No other microtool allows this, and therefore most experiments with optical tweezers in plant cell b...

No other microtool allows this, and therefore most experiments with optical tweezers in plant cell biology are on objects inside living cells,. Protocol 2[r]

Plant Cell Biology

The Practical Approach Series

Related Practical Approach Series Titles

light Microscopy in Biology 2/e

Protein Localization by Fluorescence Microscopy

Flow Cytometry 3/e

In Situ Hybridization 2/e

Cell Separation

Arabidopsis

Plant Cell Culture

Plant Molecular Biology*

* indicates a forthcoming title

Please see the Practical Approach series website at

http://www.oup.co.uk/pas

for full contents lists of all Practical Approach titles.

UNIVERSITY PRESS

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in a retrieval system, or transmitted, in any form or by any means, without the prior permission in writing of Oxford University Press, or as expressly permitted by law, or under terms agreed with the appropriate reprographics rights organization Enquiries concerning reproduction outside the scope of the above should be sent to the Rights Department, Oxford University Press, at the address above

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A catalogue record for this title is available from

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Library of Congress Cataloguing-in-Publication Data

Plant cell biology / edited by Chris Hawes and

Beatrice Satiat-Jeunemaitre.-2nd ed.

(Practical approach series; 250)

Includes bibliographical references (p.).

1 Botanical microscopy-Technique 2 Plant

cytochemistry-Technique 3 Plant cells and tissues I Hawes, C R II Jeunemaitre, Beatrice III Series.

Satiat-1 3 5 7 9 Satiat-1 0 8 6 4 2

QK673 P58 2001 571.692dc21 00-054847

ISBN 0 19 963866 7(Hbk)

ISBN 0 19 963865 9(Pbk)

Typeset in Swift by Footnote Graphics, Warminster, Wilts

Printed in Great Britain on acid-free paper

by The Bath Press, Bath, Avon

It is just seven short years since the first edition of Plant Cell Biology was published,

yet in that time there have been a number of significant advances in the

technologies used by cell biologists The start of the new millennium coincides

with the dawn of the so-called 'post-genomics' era where biologists will struggle

to cope with the almost overwhelming flow of information that will emerge from

the various sequencing projects We predict that one consequence of this will be

a surge in demand for the techniques of cell biology to aid in the interpretation of

the function and location of the myriad of proteins and macromolecules that

make up the cell Ironically this demand will necessitate the application of

technologies that have somewhat dropped out of favour in the past decade or two

such as classical histochemistry and electron microscopy These will, however,

have to be combined with new techniques such as the in vivo expression of

fluorescent markers and the subcellular manipulation of organelles and

physiological measurements from the cytoplasm

When taking over as editors of the new volume we were posed with several

serious problems As stated by Harris and Oparka in 1993, in the production of

such a volume one has to be extremely selective and miss out many important

areas of the topic This has been even more difficult considering the excellent

coverage of the original volume, so this edition contains a mixture of up-dated

chapters from original authors, new authors covering topics that were in the

original volume, and a raft of totally new chapters

The microscope in its many guises is still the workhorse of the average cell

biologist and as microscopists we offer no apologies for the techniques

surround-ing the instrument besurround-ing dominant in this volume Therefore, we start with a

general introduction to the light microscope and the associated microscopies of

confocal and multi-photon imaging This is followed by a chapter on the use of

fluorescent probes for in vivo imaging and for making physiological

measure-ments by microscopy However, for much quantitative work microscopy is not

necessary and useful information can be extracted using the technique of

cyto-metry covered in Chapter 3 As the power offered by molecular biology is easy to

harness to help solve cell biological problems, we have included new chapters

on the expression of heterologous proteins in protoplasts and on the use of the

now famous green fluorescent protein and its derivatives for in vivo imaging of

cell dynamics We now have the ability to directly manipulate cytoplasm andorganelles and Chapters 6 and 7 give an introduction to the latest technologies

in microinjection and micromanipulation followed by the use of logical techniques to complement cell biological investigations

electrophysio-Moving away from the study of living cells, in Chapter 9 we revisit some

of the more useful histological techniques that are now much in demand bymolecular and developmental biologists The following three chapters cover thevarious methods for localizing macromolecules and nucleic acids by light andelectron microscopy, whilst also including the basic techniques of specimenpreparation for electron microscopy A technology that has suffered with theinexorable rise of molecular biology and that we lose at our peril Finally, wefinish on a biochemical note covering cell fractionation and organelle isolation,useful techniques that have become ever more powerful with the development

of a wide range of marker antibodies

Obviously in many instances authors of the various chapters have only beenable to dip into the vast array of techniques at hand and we apologize in advance

if we have missed your favourite technique or propose a different protocol to theone you may routinely use And finally, we would like to thank the various con-tributors for giving their valuable time in preparing their chapters and divulgingtheir laboratory hints and tips and for succumbing to the onerous demands ofthe editors

Oxford C H

Gif-sur-Yvette B S-J.

April 2001

4 Microscope imaging modes 10

Bright field imaging 10

Phase contrast 12

Differential interference contrast (Nomarski) 24

Dark field 16

Epifluorescence and reflected light microscopy 2 7

5 Confocal and 3D microscopy 19

The problem of out-of-focus light 19

The confocal principle: explanation by ray optics 20

Practical confocal microscopes 22

Imaging and the point spread function 23

Deconvolution 24

Two photon imaging 26

6 Comparison of conventional, wide-field fluorescence imaging with confocal fluorescence imaging 27

Noise and resolution 27

When should confocal microscopy be used? 29

Objective lenses for confocal imaging 30

7 Specimen preparation for confocal imaging 30

References 33

2 Fluorescent probes for living plant cells 35

Mark Fricker, Andrew Parsons, Monika Tlalka, Elison Blancaflor, Simon Gilroy, Andreas Meyer, and Christoph Plieth

