Ebook Textbook of oral pathology (2/E): Part 1

(BQ) Part 1 book “Textbook of oral pathology” has contents: Microscope, tissue processing methods, histological staining methods, diagnostic pathology, advanced diagnostic techniques, teeth anomalies, craniofacial anomalies, dental caries, benign tumors,… and other contents.

D O N

Textbook of

ORALPATHOLOGY

Textbook of ORAL PATHOLOGY

Textbook of

Second Edition

Editors

Anil Govindrao Ghom

MDS(OralMedicine and Radiology)

Professorand Head

DepartmentofOral Medicine andRadiology

ChhattisgarhDentalCollegeand Research Center

Sundra, Rajnandgaon, Chhattisgarh,India

ShubhangiMhaske(Jedhe)

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All rights reserved No part of this book may be reproduced in any form or by any means without the prior permission of the publisher.

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This book has been published in good faith that the contents provided by the contributors contained herein are original, and is intended for educational purposes only While every effort is made to ensure accuracy of information, the publisher and the editors specifically disclaim any damage, liability, or loss incurred, directly or indirectly, from the use or application of any of the contents of this work If not specifically stated, all figures and tables are courtesy of the editors Where appropriate, the readers should consult with a specialist or contact the manufacturer of the drug or device.

Textbook of Oral Pathology

First Edition: 2009

Second Edition: 2013

ISBN 978-93-5090-171-7

Printed at

Jaypee Brothers Medical Publishers (P) Ltd.

17/1-B Babar Road, Block-B, Shaymali

Email: joe.rusko@jaypeebrothers.com

Sharad Pawar Dental College

Sawangi, Wardha, Maharashtra, India

Anil Govindrao Ghom

MDS (Oral Medicine and Radiology)

Professor and Head

Department of Oral Medicine and

VSPM Dental College and Research

Institute, Nagpur, Maharashtra, India

Ashok Mhaske

MS (General Surgery)

Vice Dean, Professor and Head

Department of Surgery

People’s College of Medical Sciences

and Research Centre

Bhopal, Madhya Pradesh, India

People’s College of Dental Academy Bhopal, Madhya Pradesh, India

Pradnya Lele

MDS (Oral Pathology)

Lecturer Department of Oral Pathology and Microbiology

Government Dental College and Hospital, Mumbai, Maharashtra, India

Pranoti Pradhan

MDS (Oral Medicine and Radiology)

Professor Department of Oral Medicine and Radiology

Maitri Dental College and Research Centre, Durg, Chhattisgarh, India

Rashmi Ekka

MDS (Oral Medicine and Radiology)

Lecturer Department of Oral Medicine and Radiology

Chhattisgarh Dental College and Research Institute

Sundra, Rajnandgaon, Chhattisgarh, India

Sangamesh Halawar

MDS (Oral Pathology)

Reader Department of Oral Pathology Vasantdada Patil Dental College and Hospital

Kavalapur, Sangli, Maharashtra, India

Sundra, Rajnandgaon, Chhattisgarh, India

Shubhangi Mhaske (Jedhe)

MDS (Oral Pathology)

Professor and Head Department of Oral Pathology People’s Dental Academy, Bhopal, Madhya Pradesh, India

Smruti Nanda BDS

Lecturer Department of Oral Medicine and Radiology

Chhattisgarh Dental College and Research Institute

Sundra, Rajnandgaon, Chhattisgarh, India

Vivek Thombre

MDS (Periodontology)

Reader Department of Periodontology Chhattisgarh Dental College and Research Institute

Sundra, Rajnandgaon, Chhattisgarh, India

Foreword to the Second Edition

It is at once a great privilege and pleasure to write a foreword for the invaluable compilation

of 2nd edition of Textbook of Oral Pathology by Dr Anil Govindrao Ghom and Dr Shubhangi

Mhaske (Jedhe) The authors have taken extraordinary pains and care in putting together

meticulously the data collected over many years This has resulted in production of superb new

edition The excellent features of this edition are that the authors covered all the topics according

to new syllabus of almost all universities Special attraction, which I found in the book, is the

multiple choice questions at the end of each chapter and the points to remember

An attempt has been made in the book, to simplify and make it easy to remember the subject

The book will help the undergraduate and postgraduate students to get a bird’s eye view of the

entire topics

I would like to congratulate the efforts of editors for designing 2nd edition of Textbook of Oral Pathology I am sure

that the book will rapidly find a place in most progressive libraries the world over

Manisha Sanjay Tijare MDS (Oral Pathology)

Professor and HeadDepartment of Oral Pathology and MicrobiologyPeople’s College of Dental Sciences and

Research CentreBhopal, Madhya Pradesh, IndiaDean, Faculty of DentistryBarkatullah University, Bhopal, Madhya Pradesh, India

PhD GuideBarkatullah University, Bhopal, Madhya Pradesh, India

Executive Committee Member Indian Association of Oral and Maxillofacial Pathologists (IAOMP)

It gives me immense pleasure to write a few words about the Textbook of Oral Pathology.

This book is the outcome of combined efforts of Dr Anil Govindrao Ghom, Professor of Oral Medicine and Radiology and Dr (Mrs) Shubhangi Mhaske (Jedhe), Associate Professor of Oral Pathology and Microbiology I know Dr Shubhangi

as undergraduate as well as postgraduate student I feel her sincerity coupled with hard work and humanly approach toward the patients during her postgraduate studies contributed in making the textbook

My heartiest congratulations to Professor (Dr) Anil Govindrao Ghom and Dr (Mrs) Shubhangi Mhaske (Jedhe) for their great endeavor in bringing out the book The book contains six sections which are divided into 37 chapters related

to oral lesions/diseases inclusive of basic topics of oral pathology The topics like microscopy, stains and routine as well

as special investigations are noteworthy The lots of updated information in the book will be helpful to undergraduates, postgraduates and also for practising dental fraternity

The total of over 600 clinical photographs, microphotographs and line diagrams incorporated in the book are definitely useful for in-depth understanding of the subject The textbooks available on these subjects are many, but only a few ones cover both the subjects: oral medicine and radiology as well as oral pathology Contribution of Indian authors toward the books in dentistry is less as compared to the foreign authors Therefore, Mrs Mhaske (Jedhe) deserves a word of appreciation for her sincere and painstaking efforts

The book is an excellent contribution to a scientific literature in Indian scenario and thereby facilitating our students

to understand various diseases

With regards and best wishes

Jagdish V Tupkari

Professor and HeadDepartment of Oral Pathology and MicrobiologyGovernment Dental College and Hospital

Mumbai, Maharashtra, India

Foreword to the First Edition

Preface to the Second Edition

I may not have gone where I intended to go, but I think I have ended up where I needed to be.

― Douglas Adams

In the study of oral and dental sciences, oral pathology is the subject that concentrates on the mechanisms of the disease process and the morphologic changes in tissue that it causes Everyday, there are new additions to the knowledge of the subject and we have to keep pace with it to update our students about it That is the reason, we are here with the 2nd

edition of Textbook of Oral Pathology

In our first edition, there is a lot of feedback which has come to improve the quality of the book We tried to incorporate all the feedback in the book As due to new Dental Council of India (DCI) guidelines, multiple choice questions (MCQs) are part and parcel of examination pattern of the Bachelor of Dental Surgery (BDS) curriculum We have incorporated MCQs at the end of every chapter so that students can practice it and have some insight into what type MCQs can be there

in the examination Also, we have included ‘points to remember’ in every disease so that with respect to time, students can revise it fast There are also many new additions of photographs—clinical, histopathological as well as radiological

Preface to the First Edition

In the study of oral and dental sciences, oral pathology is the subject that concentrates on the morphologic changes in oral tissues which cause diseases and the mechanisms of the disease process Most recently published works on the subject are the product of eminent authorities with multiple authorship with current research advances ‘Why another book in oral pathology’? This question can probably be answered by the phrase,

Nothing can be changed by changing the face But many things can be changed by facing the change.

