Contrast techniques in light microscopy

136 Pages·1996·19.19 MB·English

by  Bradbury, Savile, Evennett, Peter

MICROSCOPYHANDBOOKS34
Contrast Techniques
in Light Microscopy
Royal Microscopical Society MICROSCOPY HANDBOOKS
01 An Introductionto the OpticalMicroscope (2ndEdn)
03 SpecimenPreparationfor TransmissionElectronMicroscopyof
Materials
04 HistochemicalProteinStainingMethods
08 MaintainingandMonitoringtheTransmissionElectronMicroscope
09 QualitativePolarizedLightMicroscopy
11 An Introductionto Immunocytochemistry(2ndEdn)
15 DictionaryofLightMicroscopy
16 LightElementAnalysisintheTransmissionElectronMicroscope
17 ColloidalGold: a New Perspectivefor CytochemicalMarking
18 Autoradiography: a Comprehensive Overview
19 Introductionto Crystallography(2ndEdn)
20 The OperationofTransmissionandScanningElectronMicroscopes
21 CyropreparationofThinBiologicalSpecimensfor Electron
Microscopy
22 An Introductionto SurfaceAnalysis by ElectronSpectroscopy
23 BasicMeasurementTechniquesfor LightMicroscopy
24 The PreparationofThinSections ofRocks, Minerals and Ceramics
25 The Role ofMicroscopyinSemiconductorFailureAnalysis
26 EnzymeHistochemistry
27 In Situ Hybridization: a PracticalGuide
28 BiologicalMicrotechnique
29 FlowCytometry
30 Food Microscopy: a ManualofPracticalMethods, UsingOptical
Microscopy
31 ScientificPhotoMACROgraphy
32 MicroscopyofTextileFibres
33 ModernPhotoMICROgraphy
34 ContrastTechniquesinLightMicroscopy
Frontispiece
All the illustrations in this plate are taken with epi-illumination and various contrast
modes.Theupperfourpictures(a-d)areofthesurfaceofaglass-bondedfusedalumina
grinding wheel; all represent the same field of view and are taken with the
x40 objective, the field width in each case representing approximately 250 urn.
(a)Bright field. (b)Crossed polars. (c)Differential interferencecontrast(DIC). (d)DIC
butwith adifferentprism setting.The lowerleftandlower rightpictures (e-f)show the
surfaceofaNi-Cualloy using DICwithdifferent prismsettings toshow howthegrains
appeartobeeitherraisedaboveorsunkbelowthesurface,accordingtothemicroscope
setting. The field width of (e)and (f)is250 urn.

Contrast Techniques
in Light Microscopy
Savile Bradbury
Pembroke College,
Oxford, UK
Peter Evennett
TheUniversityofLeeds,
Leeds, UK
0
CRC Press
~
Taylor&FrancisGroup
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Firstpublished 1996byBIOSScientificPublishing Ltd.
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Contents
Preface Xl
1. Introduction 1
Why use a microscope? 1
The needfor contrastenhancement 2
Assessmentofthe image 4
References 6
2. FundamentalConsiderations 7
Light andthe specimen 7
Absorption, transmissionandreflectionoflightby the specimen 8
Scatteringand diffraction 12
Refractionand polarizedlight 13
Phase change 16
Fluorescence 17
Straylight 18
References 19
3. Bright-fieldandDark-groundTechniques 21
Manipulationoftheillumination 22
Obliquelight 24
Polishing, etchingandothersurfacetreatments 26
Enhancingcontrastby use ofcolour-naturalcolour, stainingand
illuminationchanges 26
Colourfilters 29
Dark-groundimagingfor transmittedlight 30
Dark-groundepi-illumination 35
Rheinbergillumination 36
Straylight 40
References 41
vii
viii CONTRASTTECHNIQUES INLIGHTMICROSCOPY
4. Refractive DifferencesbetweenSpecimenandMountant;
the 'BeckeLine' 43
Dispersionstaining 44
References 48
5. Polarized-lightTechniques 49
Thepolarized-lightmicroscope 50
Observations witha single polar 52
Observationsbetweencrossedpolars;first-order redplate 54
Theuse ofpolarizedlightinreflected-lightmicroscopy 57
References 58
6. Phase ContrastandModulation Contrast 59
Theoryofphase contrast 60
The constructionofthe phase-contrastmicroscope 62
Variants ofphase contrast 66
The phase-contrastimage 68
Phasecontrastinreflectedlight 69
Modulationcontrast 70
Constructionofthemodulation-contrastmicroscope 70
Setting-upphasecontrast 73
Setting-upmodulationcontrast 75
References 75
7. Interference Contrast 77
Differentialinterferencecontrast 80
TheDIC microscope 81
Thetransmitted-lightDIC image 86
DIC inreflectedlight 87
Setting-upDIC for transmittedlight 87
Setting-upDIC for reflectedlight 88
References 89
8. FluorescenceMicroscopy 91
Types offluorescence 92
Thefluorescence microscope 94
Lightsourcesfor fluorescence 95
Objectivesandfilters 96
References 100
CONTENTS ix
9. EnhancementofContrast in the Image 101
Photographictechniques ofcontrastenhancement 101
Filmchoice, negative development and printing 102
Image enhancementby video techniques 104
Imagemodificationby digitaltechniques 105
Contrastenhancementby manipulation ofthe grey-level
histogram 107
References 109
Appendix 111
Avectortheoryapproachto phase contrast 111
Reference 114
Index 115