1 Introduction 35

2 Selecting probes with high brightness 35

Spectral considerations 37

3 Fluorescence lifetime imaging microscopy (FLIM) 38

4 Fluorescence polarization anisotropy 38

5 Fluorescence resonance energy transfer (FRET) 38

6 Photobleaching and fluorescence redistribution after photobleaching (FRAP) 39

7 Optimization of fluorescent systems for live cell imaging 40 Selection of the excitation wavelength 41

The dichroic mirror 41

Selection of the emission wavelength 41

Choice of measurement system 42

8 Securing the specimen for microscopy 43

9 Perfusion systems 45

10 Loading strategies for plant cells 46

Extracellular and permeant intracellular dyes 46

Ester loading 47

Low pH loading 48

Cutinase pre-treatment and low pH loading 49

Electroporation 50

Loading via detergent permeabilization 50

Loading tissues with phloem-mobile probes 50

11 Intracellular dye concentrations, viability, and toxicity 53

12 Selection and use of fluorescent probes 54

The plasma membrane and endocytosis 64

The cell wall 65

Measurement of cytoplasmic glutathione levels 77

Reactive oxygen species 77

2 Cytometry demonstrated through cell cycle analyses 86

How to understand monoparametric DNA histograms 87

Developing multiparametric DNA histograms and immunofluorescence 90

Extracting intact plant nuclei 91

Which DNA fluorochrome is appropriate? 94

Running and reading the cytometer 94

BrdU incorporation to identify DNA synthesis by fluorescence quenching 95

3 The particular application of genome size calculation and 'DNA ploidy' 99

Terminology 99

Internal or external standards 99

Calculating base composition 100

4 Sorting of protoplasts and cellular organelles 101

5 Tests for cell viability during functional assays 104

2 Current methods of transient expression 108

Naked DNA transfer 108

Biological vectors 108

3 Application of transient expression 109

Promoter analysis 109

Cell biology and biochemistry 110

4 Practical considerations for cell biologists 111

Naked DNA transfer 112

Measurement of protein secretion and cell retention 116

Large scale transient expression for cell fractionation 118

Specialized applications 122

5 Conclusions 124

References 124

5 The green fluorescent protein (GFP) as reporter in plant cells 127

Jean-Marc Neuhaus and Petra Boevink

1 Introduction 127

2 The green fluorescent protein 127

Structure 127

GFP variants 128

3 GFP as a reporter for gene expression 128

4 GFP as a reporter for protein location 129

Cytoplasm and nucleus 129

Chloroplasts and mitochondria 130

Secretory pathway 130

Viral proteins 131

5 Transformation methods 131

PEG-mediated transient expression in protoplasts 131

Agroborterium-mediated transient expression in planta 134

Virus-mediated transient expression 136

6 Visualization and microscopy of GFP 140

Ground parenchyma cells 151

Sieve elements and companion cells 151

Algae 152

Bacteria and organelles 153

Plant tissue cultures 153

5 Material suitable for injection 154

Syringe to delete air bubbles 156

Flexible fused silica capillaries 156

Petri dishes 157

References 157

7 Micromanipulation by laser microbeam and optical tweezers 159

Karl Otto Greulich

1 Introduction 159

2 What are laser microtools? 159

3 Physical background 160

Generating extreme heat 160

Why can light be used to move microscopic objects? 160

4 How to build laser microtools 161

The choice of lasers 161

Building a laser microbeam or optical tweezers 162

5 Applications of laser microbeams in plant biology 163

Laser-induced microinjection 163

Ablation to study cell fate during plant development 164

Protoplast fusion 165

Preparation of cell membranes from root hairs 165

6 Applications of optical tweezers to plant biology 166

Capturing subcellular organelles for inspection 166

The membrane is equivalent to a capacitor 171

A cell as an equivalent circuit 172

3 Techniques for the measurement of membrane capacitance 172

Square-wave stimulation: time-domain technique 173

What kind of cells can be examined? 176

Estimation of the specific capacitance 176

Recording of single fusion and fission events 176

What kind of information can be extracted from the measurements? 179

Macroscopic measurement of membrane capacitance 181

2 Conventional chemical fixation methods 189

3 Conventional embedding methods and sectioning 191

Embedding in a matrix 192

Frozen sections 194

4 Conventional staining methods 195

General tissue stains 195

Cell wall stains 397

Carbohydrate and starch stains 200

Lipid stains 201

Nucleic acid stains 202

Miscellaneous staining methods 203

References 206

10 Immunocytochemistry for light microscopy 207

Beatrice Satiat-Jeunemaitre and Chris Howes

1 Introduction 207

2 Principles and use of immunocytochemistry 208

Direct and indirect immunostaining 208

The antibody-antigen complex 208

Whole molecules or fragments 209

Polyclonal and monoclonal antibodies 211

When to perform in situ immunoreaction 214

Antibodies to epitope tags 215

3 Basic methods for immunostaining 215

Preparing plant material 216

Attaching material to slides and coverslips 220

Accessing epitopes in cells 220

Counterstaining and mounting 230

Interpreting the immunostaining pattern 230

Low temperature methods 250

Rotary shadowing of proteins 257

3 Scanning electron microscopy 258

Ambient temperature SEM 258

Low temperature SEM 262

2 Applications of in situ hybridization 269

DNA:DNA in situ hybridization 269

RNA:RNA in situ hybridization 269

3 Background to the methods 269

4 Chromosome preparation for DNA:DNA in situ hybridization 270

Pre-treatment for DNA:DNA in situ hybridization 283

Pre-treatment for RNA:RNA in situ hybridization 284

8 In situ hybridization reaction 285

DNA:DNA in situ hybridization 285

RNA:RNA in situ hybridization 286

9 Post-hybridization washes 287

Washes for DNA:DNA in situ hybridization 288

Washes for RNA:RNA in situ hybridization 288

10 Probe detection and visualization 289

9 Isolation of endoplasmic reticulum 308

10 Isolation of Golgi apparatus 310

11 Isolation of transport vesicles 312

12 Assays for marker enzymes 325

13 Antibodies for organelle recognition 319References 320

A1 List of suppliers 325

Index 333

Protocol list

Microscope imaging modes

Adjustment for bright field imaging with Kohler illumination 11

Adjustment for phase contrast 14

Adjustment for DIC 15

Adjustment for dark field 16

Specimen preparation for confocal imaging 30

Collection of confocal images 32

Fluorescent probes for living plant cells

Growth of Arabidopsis thaliana seedlings in Phytagel for in situ observation

of roots 44

Loading strategies for plant cells 46

Loading dyes by vacuum infiltration of leaf pieces 47

Loading dyes as acetoxymethyl ester or acetate ester derivatives 47

Low pH loading of root tissues 49

Increasing the permeability of the cuticle by cutinase digestion 50

Loading root tissues with phloem-mobile probes 51

Direct observation of phloem transport in bean leaves 52

Selection and use of fluorescent probes 54

Labelling of actin filaments in living cells using fluorescent phallotoxins 63

Labelling of microtubules in epidermal cells using fluorescent analogue

cytochemistry 64

Physiological probes 66

In vitro calibration of calcium ratio dyes 68

In situ calibration of calcium dyes 71

In situ calibration of ratio pH dyes using nigericin 75

Measurement of H2O2 in situ using H2DCF 77

Data analysis 78

Processing ratio images 79

Quantitative analysis of ratio data from regions of interest 80

Measurement of signal attenuation with depth through a permeabilized specimen

infiltrated with a fluorochrome 'sea' 80

Flow cytometry

General toolkit for isolation of diverse nuclei for cytometric analysis 91

BrdU incorporation to identify DNA synthesis by fluorescence quenching 97

Sorting of protoplasts and cellular organelles

Sorting subclasses of plant nuclei 102

Practical considerations for cell biologists

Preparation of electroporation competent protoplasts from tobacco leaves 113

Electroporation and subsequent incubation 115

Harvesting cells and medium from electroporated protoplasts 116

Extraction and concentration of proteins from cells and medium 117

Isolation of vacuoles from protoplasts after short transient expression 119

Quantifying the recovery of vacuoles 120

Cell fractionation by sucrose density centrifugation 121

Assessment of membrane association 123

Assessment of membrane orientation 123

Transformation methods

Preparation of protoplasts from sterile tobacco plants 132

Preparation of protoplasts from an Arabidopsis cell suspension 134

PEG-mediated transient expression in protoplasts 135

Agrobarterium-mediated transient expression in tobacco leaves 136

PVX vector expression in whole plants 137

Biolistic inoculation of PVX vector constructs 138

Applications of laser microbeams in plant biology

Injection of DNA into plant cells 164

Applications of optical tweezers to plant biology

Basic operation of a laser tweezers set-up 166

Capacitance measurements as an assay for exo- and endocytosis: practical considerations

Measurement of single fusion and fission events in the cell-attached configurationusing a dual-phase log-in amplifier 177