The purpose of the book is mainly to provide the undergraduate and postgraduate students, an easy way of reference for covering a broad-spectrum of oral pathology in lucid and simple language In support of all the composite works, it can be stated that the progress of oral and maxillofacial pathology in its many varied specialties coming up has made the subject too vast to be covered adequately by a single author Also, a balance between the advances and the basic essentials

is need of the hour With this balanced perspective and from the viewpoint of the graduate and postgraduate students under training, the authors have endeavored to compile the oral and maxillofacial pathology with respect to the related essential clinical oral medicine and radiology The book gives an extensive coverage and emphasizes on detailed description, adequate well-labeled illustrations, flow charts, recent developments and molecular aspects Aside from oral pathology

in general, the initial phase of the book includes the basics of the embryology, anatomy and pathology The study of microscope, tissue processing, diagnostic tests and advanced techniques are also included The photomicrographs and the clinical photographs in the book signify the adage: “A picture is worth a thousand words”; as the reader is encouraged to study the details and to clarify the confusion of the topics

In spite of sincere efforts, elements of human error or shortcomings are likely; the readers are welcome to point out all such mistakes and render valuable suggestions for further improvements and shall be greatly acknowledged

Anil Govindrao Ghom Shubhangi Mhaske (Jedhe)

This book could not have been written without the encouragement and motivation of my teachers and students I would also like to express my gratitude for my contributors Drs Sangamesh Halawar, Pranoti Pradhan, Monal Yuwanati, Pradnya Lele, Aparna Thombre, Avadhoot Avadhani, Vivek Thombre, Amol Gadbail, Rashmi Ekka, Seema Vaidya, Smruti Nanda, Savita Ghom, Satish Chhugani, without whose help and cooperation writing the book would have been

an uphill task

I would like to acknowledge the efforts of my Postgraduate students Satish, Manjari, Mansi and Bharani I am also thankful to Drs Swati Arora and Varun Rastogi, Senior Lecturers, Department of Oral and Maxillofacial Pathology, Kalka Dental College, Meerut, Uttar Pradesh, India, for their contribution of different classification systems of odontogenic tumors

Last but not least, my dear wife, “As fish is without water, so is me without my wife Savita.” Her contribution to my life is beyond what I can express in words I am also thankful to my daughter Milini and son Sanvil for their love and affection

Finally, I am indebted to the almighty for presenting me with such wonderful opportunities and people in this life

Anil Govindrao Ghom

I dedicate my work to my caring devoted mother, Late Mrs Sulochana Jedhe to whom I owe special idiosyncratic gratitude throughout my life I express my profound thankfulness to my enthusiastic, dynamic and empathetic husband

Dr Ashok Mhaske (Vice Dean, Professor and Head, Department of Surgery, People’s College of Medical Sciences and Research Centre); my compassionate son Sumedh; benevolent father Shri Shrikant Jedhe (Retd), Additional Registrar, Cooperative Societies, Maharashtra Government; munificent parents-in-laws Anna and Aai (Shri NS Mhaske and Mrs Rukhmini Mhaske); knit family ties Niket, Samir (Engineer), Sharayu Tayade (Engineer), Shirin, Dr Mandakini,

Dr Maya and Advocate Dharmanand

I extend my indebted thankfulness to all who contributed especially my colleagues, relatives, friends, and dear students Each one provided extremely and distinctly valuable support for the completion of this work My special thanks

to Honorable Suresh Vijaywargiya, Chairman, People’s Group, Bhopal; Ms Megha Vijaywargiya, Director (Human Resource), People’s Group, Bhopal, Madhya Pradesh, India, for the inestimable support and my colossal inspiration;

Dr Jagdish V Tupkari, Professor and Head, Department of Oral Pathology and Microbiology, Government Dental College and Hospital, Mumbai, Maharashtra, India; Dr Suresh Barpande, Dean, Government Dental College and Hospital, Aurangabad; Dr Vinay Hazare, Dean, Government Dental College and Hospital, Nagpur, Maharashtra, India; Dr MK Gupta, Dean, People’s Dental Academy, Bhopal, Madhya Pradesh, India; Dr Alka Kale, Dean, KLE Institute of Dental Sciences, Belgaum, Karnataka, India; Dr PV Wanjari and Dr Sangeeta Wanjari, Professor and Head, Department of Oral Pathology, Modern Dental College, Indore, Madhya Pradesh, India

Shubhangi Mhaske (Jedhe)

1 MICROSCOPE

ShubhangiMhaske(Jedhe)

Definition of Microscope 1; History of Microscope 1; Simple Microscope3;

Compound Microscope 3; Parts of Microscope 3; Image Formationin

Microscope 10; Specialized MicroscopyTechniques 11; Maintenance of Laboratory

Microscope 19

1

2 TISSUE PROCESSING METHODS

ShubhangiMhaske(Jedhe) , Avadhoot Avadhani

Introduction and Terminology 22; Gross Examination 22; PreparationofTissue Specimen

for HistologicalStaining 23; RoutineMethod for Histological Study 25; Study ofHard

Tissues 30; Frozen Sections 32; Staining of Cut Sections 32; Mounting 33; Artifacts

in HistologicalSections 33

22

3 HISTOLOGICAL STAINING METHODS

ShubhangiMhaske(Jedhe) , Amol Gadbail

Chemistry of Stains 36; Classification of Stains 37; Theoriesof Staining 38; Vital

Staining 39; Factors Affecting Staining 39; StainingProcedure 40; Hematoxylin and

Eosin Stains 40; Special Stains 42; Gordon andSweets'MethodforReticulin Fibers 43

36

4 DIAGNOSTIC PATHOLOGY

ShubhangiMhaske(Jedhe) , PradnyaLele

Biopsy 47; TypesofBiopsyProcedures 48; Exfoliative Cytology 51; Oral

Mucosal BrushBiopsy 54; Liquid BasedCytology 55; FineNeedle Aspiration

Cytology 55; Frozen Section Biopsy 56

47

5 ADVANCED DIAGNOSTIC TECHNIQUES

ShubhangiMhaske(Jedhe) ,Monal Yuwanati

Histoche mi cal Techniques 57; Fixationin Histochemistry 57; EnzymeHistochemistry 58;

Immunohistochemical Methods 58; ImmunofluorescentTechniques 59;

FlowCytometry 61; Polymerase ChainReaction 61; Hybridization Methods 62;

Laser CapturesMicrodissection 63; Proteomics 63; Cytogenetics 64

57

6 HEALINGOFWOUND

ShubhangiMhaske(Jedhe)

FactorsAffecting the Wound Healing 68; Cascade of Wound Healing 72;

Healingof BiopsyWounds 74; Healing ofExtractionWounds 74; Healing of

68

Fractures 75; Healing ofOsseointegratedImplants 76; Healing of Pulp 76;

Cementum 77; Dentin 77; Enamel 77; Skin Healing and Oral Mucosal Wound

Healing 77; AClinical Approach toOptimizingWound Healing 79

82

7 HYPERPLASIA, HAMARTOMA ANDNEOPLASM

ShubhangiMhaske(Jedhe)

Dysplasia 82; Metaplasia 82; Hyperplasia 82; Hamartoma 83; Choriostoma 84;

Neoplasm 85; Carcinogenesis 87; ChemicalCarcinogenesis 87; Physical

Carcinogenesis 88; HormonalCarcinogenesis 89; BiologicCarcinogenesis 89;

Metastasis 92; GradingandStagingofTumors 93

8 TEETH ANOMALIES

AnilGovindraoGhom , ShubhangiMhaske(Jedhe) ,Savita Ghom

Disorders of Development of Teeth 97; Scale ofHumanTooth Development 98; Disorders

ofSizeof Teeth 98; Disturbances inShapeof Teeth 99; DisordersofNumberof

Teeth 109; Structureof Teeth 111

97

9 CRANIOFACIAL ANOMALIES

Anil Govindrao Ghom, Shubhangi Mhaske(Jedhe)

Developmental Anomalies ofJaws 128; Developmental Disorders of OralMucosa 138

111

10 DENTAL CARIES

AnilGovindraoGhom , ShubhangiMhaske(Jedhe)

Theories ofCariogenesis 145; Secondary Contributing Factorsin DentalCaries 150;

Classification 152; SmoothSurface Caries 153; PitandFissure Caries 156; Root

Caries 158; Recurrent Caries 160; ChemicalMeasures of CariesControl 164

144

AnilGovindraoGhom,ShubhangiMhaske(Jedhe)

