will find them invaluable.
 

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Contents

Simple Ray Optics the Kohler Program
1
12 A PRELIMINARY NOTE ON SOME CONVENTIONS AND TERMINOLOGY
2
14 REAL AND VIRTUAL IMAGES
5
15 THE POWER OF LENS COMBINATIONS
6
17 CONJUGATE PLANES
8
18 FUNCTIONS OF THE FIELD AND APERTURE DIAPHRAGMS
9
110 EPIILLUMINATION INCIDENTLIGHT MICROSCOPY
10
111 THE KOHLER PROGRAM
12
Modulation Contrast
76
Apodization
80
102 SIMULATION OF APODIZATION
81
Fluorescence
84
111 EXCITING AND BARRIER FILTERS
85
113 LOSS OF LIGHT AT AN INCIDENTLIGHT BEAMSPLITTER
87
115 FULLAPERTURE VS DARKFIELD ILLUMINATION
88
116 FADING OF FLUORESCENCE
89

112 EXERCISES USING THE PROGRAM
14
Lenses and Lens Aberrations the Snellius Program
16
21 CHROMATIC ABERRATION
17
22 SPHERICAL ABERRATION
18
23 COMA
22
25 CURVATURE OF THE FIELD
23
27 CHROMATIC DIFFERENCE OF MAGNIFICATION
24
28 TYPES OF OBJECTIVE
25
29 THE SNELLIUS PROGRAM
26
Elementary Diffraction Theory mainly due to Abbe
27
32 DIFFRACTION BY AN AMPLITUDE GRATING
29
33 DIFFRACTION BY A NONEXISTENT GRATING
30
34 ABBES THEORY OF MICROSCOPIC IMAGE FORMATION
31
35 EFFECT OF OBLIQUE COHERENT ILLUMINATION ON THE RESOLUTION OF A GRATING
34
36 EXTENSION OF THE ABBE THEORY TO NON PERIODIC OBJECTS
35
37 THE AIRY DISC
36
38 INTERACTION OF COHERENT AND INCOHERENT LIGHT WAVES
37
39 RESOLUTION OF TWO POINT OBJECTS WITH COHERENT AND INCOHERENT ILLUMINATION
38
310 EFFECT OF OBLIQUE COHERENT ILLUMINATION ON THE RESOLUTION OF TWO POINT OBJECTS
39
311 A NOTE ON FRAUNHOFER AND FRESNEL DIFFRACTION PATTERNS
40
312 OUTOFFOCUS IMAGES DECONVOLUTION AND THE BECKE LINE
41
313 APPLICATION OF THEORY TO PRACTICEEFFECT OF THE SENSITIVITY OF THE HUMAN EYE
44
Extension of the ABBE Theory to Transparent Objects Zernikes Phase Contrast
45
41 PRACTICAL PHASE CONTRAST
46
42 TERMINOLOGY OF PHASE PLATES
48
43 A SIMPLE THEORY OF IDEAL PHASE CONTRAST
48
44 A MORE GENERAL THEORY OF IDEAL PHASECONTRAST
48
45 NONIDEAL PHASE CONTRAST
48
47 SELECTED HISTORICAL NOTES
48
Function of the Microscope Condenser Partially Coherent Illumination
50
52 FUNCTION OF THE CONDENSER APERTURE DIAPHRAGM IRIS
51
53 FOCUS AND CORRECTIONS OF THE CONDENSER
54
Interference Contrast
55
THE MACHZEHNDER SYSTEM
58
DIFFERENTIAL INTERFERENCE CONTRAST
60
64 APPLICATIONS OF SHEARING INTERFERENCE MICROSCOPY
61
65 COMPARISON OF DIFFERENTIAL INTERFERENCE CONTRAST WITH OBLIQUE ILLUMINATION
62
67 INCIDENTLIGHT DIFFERENTIAL INTERFERENCE CONTRAST
63
Darkfield and Related Techniques
65
71 OBLIQUE ILLUMINATION
66
72 SYMMETRICAL PERIPHERAL DARKFIELD
68
76 SCHLIEREN MICROSCOPY
70
77 IMMERSION REFRACTOMETRY AND DISPERSION STAINING
71
Gegenfeld
73
118 COMPUTER SIMULATION OF FLUORESCENCE
91
Confocal Scanning
92
122 PRACTICAL IMPLEMENTATION OF CONFOCAL SCANNING
93
123 ADVANTAGES AND SOME LIMITATIONS OF CONFOCAL SCANNING
95
124 NONIDEAL CONFOCAL SCANNING
98
125 COMBINATION OF CONFOCAL SCANNING WITH OTHER MICROSCOPIC TECHNIQUES
99
126 EXPLAINING SUPERRESOLUTION IN CONFOCAL SCANNING
100
127 CONFOCAL SCANNING SIMULATION IN THE ZERNIKE PROGRAM
101
Microdensitometry
102
132 ERRORS IN DENSITOMETRY AND MICRODENSITOMETRY
104
133 CALIBRATION STANDARDS NONUNIFORM BACKGROUND
109
134 APPLICATIONS OF MICRODENSITOMETRY
110
135 COMPUTER SIMULATION OF MICRODENSITOMETRY
111
Polarized Light The Nicol Program
112
142 POLARIZED LIGHT MICROSCOPE AND ACCESSORIES
116
143 TYPES OF ANISOTROPIC OBJECT
122
144 THE NICOL PROGRAM
125
145 ANALYSIS OF AN UNKNOWN ANISOTROPIC OBJECT
127
146 SUGGESTED EXERCISES USING THE NICOL PROGRAM
131
How the Zernike Program Works
135
152 THE FOURIER TRANSFORM
137
153 REPRESENTATION OF FINITE OBJECTIVE NA
139
154 FOCUSSING
140
155 SPHERICAL ABERRATION
141
156 PARTIALLY COHERENT ILLUMINATION
142
157 SOME INHERENT LIMITATIONS OF THE PROGRAM
143
How to Use the Zernike Program
147
162 ENTERING INFORMATION
149
163 ERRORS AND ERROR MESSAGES
150
165 THE MAIN MENU
151
166 THE IMAGING SYSTEM
153
167 USING PARTS OF THE PROGRAM IN AN ARBITRARY ORDER
157
169 ACCESSIBLE PROGRAM VARIABLES ARRAYS AND FUNCTIONS
158
Some Suggested Exercises Using the Zernike Program
160
171 DIFFRACTION BY AN GRATING EFFECT OF THE GRATING INTERVAL
161
174 RESOLUTION EFFECT OF OBJECTIVE NA
162
177 RESOLUTION EFFECT OF FLUORESCENCE OR CONFOCAL SCANNING
163
1710 TRANSPARENT OBJECT BRIGHTFIELD IMAGING
164
1712 TRANSPARENT OBJECT OTHER METHODS OF IMAGING
165
1714 FRAUNHOFER DIFFRACTION BY A DOUBLE SLIT
166
1715 THE BECKE LINE
167
Bibliography
168
Index
178
Copyright

Common terms and phrases

About the author (1999)

D J Goldstein was a Nuffield Dominion TravellingFellow at Oxford University and a visiting Professor in Pittsburgh. He taught at the Universities of the Witwatersrand (Johannesburg, South Africa) and Sheffield (UK) while publishing research in embryology, histology, immunology, histochemistry and microscopy. Since retiring in 1989 as Reader in Anatomy at Sheffield University, he has been an independent research worker in biomedical science.

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