Measurement of single fusion and fission events in the whole-cell configuration using adual-phase log-in amplifier 179

Monitoring of macroscopic changes in surface area using whole-cell membranecapacitance measurements 181

Combined fluorescent and electrical measurements of exo- and endocytosis 186

Plant histology

Standard paraformaldehyde-glutaraldehyde fixation 190

Standard FAA fixation 191

Conventional embedding methods and screening 191

Silanized slides 191

Embedding in and sectioning polyethylene glycol (PEG) 192

Embedding in and sectioning wax 193

Embedding in and sectioning LR White acrylic resin 194

Conventional staining methods 195

Toluidine Blue 195

Acridine Orange 196

Haematoxylin with counterstains 196

Ruthenium Red for pectin 198

Calcofluor for cellulose 198

Phloroglucinol for lignin 198

Aniline Blue for callose 199

Resorcinol Blue for callose 199

PAS for total carbohydrate 200

Iodine staining for starch 201

Sudan staining for lipids 201

Nile Blue for acidic lipids 202

Methyl Green-Pyronin for RNA and DNA 202

Fluorescent stains for DNA 203

Stains for cell viability 204

Methyl Blue for fungi 204

Thionin-Orange G for fungi 204

Gram stain for bacteria 205

Principles and use of immunocytochemistry

Fractionation of IgGs from serum 211

Tissue printing for observing immunological and protein profiles 216

Generic protocol for immunofluorescence 218

Immunofluorescence on root squashes 220

Immunofluorescence on suspension culture cells or protoplasts 222

Immunostaming of pollen tubes 223

Gold labelling and silver enhancement of mung bean hypocotyl microtubules

(IGSS method) 224

Immunostaining on de-waxed sections 225

Methacrylate embedding, sectioning, and immunostaining 226

Immunofluorescence on cryosections of roots 228

Immunostaining by freeze-shattering of plant cell walls 229

Transmission electron microscopy

Preparation of a typical double aldehyde fixative (1% paraformaldehyde/

2% glutaraldehyde) 237

Fixation and dehydration 237

Resin embedding 240

Progressive lowering of temperature and acrylic resin embedding 242

Preparation of Reynold's lead citrate 244

Zinc iodide/osmium tetroxide impregnation (ZIO) 245

Phosphotungstic acid staining of plasma membranes 245

PATAg staining of carbohydrates 246

On grid negative staining 247

Standard immunogold labelling and silver enhancement 249

Typical freeze-substitution schedule 253

Cryosectioning and immunogold labelling 254

Freeze-fracture and replication including deep-etching and rotary replication 256 Rotary metal shadowing of isolated proteins 258

Basic preparation of material for ambient temperature SEM 259

Critical-point drying 260

Fracturing and osmium maceration 261

Cryo-SEM 263

Chromosome preparation for DNA:DNA In situ hybridization

Preparing mammal chromosome spreads 270

Preparing plant chromosome spreads 272

Material preparation for RNA:RNA in situ hybridization 273

Coating of slides for cryosections 274

Preparation of cryosections 274

Acridine Orange staining to check retention of nucleic acids in cryosections 275

Labelling the nucleic acids

Labelling of DNA by nick translation 277

Labelling DNA using PCR 278

Labelling RNA by in vitro transcription 280

Precipitation of probe (for DNA or RNA probes) 281

Checking label incorporation: dot blot 282

Material pre-treatment 283

Pre-treatment for DNA:DNA in situ hybridization

Pre-treatment for RNA:RNA in situ hybridization: cryosections 284

In situ hybridization reaction

DNA:DNA in situ hybridization reaction 285

RNA:RNA in situ hybridization 287

DNA:DNA post-hybridization washes 288

RNA:RNA post-hybridization washes 288

Probe detection and visualization

Probe detection 290

Organelle isolation

Detergent-assisted homogenization of protoplasts 298

Isolation of chloroplasts 300

Isolation of intact chloroplasts from spinach leaves 300

Isolation of mitochondria from spinach leaves 301

Isolation of nuclei from tomato leaves 303

Isolation of peroxisomes from spinach leaves 304

Isolation of plasma membranes by aqueous phase-partitioning 305

Large scale preparation of vacuoles from storage tissue 307

Preparation of tonoplast membranes from isolated vacuoles 308

Isolation of endoplasmic reticulum 310

The isolation of intact dictyosomes 312

Isolation of clathrin-coated vesicles 313

The isolation of secretory vesicles from pollen tubes 314

Marker enzymes 316

A surface area

AM acetoxymethyl

AOTF acousto-optic tuneable filter

BSA bovine serum albumin

c specific capacitance

CBN conjugate bond number

CCD charge coupled device

CCV clathrin-coated vesicles

CFP cyan fluorescent protein

Cm membrane capacitance

Cp capacitance of membrane patch

Cpip (stray-) capacitance of patch electrode

Cpm capacitance of whole plasma membrane

CV coefficients of variation

CWL centre wavelength

DCB 2,6-dichlorobenzonitrile

DEPC diethylpyrocarbonate

DIC differential interference contrast

DPI dots per inch

DTT dithiothreitol

DW distilled water

EM electron microscopy

ER endoplasmic reticulum

Erev reversal voltage of total plasma membrane currents

FAA formalin/acid/alcohol mixtures

FA-BSA fatty acid-free bovine serum albumin

FACS fluorescence activated cell sorter

FALS forward angle light scatter

FDA fluorescein diacetate

FLIM fluorescence lifetime imaging microscopy

FLIP fluorescence loss in photobleaching

FRAP fluorescence redistribution after photobleaching

FRET fluorescence resonance energy transfer

FWHM full width at half-maximum

GEF galinstan expansion femtosyringe

GFP green fluorescent protein

GST glutathione S-transferase

GUS B-glucuronidase

HA haemagglutinin

HBW half band width

I total current passing the plasma membrane

Ic capacitive current

Ir resistive current

LM light microscopy

LED light emitting diode

LUT look-up table

MS Murashige and Skoog salt mixture

NA numerical aperture

NLS nuclear localization signal

NRA Naturstoffreagens A

PCR polymerase chain reaction

PEG polyethylene glycol

r.m.s root mean square

ROI regions of interest

ROS reactive oxygen species

Rp resistance of membrane patch

Rpm resistance of whole plasma membrane

RS seal resistance

SDS sodium dodecyl sulfate

SEM scanning electron microscopy

S/B signal-to-background

S/N signal-to-noise

SV secretory vesicles

TEM transmission electron microscopy

TESPA 3' aminopropyl triethoxysilane

w/v weight to volume ratio

YFP yellow fluorescent protein

ZIO zinc iodide/osmium tetroxide

The development of the optical microscope in the seventeenth century opened

up new areas of study in many fields of science In particular, the observations of

plant and animal tissues and micro-organisms gave rise to cell biology, although

our modern idea of what a 'cell' actually is arose somewhat later Some of the

data recorded by the early microscopists, using only primitive microscopes

and drawing freehand what they saw, are quite remarkable Hooke, Malpighi,

and Leuwenhoek are the best known seventeenth century microscopists, but

Nehemiah Grew was the first true specialist in plant microscopy He produced

detailed descriptions of plant microanatomy which have proved remarkably

accurate An example is shown, along with Grew's original legend (1), in Figure 1.