Characteristicsof Benign Tumor 168; ClassificationofBenign Tumor 168; Epithelial

Origin 168; FibrousConnective Tissue 178; Cartilage 184; AdiposeTissue 186;

Bone 189; VascularTissue 196; NeuralTissue 203; Muscle 211; GiantCell

Lesion 213

12 PREMALIGNANT LESIONSAND CONDITIONS

AnilGovindrao Ghom, ShubhangiMhaske(Jedhe)

ConceptofPrecancer 219; Terminology andDefinitions 219; Leukoplakia 220;

Erythroplakia 229; Carcinomain situ 231; OralLesionAssociated withuseof

Tobacco 233; Lichen Planus 235; Oral Submucous Fibrosis 243; Dyskeratosis

Congenita 249; LupusErythematosus 250

219

13 MALIGNANT TUMORS

Anil Govindrao Ghom, Shubhangi Mhaske(Jedhe), AshokMhaske

Classification 256; Etiology and RiskFactorsfor OralCancer 256; RiskFactors 258;

EpithelialTumors 258; Metastatic Carcinoma 266; Basal CellCarcinoma 267;

AdenosquamousCarcinoma 269; BasaloidSquamous Carcinoma 269; Sinonasal

Undifferentiated Carcinoma 270; Verrucous Carcinoma 271; Transitional Cell

Carcinoma 273; Malignant Melanoma 273; SpindleCellCarcinoma 277;

AdenoidSquamousCellCarcinoma 277; NasopharyngealCarcinoma 278; Merkel Cell

Carcinoma 279; FibrousConnective Tissue 279; Malignant Fibrous Histiocytoma 282;

SynovialSarcoma 282; AdiposeTissue 283; Cartilage 284; Mesenchymal

Chondrosarcoma 287; Bone 287; Ewing's Sarcoma 289; Vascular 291; Neural

Tissue 292; Muscle 294

255

14 ODONTOGENIC TUMORS

AnilGovindrao Ghom , ShubhangiMhaske(Jedhe)

ClassificationofOdontogenic Tumors 299; Development of Tooth 301; Stagesof Tooth

Development 301; Ameloblastoma 303; Variant ofAmeloblastoma 313;

Squamous Odontogenic Tumor 317; CalcifyingEpithelialOdontogenic Tumor 318;

AdenomatoidOdontogenic Tumor or Cyst 321; MixedOdontogenic Tumors 325;

Continuum Concept( Cahn and Blum ) 325; Ameloblastic Fibroma 326;

Ameloblastic Fibrodentinoma 328; Ameloblastic Fibro-odontoma 328; Odontoma 329;

Odontoameloblastoma 331; OdontogenicFibroma 332; Granular CellOdontogenic

Tumor 333; OdontogenicMyxoma 333; MalignantTumors 335

299

15 CYSTOF OROFACIAL REGION

AnilGovindrao Ghom , ShubhangiMhaske(Jedhe)

Classification 343; Theoriesof CystEnlargement 343; Dentigerous Cyst 345;

EruptionCyst 349; OdontogenicKeratocyst 350; Primordial Cyst 355; Gingival Cyst

ofNewborn 355; Gingival Cyst ofAdult 356; Lateral Periodontal Cyst 357; Glandular

Odontogenic Cyst 358; CalcifyingEpithelialOdontogenicCyst 359; Inflammatory

RadicularCyst 361; ResidualCyst 364; Inflammatory CollateralCyst 365; Paradental

Cyst 365; Mandibular Buccal Infected Cyst 366; SuppuratingCyst 366; Healing

Cyst 366; Nonodontogenic Cysts 366; Median PalatineCyst 368; Nasoalveolar

Cyst 369; Median Mandibular Cyst 370; Globulomaxillary Cyst 370; Nonepithelial

Cysts 370; AneurysmalBoneCyst 371; Cystsofthe MaxillarySinus 372; Antral

Pseudocyst 374; Retention Cyst 374; SoftTissueCyst 374; BranchialCleft

Cyst 376; Oral LymphoepithelialCyst 376; ThyroglossalDuct Cyst 377; Anterior

Median LingualCyst 377; Oral Cyst withGastric orIntestinal Epithelium 377; Cystic

Hygroma 377; FollicularCystsof the Skin 378; Nasopharyngeal Cyst 378; Thymic

Cyst 378; Cysts of Salivary Glands 378; ParasiticCyst 378; Cysticercosis

Cellulose 379; Syndromes Associated withOdontogenicCysts 380; Treatment of

Cysts 381

342

16 PERIODONTAL PATHOLOGY

Vivek Thombre, AnilGovindraoGhom , ShubhangiMhaske(Jedhe)

FibromatosisGingiva 386; Retrocuspid Papilla 388; GingivalInflammationor

Gingivitis 388; NecrotizingUlcerativeGingivitis 391; Desquamative Gingivitis 392;

Plasma CellGingivitis 393; GranulomatousGingivitis 394; GingivalAbscess 394;

Pericoronal Abscess 395; ChronicInflammatoryEnlargement 396; GingivalEnlargement

due toDrugs 397; Pregnancy Tumor 398; GranulomaPyogenicum 399;

Periodontal Pockets 401; Adult Periodontitis 402; RapidlyProgressivePeriodontitis 402;

Aggressive Periodontitis / Juvenile Periodontitis 403; Papillon-LefevreSyndrome 405;

Haim-Munk Syndrome 406

386

17 SALIVARY GLAND PATHOLOGY

Anil Govindrao Ghom , ShubhangiMhaske(Jedhe)

Classificationof Salivary Gland Disorders 410; DevelopmentofSalivary

Gland 410; MajorSalivary Glands 411; Aberrancy 412; Aplasia and

Hypoplasia 412; Hyperplasia of Salivary Gland 413; Atresia 413; Accessory

Duct 414; Diverticuli 414; Sialorrhea 414; Xerostomia 414; Sialolithiasis 415;

StricturesandStenosis 417; Mucocele (Mucous ExtravasationPhenomenon ) 417; Salivary

DuctCystor Mucus RetentionCyst 419; Ranula 419; Sialosis ( Sialadenosis ) 420;

Allergic Sialadenitis 421; Mumps 421; CytomegalovirusInclusionDisease 422;

Bacterial Sialadenitis 422; Sjogren s Syndrome 424; Mikuliczs Disease or

BenignLymphoepithelialLesion 427; UveoparotidFever 428; Tumorsof

Salivary Glands 428; Histogenesis 428; TheoriesofSalivary GlandTumor

Histogenesis 429; GeneralFeatures ofSalivary GlandTumors 429;

ClinicalStagingof Salivary GlandTumors 429; Pleomorphic Adenoma 430;

Basal Cell Adenoma 433; Canalicular Adenoma 434; Warthin 's Tumor 435;

Oncocytoma 437; Myoepithelioma 438; Ductal Papillomas 438; Mucoepidermoid

Carcinoma 440; Central MucoepidermoidCarcinoma 444; Acinic Cell

Adenocarcinoma 444; AdenoidCystic Carcinoma 445; PolymorphousLow

-Grade Adenocarcinoma 447; Malignant MixedTumor 448; Connective Tissue

Tumors 449; Necrotizing Sialometaplasia 450

409

18 BACTERIAL INFECTION

AnilGovindrao Ghom , ShubhangiMhaske(Jedhe)

Impetigo 453; Erysipelas 454; Syphilis 455; Gonorrhea 460; Leprosy(Hansen

Disease) 462; Tuberculosis 465; Actinomycosis 468; Noma 471; ScarletFever 472;

Diphtheria 473; Tularemia 474; Rhinoscleroma 474; Granuloma Inguinale 475; Oral

Manifestations 475; StreptococcalTonsillitis and Pharyngitis 476; TonsillarConcretion

and Tonsillolithiasis 476; LymphogranulomaVenereum 476; Myiasis 477; CatScratch

Disease 478; PyostomatitisVegetans 479; Sinusitis 479

453

19 FUNGALOR MYOCOTIC INFECTION

Anil Govindrao Ghom , ShubhangiMhaske(Jedhe)

Candidiasis 484; Oral Candidiasis 486; ChronicMucocutaneousCandidiasis 491;