Although there is only space to show one such picture, the original volumes

contain many pages of such detailed, painstaking hand-engravings With today's

access to photography, video cameras, and computer image processing, this

should serve as a reminder of how much can be achieved with careful

observa-tion and a very simple microscope Many biologists overlook the useful

informa-tion that can be rapidly obtained by even a very simple laboratory microscope

The optical microscope has developed steadily since its invention Particularly

important innovations were the analysis of the imaging process by diffraction

theory by Abbe, and the concomitant development of the optimal bright field

condenser by Abbe and Zeiss The development of textile dyes provided many

stains which were ideal for bright field microscopy of biological specimens Later

Zernike introduced phase contrast optics, which proved particularly useful in

visualizing unstained biological material In fact one of the main reasons the

early microscopists managed to see unstained biological material was because of

the aberrations in their optical components, which introduced a pseudo-phase

contrast effect Paradoxically, as lens design improved, the objects became harder

and harder to see Phase contrast optics reintroduced this contrast in a

con-trolled way More recently Nomarski invented differential interference contrast

(DIC) which produces an image whose contrast depends on the changes in

Figure 1 A transverse section of a horse-radish root, hand-drawn and hand-engraved by

Nehemiah Grew (1) in 1673 Grew's original legend is as follows:

Fig 1 A slice of the lower part of the root of Horse-radish cut traversly, as it appeareth to the bare eye a The skin ac The bark, with the succiferous vessels therein represented by the smaller specks Within stand the air-vessels represented by the larger and blacker specks e The pith.

Fig 2 The same slice, as it appeareth through a microscope AA The skin A.8 The bark B.L The succiferous vessels therein postured in the form of a glory B.G The air-vessels postured in

a thick ring; the several conjugations whereof are radiated G.E Other succiferous vessels within the air-vessels postured in a thin ring E The pith ee The bubles of the pith.

(Reproduced from ref 1 with permission.)

refractive index within the specimen In some types of biological specimens Thishas proved a very valuable technique

The progress of development of optical methods has accelerated markedly inthe past few years In the period from 1950 onwards, the new technique ofelectron microscopy provided radically new insights into cell structure, andrevolutionized our conceptions of subcellular organization, eclipsing opticalmethods for a while In the past twenty years optical methods have undergone arenaissance, mainly because of the use of highly sensitive and specific fluor-escence probes These include monospecific antibodies to all type of biological

macromolecules, specific DNA and RNA segments used as in situ probes, and

fluorescent dyes for DNA, pH, and particular ions (see Chapters 2, 3, 10, 12) Light

is a relatively non-destructive method of probing cells, and this makes optical

studies of living cells possible, often coupled with microinjection (Chapter 5) or

other loading of fluorescent markers In the past few years the introduction of

the green fluorescent protein (GFP) has started a revolution in the microscopy of

living cells and organisms This naturally fluorescent protein can now be

ex-pressed transgenically in virtually any organism or cell of interest, and can be

used for a wide variety of in vivo studies Most exciting, the GFP gene can be fused

to the gene for another protein of interest to express a GFP-protein fusion

Surprisingly, in almost all cases, the chimeric protein behaves in the same way

and is localized as the original protein This means that it is becoming possible to

analyse virtually any protein of interest in living cells, opening up enormous

opportunities for future cell biology research

In parallel with these developments in fluorescent probes, there have been

radical developments in optical microscopy itself, notably the invention of the

confocal microscope This was suggested by Minsky in 1957 as a method for

increasing the resolution of an optical microscope It was first implemented by

Petran for reflection imaging Although confocal methods have been also

de-vised for transmission imaging, it is with fluorescence imaging that confocal

microscopy has had the greatest impact in biological microscopy Fluorescence

microscopy is a dark field imaging mode, with very bright structures contrasted

against a black background In conventional epifluorescence imaging the

con-tribution of each part of the specimen to the coarse features in the image

extends a long way either side of the focal plane whereas the fine image detail is

rapidly attenuated away from the focal plane This gives rise to a high

back-ground out-of-focus contribution that tends to obscure the fine structure and

makes the images generally hard to interpret in detail The confocal

arrange-ment, which is described below, excludes nearly all of the out-of-focus

com-ponent and thus produces clearer fluorescence images—clean 'optical sections'

It is also the best technique for measuring focal section series, and thereby

obtaining true three-dimensional reconstructions of cellular and subcellular

structures

The confocal microscope produces optical sections by manipulation of the

light in the microscope An alternative method for 3D optical imaging is to use

computer image processing to remove the out-of-focus light from each image by

calculation This method, invented nearly twenty years ago, is called

deconvolu-tion or deblurring, and is now becoming much more widespread as electronic

cameras improve, and computers become more powerful and less expensive

This chapter gives a brief introduction to the most useful optical imaging

modes with some examples of the type of images to be expected from plant

material Confocal and 3D microscopy will also be described and examples will

be given of ways in which it is proving useful in plant studies Rather than give a

complete manual on these various techniques, the intention is to show the

range of methods available and what they can offer to a plant cell biologist The

reader is referred elsewhere for more detailed instructions on the use of specific

techniques (2-7)

2 Explanation of terms

Before describing the different types of optical imaging, a brief description some

of the technical terms that are encountered in optical microscopy will be given,particularly in the description of the optical components A more complete

glossary is given in ref 8 The primary image forming lens is called the

object-ive It is invariably an assembly of several optical components Another lens

assembly, the condenser, focuses illuminating light on the specimen for mitted light microscopy The final image is produced by the eyepiece or ocular.

trans-Each objective lens has a type description, and some numbers engraved on theside of the barrel; for example 'Plan 25/0.08 160/0.17' The type description worddenotes the level of correction of aberrations of the objective

Optical lenses suffer from various aberrations, and these are corrected to

different extents in different types of objective In achromats, the chromatic

aberration (bringing light of different wavelengths to different focal planes)

is minimized for two wavelengths (usually one below 500 nm and one above

600 nm) In apochromats, the chromatic aberration is minimized for three wavelengths (generally about 450 nm, 550 nm, and 650 nm) In plan objectives

the curvature of field (most noticeable at the edge of the field of view) is

minimized In the objective terminology this is also used as a prefix—e.g

plan-achromat or plan-apochromat Other terms are used by individual

manu-facturers to describe special features; for example, Zeiss call objectives which are

designed for UV transmission neofluar and plan-neofluar, whereas other manufacturers use different terms such as fluor, UV UV transmission is neces-

sary for the excitation of some fluorochromes, such as the DNA dye DAPI apochromats from some manufacturers do transmit light in the near UV and can

Plan-be used for DAPI, those from others do not Generally plan-apochromats are thebest objectives (and the most expensive)

The first number after the type is usually the magnification Objective

magnifications range from X l to X l00 The most useful magnifications for cellbiological work are: a low magnification (X16 or X25); intermediate X40; and

high X63 (or X60) Next to the magnification is the numerical aperture, a

number greater than zero and less than 1.5 Technically it is the refractive index

of the immersion medium multiplied by the sine of the aperture angle of thelens—in essence it tells you the angle of the cone of scattered light the objectivecollects The larger the numerical aperture, the greater the light collectionefficiency and the greater the resolution obtainable

Resolution is, loosely, the extent to which fine image detail is observable.

There are several more rigorous definitions; the one most often used in scopy is the minimum distance between two points such that they can be recog-nized as distinct The Rayleigh resolution criterion gives this value as 0.61*Y/NA,where lambda is the wavelength of the light used and NA is the numericalaperture of the objective However in imaging modes which use a condenser, the

micro-NA of the condenser also contributes to the resolution (see ref 3 for a fullerexplanation) With the highest numerical aperture objectives, the resolution is

approximately 0.25 um (depending on the wavelength of the light) Some

object-ives have an adjustment collar to allow the numerical aperture to be changed

The highest available numerical aperture should normally be used, but it is

occasionally useful to be able to decrease it, for example for dark field imaging

(see below), or to increase the depth of field Many objectives are designed for a

tube length of 160 mm, which is usually the next number engraved on the side

of the objective This is the distance between the objective and the eyepiece

Some objectives are termed infinity-corrected, which has the advantage of

allow-ing an arbitrarily long distance between the objective and the eyepiece As far as

the optics are concerned, 160 mm tube length objectives are interchangeable

between different microscopes, although some of the correction of chromatic

aberration is often in the eyepiece, so eyepiece and objective should strictly be

matched for optimal performance But infinity-corrected objectives cannot be

interchanged with 160 mm ones, unless a special correction lens is used Thus,

until recently objectives could be interchanged between microscopes fairly easily,

because all the manufacturers used a common thread (defined by the Royal

Microscopical Society—RMS) for mounting them Unfortunately all the major

manufacturers now use their own, different, threads on their objectives, making

it virtually impossible to exchange objectives between different microscopes

This development is anti-competitive and regrettable

A final number may be engraved on the objective, usually 0.17 This

corres-ponds to a coverglass thickness of 0.17 mm It means the objective has been

designed for a coverglass of this thickness, and you should not use any other

thickness—using any other coverglass thickness, whether thinner or thicker,

will degrade the optical performance of the objective, mainly by increasing the

spherical aberration It is a good idea never to have any other thicknesses in the

laboratory to avoid confusion

Many objective lenses are immersion lenses This means that they are

de-signed to have a liquid of a certain refractive index between the lens and the

coverglass These objectives will have 'Oil' or 'Oel' or 'Imm' written on them

Some objectives are designed for other immersion liquids—usually either

glycerol ('Glyc') or water ('W') Still other objectives have a variable correction

collar for different media—it is important to make sure the collar is in the right

position before use Objectives which are not designed for immersion are often

called 'high dry' lenses If possible, it is useful not to mix immersion and

non-immersion objectives on a microscope This will avoid confusion and simplify

changing from one objective to another Using the wrong immersion medium or

the wrong coverglass thickness will increase the spherical aberration This

means that different levels of image detail are brought to a focus at different

distances from the objective In general this will make the images more blurred

In fact in normal biological use the spherical aberration is almost always worse

than it should be This is because the objectives are designed to image specimens

immediately below the coverglass In most biological specimens there is a layer

of mounting medium between the underside of the coverglass and the

speci-men, and this extra optical path length increases the spherical aberration This is

particularly easy to see with epifluorescence Focus on a small bright spot andobserve how the spot changes either side of focus In the absence of sphericalaberration it should expand and fade equally either side Almost invariably youwill find it is asymmetric, disappearing quickly one side, but slowly the otherside An image of a single point such as this is called the point spread function It

is an important characteristic of a microscope, especially if you are interested indetailed 3D imaging or image processing Within certain limits, if you know how

a single point is imaged, then you can determine the image which should resultfrom any specimen (at least for fluorescence and bright field imaging)

A final objective characteristic which is often important is the working

distance This is the distance between the focal plane and the front of the

objective It is usually very small (perhaps 0.2 mm) for high numerical aperturelenses, since to obtain a large collection angle it is necessary either to have thelens close to the specimen or to have very large diameter lenses, which are expen-sive It is possible to obtain objectives with particularly long working distances, ofthe order of tens of millimetres (for example the Nikon SLWD range, which stillhave quite large numerical apertures) This type of lens is normally used oninverted microscopes for observation of samples in containers such as Petridishes, but is also very useful for micromanipulation and microinjection on anon-inverted microscope, since it gives reasonable access to the specimen duringobservation

The condenser is crucially important in transmission imaging Its role isobvious in phase contrast and dark field imaging (see below), but it is often notappreciated that its contribution to the overall resolution is equal to that of theobjective in bright field and DIC imaging For high resolution, you should use ahigh quality plan-apochromat condenser If the objective is an oil immersionone, then the condenser should also be an oil immersion lens if possible A largeresearch microscope will have a number of different condenser positions A wheel

in the condenser is rotated to bring different phase rings, dark field apertures,and DIC prisms into the optical path

The eyepieces on a microscope are rarely, if ever, changed Their tion is calculated so that the image is magnified enough for the human eye to seeall the significant detail This is usually X10 or X20 In binocular microscopesthere is a focusing collar on one or both eyepieces If the microscope has a

magnifica-camera attached, one eyepiece will probably have a photoscreen graticule in

it, or there may be an eyepiece graticule If so, focus on the specimen and,looking only through this eyepiece (shutting the other eye), adjust the eyepiececollar until the graticule is sharply in focus Then the specimen and graticuleshould both be sharply in focus Now look through the other eyepiece and adjust

it to bring the specimen sharply into focus This will have adjusted the eyepiecesfor your eyes, and ensured that when the image looks in focus to you, it will be

in focus at the camera (provided the camera has been set up correctly)

The overall magnification of the microscope is the product of the eyepiece (orcamera projection lens) magnification and the objective magnification On somemicroscopes this has to be multiplied by an additional factor, and occasionally

there is another intermediate magnification-changing lens (sometimes called an

optovar) It is best to measure the actual magnification of your microscope To do

this you need a stage micrometer—a microscope slide with a scale engraved

on it An eyepiece graticule is also useful This is a glass plate with a

dimen-sionless scale engraved on it, which is inserted in the eyepiece It is calibrated for

each objective magnification using a stage micrometer, and is used to estimate

the size of objects through the eyepiece

3 Recording images

For all but very routine microscopy it is a good idea to use photography or other

image recording almost as a matter of course Somehow you can never find such

a good field of view as the one you saw when taking a quick look! The moral is—

record it, and take each image as if it was a picture for publication, because you

never know in advance which ones you will actually want to publish

3.1 Image resolution

The microscope eyepiece in its normal configuration produces a virtual image,

which is then projected as a real image onto the retina by the lens of the eye

Recording the image onto film or electronically requires that a real image is

produced by the microscope This is usually achieved by a projector lens, which

is optically often the same as an eyepiece ocular However this lens needs to be

at a slightly different distance from the objective in order to form a real image of

the same focal plane as the eye sees through the regular eyepiece The position

of the projection ocular is usually adjustable, so that the plane of focus seen

directly and that projected onto the camera are the same and are both accurately

in focus The total magnification produced by the microscope is the product of

the objective magnification, the eyepiece or projector magnification, and the

microscope intermediate magnification factor (including optovar magnification

if present) The magnification of the regular eyepiece is chosen so that the total

magnification produced is sufficient to enable the human eye to see the finest

details that the objective can produce Any further magnification will not

pro-duce any more image detail, and is often called 'empty' magnification However,

an image recording device will have different resolution characteristics from the

human eye, and a different projector magnification will be needed, which may

be more or less than the human eye requires

As an example, consider the image produced by the highest resolution X60

oil immersion objective available, which will probably have an NA of 1.4 The

resolution of this objective will be approximately half the wavelength of the

light used—say 0.25 um A good rule-of-thumb from image processing theory is

that the distance between adjacent picture elements, or pixels, in a recorded

image should be about one-third the minimum image detail to be recorded Thus

to record the maximum resolution of the objective, we need to have a pixel

spacing of about 0.08 um The effective pixel spacing that can be recorded

on photographic films varies, but is somewhere around 10 um Thus a total

magnification of XI25 is needed Given an objective magnification of X60, therest of the optics—the intermediate magnification and the projector—need toproduce a total of about X2 It is not always easy to find the intermediatemagnification figures needed for the foregoing calculation Therefore it is best touse a stage micrometer to measure the magnification of the whole microscopeoptical system directly.