FormsofCandidiasis 491; Histoplasmosis 492; Blastomycosis 493; Mucormycosis 495;

484

Cryptococcosis 496; Coccidioidomycosis 497; Geotrichosis 498; Sporotrichosis 498;

Rhinosporidiosis 499; Aspergillosis 500; Paracoccidioidomycosis 501;

Toxoplasmosis 502; Leishmaniasis 502; Trichinosis 503

20 VIRAL INFECTION

AnilGovindraoGhom , ShubhangiMhaske(Jedhe)

Human Herpes Virus 505; Herpes SimplexInfection 505; Measles 509; VaricellaZoster

Infection 510; Herpes Zoster 512; JamesRamseyHunt Syndrome 514; Rubella 514;

Enteroviruses 514; Footand MouthDisease 516; Condyloma Acuminatum 517;

VerrucaVulgaris 518; Focal Epithelial Hyperplasia 518; MolluscumContagiosum

Infection 519; CytomegalovirusInfection 520; InfectiousMononucleosis 521

505

AnilGovindraoGhom , ShubhangiMhaske(Jedhe)

Classification 525; AIDSRelatedComplex 526; Prevalence 526; Characteristic of

HIV Virus 527; ClinicalFeatures 527; OralManifestations 528; UncommonOral

Manifestation of HIV 534; Diagnostic Tests 536; Screening Test forAIDS 536;

Management 537; Prevention 538

524

22 ODONTOGENIC INFECTIONANDPULP PATHOLOGY

Anil Govindrao Ghom, Shubhangi Mhaske(Jedhe) ,SeemaVaidya

Effect of Infectionon Host 541; Pathophysiology of Infection 541; Pulp 542;

Classificationof Pulpitis 542; Pulpitis 542; PulpDegeneration 545; Pulp

Calcifications 546; Necrosis ofPulp 548; Cracked Tooth Syndrome 549; Periapical

Abscess 549; Periodontal Abscess 551; Acute Exacerbation of a ChronicLesion 552;

Periapical Granuloma 553; Osteomyelitis 555; Acute SuppurativeOsteomyelitis 557;

ChronicSuppurativeOsteomyelitis 559; Infantile Osteomyelitis 560; Synovitis, Acne,

Pustulosis,Hyperostosisand OsteomyelitisSyndrome 562; ChronicRecurrentMultifocal

Osteomyelitis 563; Cellulitis 565; Ludwig 'sAngina 566; FatalComplications ofOral

Infection 568; OralFociof Infections 571; Dry Socket 573

540

23 BONE DISEASE MANIFESTEDIN JAW

Anil GovindraoGhom , ShubhangiMhaske(Jedhe) , PranotiPradhan

Fibro- osseous Lesions 576; Classification 576; Fibrous Dysplasia 577; Cherubism 580;

CentralGiantCell Granuloma 582; Paget's Disease 584; FamilialGigantiform

Cementoma 587; OssifyingFibroma,CementifyingFibroma andCemento - ossifying

Fibroma 588; JuvenileOssifyingFibroma 590; Osteoporosis 591; InfantileCortical

Hyperostosis 593; Osteopetrosis 594; Osteogenesis Imperfecta 596; PierreRobin

Syndrome 597; Marfan'sSyndrome 597; Down'sSyndrome 598; Achondroplasia 599;

Osteosclerosis 600; MassiveOsteolysis 600; Gardner 'sSyndrome 601

576

24 DISEASES OF LIP

Anil GovindraoGhom , ShubhangiMhaske(Jedhe)

ClassificationofLipDisorders 604; Anatomy 604; Developmental Disturbance of

Lip 605; Cheilitis 610; Etiology 610; Miscellaneous 617

604

25 TONGUE DISORDERS

AnilGovindraoGhom , ShubhangiMhaske(Jedhe)

Embryology ofTongue 619; Anatomy ofTongue 620; Papillae 620; Muscle 621;

ArterialSupply 621; Venous Drainage 621; Nerve Supply 621;

LymphaticDrainage 622; FunctionsofTongue 622; Classification of Tongue

Disorders 623; Aglossia and Microglossia 623; Macroglossia 624;

Ankyloglossia 625; Cleft Tongue 626; AnkyloglossumSuperius Syndrome 626; Lingual

Varicosities 627; Lingual Thyroid Nodule 627; VariationsinTongue Movement 628;

PatentThyroglossalDuct Cyst 628; Lingual Polyp 629; LingualCyst 629; Fissured

Tongue 629; Median Rhomboid Glossitis 630; Benign MigratoryGlossitis 631; Hairy

Tongue 633; CrenatedTongue 634; Foliate Papillitis 634; LeukokeratosisNicotina

Glossi 634; Depapillation of theTongue 635; DysgeusiaandHypogeusia 637;

Dyskinesia 638; Paralysis ofTongue 638; SquamousCellCarcinoma 639;

PigmentationofTongue 640

619

26 TEMPOROMANDIBULAR JOINT PATHOLOGY

AnilGovindraoGhom, ShubhangiMhaske(Jedhe)

Coronoid Hyperplasia 643; CondylarHyperplasia 643; CondylarHypoplasia 644;

BifidCondyle 645; Osteoarthritis 645; Rheumatoid Arthritis 646;

Ankylosis 648; Subluxation ( Hypermobility ) 650; Gout 651; Synovial

Chondromatosis 652; TemporomandibularJoint Dysfunction 652

643

27 CHEMICAL ANDPHYSICAL INJURIES

Anil GovindraoGhom , ShubhangiMhaske(Jedhe)

LineaAlba 655; Habitual Cheekor Lip Biting 656; TraumaticUlcer 657; Electrical

and ThermalBurns 658; AnestheticNecrosis 659; ChemicalBurns 659; Smoker

Melanosis 661; DrugInduced Discoloration of OralMucosa 661; Outright

Lesion 662; Traumatic Sequestration 662; Methamphetamine Abuse

Lesion 663; Submucosal Hemorrhage 663; OralLesion as Complicationto Anti

-Neoplastic Therapy (Non -Infectious ) 664; CervicofacialEmphysema 665;

Myospherulosis 666; Attrition 666; Abrasion 668; Erosion 669; Abfraction 670;

Dentinal Sclerosis 671; Secondary and TertiaryDentin 671; Resorptionof Teeth 672;

Hypercementosis 675; Cementicles 676; Bruxism 676; Traumatic Lesion

DueSexual Habit 678; OralPiercingand other BodyModification 679;

Fractureof Teeth 680; AmalgamTattoo 681; Bismuthism 681; Plumbism 682;

Mercurialism 683; Argyria 684; Arsenism 685 AuricStomatitis 685; Inflammatory

Fibrous Hyperplasia 685; Inflammatory Papillary Hyperplasia 686; Epulis

Granulomatosum 687; NodularFasciitis 688; Uremic Stomatitis 688; Traumatic

Keratosis 689; Bisphosphonates AssociatedOsteonecrosis 689

655

28 BLOOD PATHOLOGY

AnilGovindraoGhom , ShubhangiMhaske(Jedhe)

Disease ofLymphTissue 693; Diseaseof Red Blood Cells 694; White

Blood Cell Disorders 705; Disease ofPlatelet 708; Diseasedue toClotting

Defect 710; Dysfibrinogenemia 713; Macroglobulinemia 714; Malignancy Involving

693

BloodTissue 714; Primary Reticular CellSarcoma 717; Mycosis Fungoides 718;

Burkitt’sLymphoma 718; Chronic Myeloid Leukemia 723; Chronic Lymphatic

Leukemia 724; Multiple Myeloma 725; Plasmacytoma 727; ExtranodalNK/T -Cell

Lymphoma 728

29 SKIN DISORDERS

Anil GovindraoGhom , ShubhangiMhaske(Jedhe)

Erythema Multiforme 731; Pemphigus 734; Paraneoplastic Pemphigus 737;

Bullous Pemphigoid 737; Benign MucousMembrane Pemphigoid 738; Familial

BenignChronic Pemphigus 740; DermatitisHerpetiformis 742; Pityriasis

Rosea 743; Incontinentia Pigmenti 743; AcanthosisNigricans 744; Ehlers Danlos

Syndrome 745; Psoriasis 746; PachyonychiaCongenita 747; Porokeratosis 748;