Although photographic film is not as sensitive as the best low light levelcameras, it does have an extremely high capacity as an image recording medium.With a pixel resolution of 10 um, a 35 mm film frame records 3500 x 2400 pixels.This is much more than most cameras A typical video camera records 512 X 768pixels, and a 'high resolution' camera may record something around 1024 X

1200 pixels Thus in calculating the projector magnification needed for an tronic camera, a compromise must be made If it is necessary to retain the fullresolution of the image, only a small part of the field of view visible through theeyepiece can be recorded At 512 x 768 pixels this would be a field of around

elec-40 x 60 um Alternatively, if the projector is chosen to keep the full field of view,the resolution will be seriously degraded The only real solution is to usedifferent projector lenses for different purposes

3.2 Film recording

For immunofluorescence, the author's laboratory uses Kodak TMAX film quiteextensively for black and white photography; it has good contrast and low grain,and is available in a range of speeds (TMAX 400 is the most often used in theauthor's laboratory) Kodak Technical Pan film is much slower and has lowercontrast, but is more suitable for bright field or DIC images Technical Pan filmwith liquid Technidol low contrast developer is useful for photographing videoand computer displays; most other films have too high a contrast for this applica-tion For colour slides, use tungsten colour rated films if using a tungsten lamp,daylight rated films for a mercury arc lamp (for example when photographingimmunofluorescence specimens in their original colours, or photographingcolour video or computer screens) For colour prints, it is recommended that slidefilm is used and prints from the diapositives (Cibachrome prints)

Most modern exposure meters use centre-weighted average metering This isusually fine for bright field types of image, since the brightness of the area ofinterest is about the same as the rest of the field However, it is not appropriatefor other types of image, particularly fluorescence, since the objects of interestare much brighter than the dark background, and the average brightness is near

to the background value Here, the best solution is to use spot metering, wherethe exposure is set in a small area in the centre of the field Move the object ofinterest into the central area to set the exposure If you do not have spot meter-ing you will have to use trial and error As a guide, using 800 ASA film, a reason-ably bright cytoskeleton immunofluorescence image might need an exposure of

30 seconds, DAPI stained nuclei may need only 1 second

Often a photographic negative needs to be digitized so that the image can be

handled in a computer and later printed for publication or made into a slide To

obtain the maximum amount of information on the film, it will need to be

scanned at something like a 10 um raster Film scanners and printers are usually

rated in dots per inch (DPI) Thus scanning at 10 um requires about 2500 DPI or

better Specialized negative scanners will provide this resolution, although most

cheaper flat-bed scanners will not However, it is not always necessary to scan

images at the maximum resolution, and handling the very large files that result

can cause problems The best approach is to work back from whatever final

out-put is needed So, for example, if the final image is to be on a printer which has

400 DPI resolution and is to be 2 inches X 2 inches, this will require 800 x 800

pixels in the image file If the area to be printed is 20 mm X 20 mm on the

original negative, it will only need to be scanned at 40 pixels/mm, which is about

1000 DPI

3.3 Electronic cameras

The first video cameras used a semiconductor target, such as silicon, to record

the incoming light The pattern of light projected onto the target left a

corres-ponding pattern of charge distribution which was read out by scanning an

electron beam across it and amplifying the resulting current This raster scan

was defined by the broadcast television standards—typically 25 frames per

second at 625 horizontal lines The resulting analogue signal could be displayed

directly to a monitor To convert it to a form that could be handled by a

com-puter, it had to be digitized by a device called a frame-grabber—a fast A to D

converter and computer image memory

Alternative electronic imaging devices began to appear in the early 1980s

These devices, called charge coupled device (CCD) cameras effectively record the

incident light digitally from the start Made by semiconductor microfabrication

methods, CCD chips contain an array of 'charge wells' Photons falling on the

chip cause electrons to be produced and trapped in the wells After a given

exposure time, the charge in each well is read out and recorded The early CCD

cameras used a direct digital interface to send the electron counts for each well

directly to computer memory This is still the circuitry used in expensive

scien-tific slow scan CCD cameras Today virtually all video cameras use CCD chips as

the recording element However, to make them compatible with CCTV and

broadcast standards, the image is read out at video frame rate and converted to

an analogue video signal Thus, ironically, what was originally a digital image is

converted to a poorer analogue signal, and must still be digitized into a

computer by a frame-grabber

CCD cameras are extremely good imaging devices; they are geometrically

very accurate and photometrically highly linear In principle they are also very

sensitive, since each incident photon causes an electron to be produced The

limits to sensitivity come from two sources First, the measurement circuit

intro-duces 'readout' noise In the best slow scan CCD cameras, readout noise can

be reduced to 2-3 electrons per pixel, although a more typical value is 10-20

electrons The readout noise increases with the readout speed, and so is verymuch higher in video rate cameras The noise can be reduced by accumulatingmultiple frames in a computer frame-grabber/frame-store, but the readout noise

is introduced into every video frame read, so it is better to have 'on-chip'integration, where the chip is exposed for a longer period of time before theimage is read out Thus, readout noise is only introduced once Secondly, CCDchips accumulate 'dark current'—thermal electrons are produced and are trapped

in the charge wells along with the photon-produced electrons In video ratecameras, the dark current is small because of the short exposure time of eachframe, but with long on-chip exposures, the dark current can become very large.The solution to this problem is to cool the CCD chip The dark current can bereduced to insignificant levels for microscopical imaging using thermoelectriccooling to -40°C

A final application of CCD chips is in the current computer digital cameras Inthese cameras the chip is exposed and then read out as a digital image They aretherefore intermediate between slow scan, scientific cameras and video cameras.Currently even relatively cheap consumer cameras are available with resolutionsaround 2000*2000 pixels Since they are not cooled, they will suffer from darkcurrent, and the maximum exposure will be limited compared to a cooled CCDcamera However they are becoming an attractive option for a cheap and simpleway of recording images electronically from a microscope The current genera-tion is capable of recording bright field microscope images, and fluorescenceimages bright enough to be visible by eye, but not very weak fluorescence imagesand definitely not bioluminescence images The main problem is that most ofthe current consumer cameras have a built-in lens for standard photography, andthis makes it hard to mount them satisfactorily on a microscope What is needed

is a camera without a lens which has a standard bayonet or C-mount fitting

4 Microscope imaging modes

Microscope imaging may be divided into transmission modes, such as bright field,dark field, phase contrast, and differential interference contrast (Nomarski), andepi-illumination modes, primarily epifluorescence and reflection contrast Con-focal microscopes have been developed which use either type of illumination,but epifluorescence and reflection contrast are much simpler to implement in aconfocal arrangement and to date have been by far the most widely used inbiology Deciding what type of imaging to use is sometimes straightforward andsometimes difficult It is important to be aware of the different imaging modesavailable and of their capabilities, and to be sufficiently familiar with themicroscope to try different imaging methods with a specimen