KeratosisFollicularis 749; Warty Dyskeratoma 750; SeborrheicKeratosis 750;

Hereditary Mucoepithelial Dysplasia 751; Pseudoxanthoma Elasticum 751; Hyalinosis

Cutis Et Mucosa Oris 752; White Sponge Nevus 753; HereditaryBenignIntraepithelial

Dyskeratosis 754; Hereditary Hemorrhagic Telangiectasia 754; Peutz -Jeghers

Syndrome 755; Ephelis 755; ActinicLentigo 756; Lentigo Simplex 756; Sebaceous

Hyperplasia 756; XerodermaPigmentosum 757; Tuberous Sclerosis 757; Ectodermal

Dysplasia 758; Cowden Syndrome 760; Graftversus Host Resistance 760; Crest

Syndrome 761; Scleroderma 761; Kawasaki Disease 763

731

30 ALLERGICAND IMMUNOLOGIC DISEASES OF ORAL CAVITY

Anil GovindraoGhom , ShubhangiMhaske(Jedhe)

Introduction /Overview 766; Hypersensitivity Reaction 766; Wegner’s

Granulomatosis 767; Sarcoidosis 769; DrugAllergy 770; AllergicContact

Stomatitis 771; SecondaryVaccinia 772; Angioedema 773; AphthousStomatitis

(RecurrentAphthous Ulcers ( RAUs )orCankerSores) 774; Behqet ’s Syndrome 776;

TransientLingual Papillitis 777; PerioralDermatitis 778; Reiter’sSyndrome 778;

LichenoidContact Stomatitis/ LichenoidTissue Reaction 779; Chronic Ulcerative

Stomatitis 781; Crohn ’s Disease 782

766

31 ENDOCRINE DISORDERS

AnilGovindraoGhom , ShubhangiMhaske(Jedhe)

Anatomy and Physiology 784; Diseasesof Pituitary Gland 785; Progeria 788;

Hyperthyroidism 788; Hypothyroidism 790; Hyperparathyroidism 791;

Hypoparathyroidism 793; Pseudohypoparathyroidism 793; Diabetes Mellitus 794;

Addison 's Disease 796; Adrenogenital Syndrome 797; Melasma 797; Cushing’s

Syndrome 797

784

32 NUTRITION ANDORAL CAVITY

AnilGovindraoGhom , ShubhangiMhaske(Jedhe)

Disturbances inProteinMetabolism 800; Disturbances in Lipid

Metabolism 804; Disturbances in Carbohydrate Metabolism 807; Disturbances in

Mineral Metabolism 809; Miscellaneous Disorders 811; FatSolubleVitamins 821;

Disorders of Bilirubin 825

800

33 NEUROMUSCULAR DISORDERS AND OROFACIAL PAIN

AnilGovindraoGhom , ShubhangiMhaske(Jedhe)

Muscle Disorders 830; Neuromuscular Disorders 834; FacialPain 839

830

34 FORENSIC ODONTOLOGY

Anil Govindrao Ghom,Savita Ghom

RecordManagement 849; Identification 850; Dental Evaluation 850; Personal

Recognition 853; Fingerprinting 853; Physical AnthropologicExamination of Bonesand

Teeth 853; Postmortem Serologyand DNAProfiling 854; BiteMarks 854; Human

Abuse 858; Dentist as Expert Witness 859

849

35 SYNDROMES OF THE OROFACIAL REGION

ShubhangiMhaske(Jedhe)

Syndromes Associated with Craniofacial Anomalies ofGenetic Origin 862; Syndromes

Associated with Skin andPigmentation 868; BroadGroupsofPigmentaryDisorders 871;

Syndromes Associated with Salivary and Lacrimal Glands 872; SyndromesAffecting

Teeth 873; Syndromes Associated withLipsand Cheek 875; Syndromes Associated

withTongue 876; Syndromes Associated withGingiva 877; Syndromes Associated with

Nerves 878; SyndromesAssociated with Blood 880; Syndromes Associated with Vascular

Malformations 882; SyndromesAssociated withImmunodeficiency 882; Syndromes

Associated with Hormonal Disturbances 883; Syndromes withBenignOral Neoplasticor

3 Imageformationin microscope

3 Specialized microscopytechniques

• Stereomicroscope

• Darkfield microscope

• Phasecontrastmicroscope

Thelight microscope, now 400 years old,is the standard

instrumentforthe examinationof histological preparations.

The word microscope is derived from two Greek words

micro meaning small and scope meaningto view Thus,it

is an instrument which enables usto view small objects.

Itmagnifies (enlarges)the image ofthat small objectand

thus makes it possibletobeseen by theviewer.

HISTORY OF MICROSCOPE

( FIGS 1.1 AND 1.2 )

The early pioneers in the history of the microscope are

Digges of England and Hans and Zachcharias Janssen

(1590) of Holland, Robert Hooke (1665), John Marshal

(1700), Martin Frobenius Ledermuller (1768), Louis

Jablot (1755), Meyen (1747). But it was Antony van

Leeuwenhoek who was the first to make and use a real

microscope (Table1.1)

Early microscopes were simple microscopes but withadvanceof science compound microscopes werebuilt

DEFINITION OF MICROSCOPE

An optical instrument that uses a lens or a combination

of lenses to produce magnified images of small objects,

especiallyofobjectstoosmalltobeseenby the unaidedeye.

Figure 1.2 Robert Hooke microscope

Table 1.1 Contributors in history of microscope

Year Inventor/Scientist Contribution

Circa 1000AD Unknown Reading stone, A glass sphere that magnified when laid on top of

reading materialCirca 1284 Salvino D’Armate (Italy) Inventing the first wearable eye glasses

Hans and Zachcharias Janssen (Holland)

Multiple lenses placed in a tube magnified object kept in front

Robert Hooke (England)

Looked at a sliver of cork through a microscope lens and noticed some “pores” or “cells” in it

1674 Antony van Leeuwenhoek

(Father of Microscopy)

Built a simple microscope with only one lens to examine blood, yeast, insects and many other tiny objects

1830 Joseph Jackson Lister Reduction of spherical aberration or the “chromatic effect” by lens

combinations without blurring the image for compound microscope

microscopes

1981 Gerd Binnig and Heinrich Rohrer Scanning tunneling microscope

Figure 1.1 Antony van Leeuwenhoek microscope

Fluorescent microscope: Uses ultraviolet light with a

shorter wavelength below 400 mm which a light microscope cannot It can demonstrate, with the help of fluorochrome dyes High pressure mercury lamp, halogen lamps are used generate ultraviolet light Light source used in fluorescent microscope is different, i.e in modern microscope high intensity illumination systems are used They should to be used with specialized filters for protection of eyes

Electron microscope: This technique of microscopy

is different from light microscopy as it uses a stream of electrons in a magnetic field This stream of electrons has

a very short wavelength (a 50 KV electron beam produces light of 0.0055 nm) This is one hundred thousandth that of the visible light

Illumination

In light microscope two different types of illuminations are used (Figs 1.4A and B)

Critical illumination: When the object and light source

from the substage condenser is in the same plane it is called

as the critical illumination, as commonly used in simple equipments, but this produces uneven illumination of the object though modern filament lamps are used

Kohler illumination: This is used for specialized type of

microscopy where an image of the light source is focused

by the lamp collector or field lens in the focal plane of the condenser The image of the field or lamp diaphragm is

SIMPLE MICROSCOPE

A simple microscope consists of a single lens or a

magnifying glass

Principle: A lens of short focal length is used to produce an

enlarged image of an illuminated object at a short distance;

the lens fixed in a frame is adjustable to view the object

The shorter the focal length, the larger the magnified image

COMPOUND MICROSCOPE

(FIGS 1.3A AND B)

A compound microscope consists of two or more lenses

Principle: If a lens of short focal length is used to produce

an enlarged image of an illuminated object at a short

distance, then another lens can be so fixed that it would

produce a further enlargement of that image

The light microscope uses natural daylight or artificial

visible light (The resolution of light microscope is limited

by wavelength of its light source) A progression of light

sources has developed from oil lamps to the low voltage

electric lights of today

In histopathology laboratories microscopes with three

different types of light sources are found, the conventional

light microscope using natural or artificial visible light,

the fluorescence microscope using ultraviolet light, and

electron microscope using a beam of electrons

Light microscope: Uses a visible light of 400 to 800 nm

wavelengths to illuminate an object up to 0.2 mm made

visible with perfect optics given in a microscope In light

microscope two different types of illuminations are used

Figures 1.3A and B The first compound microscope

focused in the object plane and the aperture diaphragm is

in turn focused at the back focal plane of the objective and

can be examined with the eyepiece removed

Lens

It is named lenses because shaped like the seeds of lentil

Piece of glass or other transparent material, usually circular,

having two surface ground or polished in a specific form in

order that light ray passing through it either converges or

diverges There are two types of lens which are used They

are (Fig 1.5):

∙ Positive: It can be convex shaped and converges rays

of light forming real image

∙ Negative: It is concave shaped and diverge rays and

forms virtual image

Condensers

Light from the lamp is directed into the first major optical component—the sub stage condenser-either directly or from a mirror or prism

The main purpose of the condenser is to focus or concentrate the available light into the plane of the object, i.e the condenser collects the maximum possible light reflected by the mirror or the inbuilt light source and condenses or converges it to a very small area at the position of the specimen

Condensers used for routine microscopy should have the same numerical aperture The ideal condenser should form a true image of the light source It is practically useful

to have a condenser with a top lens that can be swung out

of the path of light, thus filling the whole field with light when very low power objective are used (Fig 1.6) Three types of condensers are used

Abbe condenser: Named after Ernst Abbe It is simplest

and least expensive type Because of its simplicity and good light gathering capacity, it is used with most microscopes unless specified otherwise; it has an NA of 0.25 It consists

of two lens elements (Figs 1.7A to C) Abbe condenser is not corrected for spherical and chromatic aberration but serves well for general observation Some types of Abbe condensers are “variable focus condensers” in which the upper lens element is fixed and lower lens is focusable When the lower element is raised to it is to position it is

Figure 1.5 Types of lenses used in optical components of

microscope

Figure 1.6 Parts of condenser

Figures 1.4A and B Critical and Kohler illumination

similar to the above condenser But when its position is

lowered, light is focused in between the elements, thus the

light can emerge as a large diameter parallel bundle For

10x the field area is larger To illuminate this large area the

top lens element is removed to achieve the illumination of

entire field For medium and high magnification the top lens

element of the variable focus condenser remains in place

Aplanatic condensers: These types of condensers are

optically corrected for spherical aberration These are not

available form all microscope manufacturers, but are of

better quality than Abbe condensers (Figs 1.7A to C)

The achromatic condensers: These are corrected for both

spherical and chromatic aberrations It has NA of 1.40

Because of its high degree of correction, it is recommended

for research microscopy and for color photomicrography

where the highest degree of perfection in the image is

desired (Figs 1.7A to C)

Object Stage

A rigid platform above the condenser which supports the

glass slide is object stage This object stage has an aperture

in the center through which the light can pass to illuminate

the specimen on the glass slide (Fig 1.8)

The stage holds the slide firmly and allows the slide

movements with a mechanical vertical and horizontal

adjustment screws The mechanical stage is graduated

with Vernier scales and the x and y movements assist

the operator to return to an exact desired location in the

specimen Traveling range in most of the microscopes is

Every objective has a fixed working distance, focal length, magnification and numerical aperture (NA) (Fig 1.9)

The working distance is the distance between an object

in focus and the front of the lens system

The focal length is the distance from the center of

a simple lens to the point at which parallel rays of light are brought to a sharp focus; in the compound lens it

is the distance between an object in focus and a point approximately halfway between the component lenses

Figure 1.8 Parts of mechanical stage

Magnification

It is product of magnification values of eyepiece and

objective in a standard microscope

Magnification in a standard microscope with tube

length of 160 mm is calculated using the formula:

Magnification = Tube length

Focal length of objectiveFor microscopes with tube length other than 160 mm:

Magnification = Tubelength × eyepiece magnification

Focal length of the objectiveMagnification for low power objective with focal

length 16 mm and standard tube length of 160 mm is:

Magnification = 160/16 = 10

Color Codes

Microscope manufacturers label their objectives with color

codes to help in rapid identification of the magnification In

addition to color coding other information is also embossed

on the objective (Table 1.2)

Numerical Aperture

The ability of the lens to distinguish fine structural adjacent

details in a specimen is known as the resolving power This

ability is expressed in terms of numerical aperture, as NA,

as it is usually called

Numerical aperture depends primarily on the extreme range of the divergent rays that can be made to admit

into the lens (angular aperture) and secondarily on the

refractive index of the medium between the object and the objective The relation between numerical aperture, angular aperture and refractive index is

NA = Refractive index × sine angular apertureThe numerical aperture for any objective is always imprinted on its mount A 10x achromatic objective usually has a numerical aperture of 0.25 and a 20x achromat will usually have a numerical aperture of 0.50; apochromatic objectives have higher numerical apertures than achromats (Fig 1.9 )

Resolution

It is the smallest distance between two dots or lines that can

be seen as separate entities It depends on the wavelength

of light and the NA of the lens As the NA of the objective increases, the resolving power increases It is calculated as:

Types of Objectives (Figs 1.10A to C)

In most modern microscopes objectives are usually made

up more than one lens This series of lenses is used to overcome certain limitations in the lenses, i.e

Optical aberrations: Aberration is the failure of a lens to

produce exact point to point correspondence between an object and its image Every lens system has an aberration

to a greater or lesser extent To improve the image quality, the lenses are designed by combining different lens shapes

Table 1.2 Color codes used for objectives

Objective color codes Magnification Color code

and glass materials It is possible to construct compound

lenses of different glass elements to correct this fault An

achromat lens is corrected for two colors, blue and red,

producing a secondary spectrum of yellow/green This

secondary spectrum can be reduced by adding fluorite to

objective Such a lens is called as fluorite lens Fluorite

lenses need to be corrected for yellow green, which is done

by adding more lens components Such type of lens is

apochromat which is most expensive.

Chromatic aberration: White light is composed of all

the spectral colors on passing through a simple lens, each

wavelength will be refracted to a different extent, with blue

being brought to a shorter focus than red This defect of

lens is ‘chromatic aberration’ and results in an unsharp

image with colored fringes (Figs 1.11A and B)

The objective can be both ‘apochromat’ and ‘achromat’

types to correct these optical aberrations

Spherical aberration: It is caused when light rays entering

a curved lens at its periphery which are refracted more

than those rays entering the center of the lens and are not

brought to a common focus (Fig 1.12)

Different types of objectives are as followes:

Achromatic: Corrected for two colors red and blue It is

the most widely used for routine purposes

Fluorite: Green light is brought to a shorter focus and

violet light to a longer focus

Apochromat: All colors are brought into same focus It is

fully corrected for three colors By the design of the lens

and use of fluorite, the formation of a secondary spectrum

is almost completely eliminated and all colors are brought

to the same focus These lenses are used especially for

photomicrography and for screening cytological smears

Plan-achromat: Although histological sections are flat the

image produced by the microscope is not flat It is saucer shaped; it is not possible to focus the whole of the field sharply at any one time This aberration is corrected using flat-field objectives also called plan-achromat lenses

Nosepiece

In most modern microscopes up to six objectives are mounted on resolving nosepiece For rapid change of all objectives they should be at focus and they should focus the same central area of the section when brought into the position Such nosepieces are known as ‘par-focal’ and

‘par-central’

Mechanical Tube

Light from the objective is received into the bottom of the microscope body-tube From there it travels to the eyepiece