4.1 Bright field imaging

This is the most basic imaging technique Adjustment for optimal bright fieldimaging (Kohler illumination) is fundamental for all the other transmissionimaging modes, and familiarizing yourself with it should be the first step in

using any microscope Always check this adjustment before using the

micro-scope for anything else (except epifluorescence, dark field, and reflection), and

check it every time you change objectives or insert any other element into the

light path

Light from each point on the bulb filament is focused by the collector lens to a

point at the condenser aperture At the field aperture this gives a wide area of

even illumination Since the field aperture is at an equivalent focal plane to the

specimen, the specimen is evenly illuminated Light from the specimen is

focused by the objective at a plane known as the primary image plane The

eye-piece then forms an image a small distance from the top lens of the eyeeye-piece

You should refer to the handbook for your microscope for a detailed

descrip-tion of the parts of your microscope and how to adjust it A descripdescrip-tion is given

here which should apply in general to any microscope, Examples of bright field

images are shown in Figures 2A and 3A.

Adjustment for bright field Imaging with Kohler illumination

Equipment

• All microscopes with a standard bright field condenser should be capable of this type ofimaging

Method

1 Focus on a specimen with the condenser and field apertures wide open,

2 Close the field aperture until you can see its edges

3 Focus the condenser up or down until the edges of the field aperture are sharp

4 If necessary, centre the image of the field aperture with the condenser centringadjustments

5 Close the condenser aperture until the glare around the field aperture just appears, and then reopen it a little (if in doubt, open the aperture about halfway).Alternatively readjust the condenser aperture during observation of the specimen

dis-to obtain the best linage Opening the condenser aperture increases the resolution,but decreases image contrast and increases bright flare around objects Closing itdecreases flare and increases image contrast, but decreases resolution

6 Finally open the field aperture until it just disappears outside the field of view Forlower power objectives (e.g x 16) the illumination may not be uniform, but brighter

in the middle In this case there is generally an accessory lens in the swing this in and readjust the condenser In the case of very low power (e.g X2.5)even this will not give an even illumination Lower the condenser right down andopen up both apertures—this will not be Kohler illumination, but it should be moreeven, and optimal resolution is not a consideration at such low magnifications

condenser-Figure 2 Filament of the alga Spirogyra grevettiana (A) Bright field image The contrast is fairly

low but the characteristic spiral chloroplasts are visible (B) Phase contrast image of the same cells The contrast is greatly enhanced, but note the prominent haloes around all the

structures, particularly the cell wall (C) DIC image of the same cells This is probably the optimal imaging mode for this specimen, showing fine structure inside the cell, good contrast, without the haloes present in the phase image (Reproduced from ref 2 with permission.)

a quarter wavelength, that passing through the phase ring is unretarded Lightwhich is not scattered by the specimen passes through the condenser apertureand through the phase ring Any light which interacts with the specimen will

Figure 3 A wax section of wild-type flower of Antirrhinum majus labelled with an in situ probe

to the transcript of the gene floricaula detected by alkaline phosphatase-conjugated antibody.

(A) Bright field image, showing the distribution of the transcript as revealed by the insoluble

dark-coloured product of the alkaline phosphatase reaction, but poor detail of the cell structure.

(B)The equivalent dark field image The crystalline product of the alkaline phosphatase

reaction reflects light strongly, and is thus seen very clearly (C) For comparison the phase

contrast image of a portion of the same specimen is shown The contrast is better than (A) but

poorer than (B) (Reproduced from ref 2 with permission.)

have- had a small specimen-dependent phase change introduced and most of it

will be scattered so that it no longer passes through the phase ring—thus it will

be additionally retarded by another quarter wavelength, and will interfere

de-structively with the unscattereci light So the specimen-dependent phase changes

will be turned into changes in amplitude, and be visible

Remember that phase contrast is an interference phenomenon; light and dark

areas are related to the refractive index within the specimen Small structurescan appear bright or dark at slightly different focus levels A dark structuremight be a dense object, or it might be something else like a hole or a vacuole.Rings and haloes around structures are also highly visible, and the resolution isless than in bright field For these reasons, although phase contrast can often be

very useful, the resulting images should be interpreted with caution Sec Figures

2B and 3C for examples of phase contrast images.

Adjustment for phase contrast

Equipment

• Phase contrast requires special objectives

having a phase ring and a matching

condenser annular aperture—it cannot bedone on other types of objectives

Method

1 Set the microscope up for Kohler illumination in bright field (Protocol 1).

2 Open the condenser aperture fully, and either replace one eyepiece with a phase

telescope, or, more usually, move a Bertrand lens into the light path (usually by arotating wheel somewhere between the lens turret and the eyepiece)

3 Observe the back focal plane of the objective, where the phase ring is located Youshould see two rings—a bright one corresponding to the condenser annulus and adark one corresponding to the phase ring,

4 Adjust the position of the condenser annulus, so that the phase rings overlap metrically This is accomplished by a centring device (not the same as the condensercentring mechanism) On some modern microscopes the annuli are pre-centred andwill not require adjusting If the two rings are of very different radii then thecondenser annulus is not the correct one for the objective

sym-5 Remove the phase telescope and return to normal viewing; you should now have

phase contrast imaging,

4.3 Differential interference contrast (Nomarski)

Differential interference contrast (DIC) was invented by Nomarski and so oftengoes by that name Another similar technique is called Hoffman modulationmicroscopy, which is less expensive to implement DIC uses two polarizers at rightangles to each other, one before the condenser and the other after the objective.Two special optical components called Wollaston prisms are introduced betweenthe crossed polarizers, one in the back focal plane of the condenser, the other inthe back focal plane of the objective The Wollaston prisms have the effect ofproducing, instead of a single image, two images slightly displaced relative to eachother This displacement is less than the resolution limit of the objective Thecrossed polarizers are used so that the two displaced images are added together

vectorially The overall effect of this arrangement is to produce a resultant image

which is the sum of the two displaced images Where two points summed have the

same phase, the resultant summed image point will have the same plane of

polarization as the original incident polarized light, and thus be blocked by the

second polarizing filter However, where there is a phase difference between the

two points the resultant will have a rotated plane of polarization, and a

com-ponent of this light will pass through the analyser The result is an image which

maps changes in refractive index within the specimen The Wollaston prisms also

introduce a specimen-independent relative phase difference between the two

images; this additional phase difference can be changed by displacing one prism,

which alters the final image seen This displacement is provided via a knurled

knob/lead screw device on most microscopes In some microscopes a quarter-wave

plate compensator is used rather than displacing a Wollaston prism

DIC images can be difficult to interpret The method displays an image of

changes in refractive index in the direction defined by the orientation of the

prisms For this reason it is particularly good at revealing edges in biological

structures—for example, organelle and nuclear boundaries, cell boundaries, and

cell walls It has also been used very effectively to image fibrous subcellular

com-ponents such as microtubules, often in combination with video enhancement

One side of an edge will be brighter than the background, the other side will be

darker For this reason the images have a false 'shadowed' appearance This is

visually very attractive and can be very informative However it can also be very

misleading, since it makes the images seem three-dimensional because of the

way the human brain interprets lighting effects It is important to realize that

these images are not three-dimensional ones, they merely give that impression

To obtain true three-dimensional information focal sectioning must be used In a

simple way this can be achieved by focusing up and down through the specimen

Because DIC can use the highest condenser aperture available it can give smaller

depths of field than other conventional imaging modes, and so is better for focal

sectioning For highest resolution with DIC, a condenser with a numerical

aperture as great as that of the objective should be used If the objective is a high

numerical aperture oil immersion one, the condenser should be also, and should

be used with oil—this is rarely appreciated, still less actually done A DIC image

of a Spirogyra filament is shown in Figure 2C

Adjustment for DIC

Equipment

• For DIC a microscope must have provision

for the two polarizing filters and the two

prisms in addition to standard bright field

optics In principle DIC should work for

any high quality objective lens It was

originally necessary to use special free objectives, but modern high qualityplan-apochromat lenses are usually goodenough for most DIC applications