Figure 1.12 Spherical aberration

Figures 1.11A and B Chromatic aberrations and its

in a tube called mechanical tube The distance from

objective to eyepiece is called “mechanical tube length”

and it is defined as “the distance from the nosepiece

opening, where the objective is mounted, to the top edge

of the observation tubes where the eyepieces (oculars) are

inserted” In standard microscopes this is 160 mm, while in

few special purpose microscopes it is 170 mm

In most microscopes tube length cannot be altered Such

microscopes are called as finite length microscope In these

microscopes if additional filters such as polarizer, analyzer

fluorescent filters are used, tube length becomes more than

160 mm and aberrations will be introduced in the image

formed To overcome this limitation, most of the modern

microscopes use infinity corrected optics where image is

projected to the infinity In this system, tube length can be

altered without affecting the quality of the image

Infinity-corrected systems have the advantage of being easier to

design and also make possible the insertion of less costly

accessories in the “parallel” light path This advanced new

optical system allows microscopes to support complex

optical component clusters in the optical pathway between

the objective and the lens tube This is especially useful

for techniques such as confocal, polarized, DIC, and

fluorescence microscopy where specialized lens systems

must be employed for optimum results

Eyepieces

The purpose of an eyepiece in a compound microscope is

to enlarge the primary image formed by the objective, and

to render it visible as a virtual image in the microscope and

also to correct some of the defects of the objective

Huygenian eyepieces are the simplest form of

eyepiece in common use; they are cheap, but they are

not corrected for chromatic difference of magnification

Although, Huygenian eyepieces can be used with

low-power achromats, they give under-corrected curvature of

field and lateral colors with intermediate and higher power

objectives The other main kind of eyepiece is the positive

eyepiece with a diaphragm below its lenses, commonly

known as the Ramsden eyepiece These eyepieces are

corrected for chromatic aberration of magnification (Figs

1.13A and B)

Different Types of Eyepieces

Compensating eyepieces are compound lenses with

a chromatic difference of magnification which is equal

and opposite to that of high-power objectives They

Figure 1.14 Compensating eyepieces are required in

binocular microscopes

are essential for use with apochromatic objectives, but they also improve the performance of most high-power achromatic objectives Eyepieces for binocular microscopes must be accurately paired, with equal centration, magnification, and field in order to reduce eye strain Interocular distance should be accurately adjusted, and the microscopist should sit at the correct height for the eyepieces to come to the exact height of the observer’s eyes (Fig 1.14) Eyepieces, generally, are produced with different magnifying powers, ranging from about 4x to 25x The most common in use are those with

a magnifying power of 10x or 15x

Figures 1.13A and B Position of field lens and eye lens in

Ramsden and Huygenian eyepieces

Pointer eyepieces: A fine pointer could be incorporated

in the eyepiece in order to enable us to point out a certain

portion of the specimen Such types of eyepieces are called

as pointer eyepieces (Figs 1.15A and B)

Multihead demonstration eyepieces: Some types of

eyepieces are such that 4 to 5 people can view the same

portion of an object at a time This type is called ‘double

demonstration eyepieces’ (Fig 1.16)

Micrometry

The most common method of making such measurements

is the use of ocular micrometer and stage micrometer We

can make measurements in compound microscopes only in

the range of 0.2 to 25 mm We cannot measure dimensions

smaller than 0.2 mm because it is less than the resolving

power of a compound microscope Likewise, measurement

Figure 1.16 Multihead viewing microscope

Figure 1.17 Ocular micrometer/Graduated reticle

Figures 1.15A and B Pointer eyepieces are helpful in

locating area of interest

A

B

above 25 mm is also not practical because it will be above the average field diameter of a wide field eyepiece Larger objects can, however, be measured with a stereomicroscope.The ocular micrometer (OM) (Fig 1.17) is a glass disk with a diameter of 1 cm It is engraved with an arbitrary scale of 100 divisions or less It is also referred to as a reticle, reticule or graticule Since it is fitted into the eyepiece of the compound microscope it is more appropriate to call

it an ocular micrometer This is the scale that is used for all measurements Since the scale is arbitrary, is to be calibrated (standardize) using a known standard scale, the stage micrometer (SM)

A stage micrometer is a standard microscope slide having a scale of defined length Usually, the scale is

1 mm (1000 mm) divided into 100 divisions, so that one

stage micrometer division = 10 mm Such a microlevel

scale is made by methods such as photographic process,

physical engraving or electro-deposition of a metallic film

directly onto the glass surface A protective cover glass slip

is usually mounted on the scale The scale may be encircled

by a black line during use for easy location and focusing

under the microscope

The calibration of the ocular micrometer refers to

determination of the distance of one division in terms of the

absolute distance of a stage micrometer A simple Vernier

principle is used for this purpose How many of OM

divisions are equal to how many of the SM divisions under

a particular microscope–eyepiece–objective combination

is found out Suppose, it is found that 2 OM divisions are

equal to 1 SM division which means that 2 OM divisions

have a value equivalent to the absolute distance of 1 SM

division, i.e 10 mm This is given by 1 OM division = 10

mm/2 = 5 mm

This value is often known as micrometer value or

calibration factor Once this value has been determined, the

dimension of any specimen can be calculated by multiplying

the number of OM divisions spanned by the specimen

with the calibration factor It must be remembered that a

calibration factor only applies to a specific microscope–

eyepiece–objective combination (Fig 1.18)

IMAGE FORMATION IN MICROSCOPE

Real image: The real image in a microscope is formed

by the objective lens The image is formed at a greater

magnification, and is inverted This is when the object is

moved nearer the lens

Virtual image: If the object is placed still nearer the lens

within the principal focus, the image is formed on the same

side as the object, is enlarged, the right way up, and cannot

be projected onto the screen This is the virtual image The

eyepiece in a microscope forms the virtual image of the

real image projected by the objective

The microscopy utilizes transmitted and reflected

light for image formation Transmitted light is light which

passes through the object or specimen from source below

and image is create in eyepiece after passing through the

objective Whereas when light reflects from a smooth

surface, the incoming light is referred to as an incident

light and the light that is bounced away from the surface

is termed the reflected light Reflection of light occurs

when the light come upon an object surface that does

not absorb the light and bounces the light away from the

surface The reflection of visible light is a property of the behavior of light that is fundamental in the function of majority of today microscopes Light is often reflected by one or more plane or flat mirrors within the microscope

to direct the light path through lenses that form the virtual images which is visible in the eyepieces Other optical components in the microscope, such as prisms, filters, and lens coatings, also carry out their functions in forming the image with a crucial dependence on the phenomenon of light reflection

A transmitted light microscope will typically be of little use to anyone wanting to examine the structure of biological specimen As a result, the reflected light microscope has been developed for these purposes Reflected light microscopy

is often referred to as incident light, epi-illumination, or metallurgical microscopy (Mostly used in Metallurgy studies), and is the method of choice for fluorescence and for imaging specimens that remain opaque (Fig 1.19)

In reflected light microscopy, the pathway for reflected light begins with illuminating rays originating in the lamp housing for reflected light This light next passes through the collector lens and into the vertical illuminator where

it is controlled by the aperture and field diaphragms After passing through the vertical illuminator, the light is

Figure 1.18 OM (Ocular micrometer) (eyepiece scale) is coincided with the SM (Stage micrometer)

then reflected by a beamsplitter through the objective to

illuminate the specimen Light reflected from the surface

of the specimen re-enters the objective and passes into the

binocular head where it is directed either to the eyepieces

or to a port for photomicrography (Fig 1.20)

The principal focus or focal point is the single point

where the parallel rays of light entering the lens are

brought together by refraction The focal point is the point

where the clear image of an object is formed Focal length

is the distance between the optical center of the lens and the

Stereomicroscopy (Figs 1.21A and B)

It is optical microscope designed for low magnification observation or a sample using incident light illumination rather than transillumination It uses two separate optical paths with two objectives and two eyepieces to provide slightly different viewing angles to the left and right eyes Stereomicroscopy overlaps macro photography for recording and examining solid samples with complex surface topography where a three-dimensional view is essential for analyzing the detail The stereo microscope

is often used to study the surfaces of solid specimens or to carry out close work such as grossing of specimen, etc

Dark Field Microscopy

Dark field microscopy is an optical microscopy illumination technique used to enhance the contrast in unstained samples It works on the principle of illuminating the sample with light that will not be collected by the objective lens, so not form part of the image This produces the classic appearance of a dark, almost black, background with bright objects on it

For the dark field method, the cone of light normally

illuminating the specimen should not enter the microscope objective, only light that is scattered or reflected by the specimen is seen by the objective This is achieved in the conventional microscope by use of dark field diaphragm stops or a special dark field substage condenser like Abbe, paraboloid or cardioid condensers A dark field stop is inserting the stop below the condenser The light rays from the condenser pass outside the objective and thus form a hollow cone Now any object with a refractive index different from the surrounding medium, placed within this hollow cone, will only reflect light into the objective and thus the object will appear ‘bright’ against the dark background The condensers used for this type of microscopy are (Figs 1.22A to C)

Figure 1.19 Image formation in reflected light microscope

Figure 1.20 Image formation in light microscopes

Types of Condenser Used in Dark Field Microscopy

• Abbe c ondenser: Used within low power objective

• Paraboloid condenser: High power oil immersion

objective

• Cardioid condenser: More refined and corrected for

aberrations than the paraboloid condenser It is used

with oil immersion objective

Two lenses, the objective and eyepiece, are responsible

for the formation of the image The object or specimen

is illuminated by the light passing through the condenser

Figures 1.22A to C Condenser used for dark field microscopy (A) Cardioid condenser; (B) Abbe condenser;

in dark background

Phase Contrast Microscopy

(Figs 1.23 and 1.24)

Phase contrast microscopy, first described in 1934 by

Dutch physicist Frits Zernike, is a contrast-enhancing

optical technique which is utilized to produce

high-contrast images of transparent specimens, such as living

cells (usually in culture), microorganisms, thin tissue

slices, lithographic patterns, fibers, latex dispersions, glass

fragments, and subcellular particles (including nuclei and

other organelles)

One of the major advantages of phase contrast

microscopy is that living cells can be examined in their

natural state without previously being killed, fixed, and

stained As a result, the dynamics of ongoing biological

processes can be observed and recorded in high contrast with sharp clarity of minute specimen detail

Phase contrast microscopy is widely employed in diagnosis of tumor cells and the growth, dynamics, and behavior of a wide variety of living cells in culture

Brightfield microscope can be converted into phase contrast by two specialized accessories A specially

designed annular diaphragm, which is matched in diameter and optically conjugates to an internal phase plate residing

in the objective rear focal plane, is placed in the condenser front focal plane

The phase contrast microscope is probably the most outstanding contribution to microscopy in recent years It can be used to produce excellent contrast effects, with a wide variety of otherwise transparent specimens Since it permits visualization of interior details in cell structures,

it has a definite advantage over the dark field microscope Probably its widest application is in the field of tissue culture, where it permits one to examine and photograph living, growing cell

Phase contrast microscope is standard biological microscope and is equiped with modified objective and condensers

∙ Condensers: Condenser has a series of annular

diaphragm made of opaque glass with a clear narrow ring, to produce a controlled, hollow cone of light

∙ Objective: It requires a different size of annulus an image of which is formed by the condenser in the basic focal plane of the objective as a bright ring of light The Objective has a phase shifting plate or positive phase plate which is a clear glass disk with a circular trough etched in it to half the depth of disk The trough also contains a neutral density light absorbing material to reduce the brightness of the direct rays which could otherwise obscure the contrast obtained

The light passing through the trough has a phase difference of 1/4 of wavelength (Figs 1.25A and B)

Principles of Phase Contrast Microscope

The basis of phase contrast microscope is the exaggeration

of minute differences in the refractive indices by advancing or retarding light waves, thus converting them into difference of amplitude, which are seen as variation

in brightness Thus if two unstained structures of almost the same refractive index are examined by ordinary illumination it will be found that they are indistinguishable from each other

Figure 1.23 Phase contrast objective

Figure 1.24 Phase plate of phase contrast microscope

showing different objectives

For each transparent or translucent particle in the

object, two rays result from an incident light, The direct,

or undiffracted ray comes through the angular diaphragm,

passes through the object and is focused on the phase

shifting ring which either retards or advances the ray 1/4th

wavelength with respect to the secondary

The second ray of the incident beam is modified by

being scattered and diffracted in passing around the margin

of the object This ray does not pass through the phase

shifting ring but traverses the other areas of the transparent

disk, and the wavelength is neither advanced nor retarded

Thus there is an optical difference of 1/4th wavelength,

which causes a phase difference with the asynchronous

waves producing reinforced darkness or brightness at

certain points Thus ‘phase contrast’ is made visible to

the observer’s eye with the help of ‘phase shifting plate’,

which enhances the optical effect of the difference

Applications

Phase contrast microscope is valuable in examination of wet

mounts and hanging drop preparations It can reveal cellular

structure of living cell due to difference in the refractive index of the components of the cell and is of great value in cytology, hematology and microbiology (Figs 1.25A and B)

Polarized Light Microscopy (Fig 1.26)

Birefringence is a property, which is shown by crystalline structures, amyloid deposits, proteins, pigments and lipids When such substances are viewed in a microscope with polarized filters, they may appear bright or even colored against a dark background

In this type of microscope is used two ‘polarizers’ made up of Nicole Prisms are used One is placed beneath the substage condenser and is held in a rotatable graduated mount, and can be removed from the light path when not required The other called ‘analyzer’ is placed between objective and eyepiece and is also graduated for measurements to be taken

When a birefringent substance is rotated between two polarizers, which are crossed, the image appears and disappears alternately at each 45° of rotation In a complete revolution of 360° the image appears four times

Only, the polarizer is used, and if no rotating stage

is available, the polarizer itself can be rotated Changes

in intensity and color are seen during rotation The color changes in a rotation of 90°, and back to its original color

in the next 90° This is due to differential absorption of light, depending upon the vibration direction of two rays in

a birefringent substance

Principle of polarized microscopy: A ray of light consists

of electromagnetic waves vibrating in all directions at right

Figure 1.26 Different types of polarizing filters for polarizing

microscope

Figures 1.25A and B Image formation and photomicrograph

in phase contrast microscope

A

B

Figure 1.27 Polarized microscopy can be applied to see

enamel hypoplasia and dental caries

Figure 1.28 Fluorescent microscope principle showing filters,

arc lamp and the image formed

angles to the path of the ray of light itself In polarized light

the waves are made to vibrate in one plane only This is

achieved by the rotating Nicole prisms, i.e polarizer and

analyzers, which is interrupted in the beam of light

The specimen are labeled into two categories isotropic

or anisotropic

Isotropic (Singly refractive): The substance which are not

illuminated by the change in direction of the beam or 90°

rotation of the analyzer, the rays transmitted by the lower

prism will not pass through the upper, the field is now dark

and the position is called crossed Nicole

Anisotropic (Birefringent or doubly refractive): These

substances are seen as positive after the changes in

direction of the beam of light, i.e 90° rotation of analyzer,

the objects are seen as bright against a dark background

Collagen fibers, bone matrix, striated muscle, cholesterol,

Zenker fixed RBC, pigments such as formalin pigment,

crystal such as talc and vegetable, fibers like cotton and

linen are anisotropic”

Applications: The polarizing microscope can be a

useful means of identification of tissue components and

of exogenous an endogenous crystal specially when

combined with special staining techniques and with

histochemistry” With this—“ The polarizing microscope

can be a useful in identification of exogenous an

endogenous tissue components and crystal It is more

effective when combined with special staining techniques

and histochemistry” (Fig 1.27)

Fluorescence Microscopy

(Figs 1.28 to 1.30)

Objects invisible by ultraviolet light may become

brilliantly luminous if coated with a fluorescent substance,

like fluorchromes Fluorchromes are the dyes which absorb

radiation (e.g Ultraviolet light) and become excited; these

excited molecules are then capable of emitting radiation

of longer wavelength and which disappear almost

immediately after withdrawal of the exciting radiation

This is called ‘fluorescence’ Thus fluorescence is the

property of some substances, which illuminated by light of

certain wavelength causes them to emit the rays of different

and longer one In fluorescence microscopy a fluorescent

specimen is illuminated with invisible ‘ultraviolet light’

(UV light has wavelength below 400), the light rays of

longer wavelength within the spectrum of visible light are

given off and those are seen as various colors of on dark

background A completely dark room is desirable Brilliant fluorescence depends upon maximum contrast and is reduced if there is background light in the room

The equipment consists of:

Light source: Halogen lamps which give off sufficient

blue light (9400–500 nm) Ultraviolet lamps need to be warmed up before use and have short life

Filters: Two filters are place, each in between condenser

and source (Exciter filter); other is placed between the objective and eyepiece (Barrier filter)