1 First adjust the microscope for Kohler bright field illumination (Protocol 1) with thecondenser set to the DIC position (condenser prism in place), but with the objectiveprism out of the optical path

2 Open the condenser aperture, and insert the polarizing filters On some scopes the polarizing filters are fixed, on others they can be rotated If the polar-izing filters are rotatable, rotate one of them until the field is maximally dark

micro-3 Insert the objective prism, and adjust the translation to obtain the 'best' image It is

up to the microscopist to judge what the best image is However, it is generally best

to have the condenser aperture as far open as possible Closing the aperture, whileincreasing the contrast, will decrease the resolution

4.4 Dark field

All the foregoing techniques, with the exception of polarized light microscopy,are essentially bright field methods; that is, darker structure in the specimen isseen imaged against a bright background This is very familiar and readilyinterpretable However it can be difficult to see small, weakly imaged features,since they represent small changes against a large background, and thereforeprovide inherently low contrast In dark field techniques, the background is lowand the specimen structure is bright, and so the contrast is much greater It isparticularly good for reflective structures such as silver grains in autoradiographsand silver-enhanced, gold-labelled immunocytological specimens (see Chapter 10).Plant cell walls also show up brightly in dark field A dark field image is shown in

Figure 3B.

Adjustment for dark field

Equipment

• A microscope with a dark field condenser

This is generally a 'patch stop' condenser,

which has a central disc to block the direct

light beam If the condenser is an oilimmersion type, use oil and use themaximum condenser aperture

3 If you are using a patch stop condenser which requires centring, use the phasetelescope or Bertrand lens to view the back focal plane of the objective Focus thecondenser up and down until the patch stop is seen as clearly as possible, thencentre it.

4 Switch back to normal viewing and adjust the condenser focus as before for the bestdark field image.b,c

a It may be necessary to readjust the centring of the patch stop; look for the location of the

darkest part of the image as the condenser is focused, check the centring of the condenserpatch stop, and recentre if it is not central

bIn contrast to bright field, the angle of the illuminating cone of light must be greater thanthat of the objective aperture, otherwise the unscattered light will not be excluded from theimage This may mean that dark field will not work effectively with very high numericalaperture objectives, unless the condenser can provide an even greater effective numericalaperture, and it may therefore not be possible to get a dark background unless you can stopdown the objective numerical aperture

cAt low magnification, the largest phase annulus may work as a patch stop Otherwise it ispossible to improvise one with a disk of card underneath the bright field condenser—there isusually a suitable filter holder above the condenser accessory lens

4,5 Epifluorescence and reflected light microscopy

The previous methods described are all transmission imaging modes—the

speci-men is illuminated from the side opposite the objective by means of a condenser

lens In epifluorescence and reflection imaging, the specimen is illuminated by

light which is introduced from the same side of the specimen as the objective,

usually through the objective, which therefore acts as its own condenser This

means that it is not necessary to adjust the condenser for these modes (although

it is a good idea to get into the habit of always checking the bright field

adjustment before using a microscope)

Reflection imaging is particularly useful for imaging highly reflective particles,

such as silver grains in autoradiographs Additional microscope components

needed are an epi-illumination source, a 50% semi-silvered mirror, and two

polar-izing filters, one in the incident light and a second with its plane of polarization

at right angles to the first in the path of the reflected light The polarized

illuminating light is reflected down through the objective by the semi-silvered

mirror at 45° Reflected light from the specimen then passes up and straight

through the mirror Light which is reflected, as opposed to back-scattered light,

will have its plane of polarization rotated, and will therefore pass through the

second polarizing filter Back-scattered light will be blocked by the filter Most

epifluorescence microscopes can be converted to reflection imaging, simply by

substituting a semi-silvered mirror for a dichroic one, and adding polarizing

filters in appropriate positions The normal mercury lamp epifluorescence source

can be used, but be sure to include an visible wavelength excitation filter, so as

to cut out UV light, and a heat filter to prevent damage to the polarizer mercially available reflection systems use circularly polarized light, which hassome advantages, but cost more For very low magnification reflection imaging adissecting microscope with an annular illumination collar which fits around theobjective lens works well.

Com-Epifluorescence is probably the most important optical technique in use insubcellular imaging studies at the moment The details are described in laterchapters; only the principles will be discussed here Fluorescent molecules absorblight of particular wavelengths, and then re-emit light at longer wavelengths and

in all directions, the energy difference ultimately heating the specimen fluorescence achieves extremely high specificity and low backgrounds (and thussensitivity) by a double discrimination—in frequency and direction—against theincident light and in favour of the emitted light A mercury or xenon arc lamp isthe most usual light source The light passes through an excitation filter, whichmatches the absorption characteristics of the fluorescent label being used Thelight is then reflected down through the objective lens by a dichroic mirror; this

Epi-is a special type of mirror which has wavelength-dependent reflectivity Light atshorter wavelengths than the mirror's cut-off is reflected, whereas light at longerwavelengths passes through It is thus both more efficient than a semi-silveredmirror and more wavelength selective Back-scattered light from the specimenwill have the same wavelength as the incident light and will therefore be re-flected again by the dichroic mirror and will not reach the eyepiece Fluorescentlyemitted light from the specimen passes through the dichroic mirror then reachesanother specific filter, the emission filter, which only passes light of the emissionwavelength of the fluorescent marker, and further discriminates against non-fluorescent light

Provided you have the correct components on your microscope, and providedthe mercury lamp has been correctly set up, there is no alignment necessary forepifluorescence Some precautions are advisable with the mercury lamps gener-ally used as the light source Many mercury bulbs have a rather short life, andare also expensive The life is considerably shortened by frequent switching onand off It is a good rule not to switch the bulb off for at least 30 minutes afterswitching on, and not to switch it on again until at least 30 minutes after switch-ing off The recommended life is only 100 hours for the older types of bulb,although it is much longer for some more recent mercury bulbs Resist thetemptation to run the bulbs longer than their recommended time; they canexplode if this is done, which is highly dangerous since mercury vapour is thenreleased into the air If this happens, evacuate and close the room until themercury vapour has had a chance to disperse

It is important to understand the properties of the optical components andfluorescent probes being used For example, the commonly used fluorescein andrhodamine probes are excited with visible light, and therefore all objective lenseswill be suitable Use plan-apochromats if available However, other fluorescentdyes, such as the DNA dye DAPI, need near UV excitation, and not all objectivestransmit these wavelengths; plan-apochromats from some manufacturers work