A Level Biology for OCR Series Editor: Ann Fullick Ann Fullick « Jo Locke « Paul Bircher OXFORDA Level Biology for OCR Series Editor Ann Fullick Authors Ann Fullick Jo Locke Paul Bircher OXFORD UNIVERSITY PRESSOXFORD Great Clarendon Street, Oxford, OX2 6DP, United Kingdom (Oxford University Press is a department of the University of (Oxford. It furthers the University’s objective of excellence in research, scholarship, and education by publishing worldwide, (Oxford is a registered trade mark of Oxford University Press in the UK and in certain other countries © Ann Fullick, Paul Bircher, and Jo Locke 2015, ‘The moral rights of the authors have been asserted First published in 2015 All rights reserved. No part of this publication may be reproctuced, stored 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 hy law, hy licence or under terms agrood with the apprapriate 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. ‘You must not circulate this work in any other form and you must impose this same condition on any acquirer British Library Cataloguing in Publication Data Data available 9780-19835192-4 10987654321 Paper used in the production of this book is a natural recyclable product made from wood grown in sustainable forests. The manufacturing process conforms to the environmental regulations of the country of origin. Printed in Great Britain ‘This resource is endorsed by OCR for use with specification 020 AS Level GCE Biology A and H420 A Level GCE Biology A In order to gain endorsement this resource has undergone an independent quality check. OCR has not paid for the production of this resource, nor does OCR receive any royalties from its sale. For more information about the endorsement process please visit the OCR website www.ocr.org.tik Acknowledgements The authors would like to thank John Beazley for his reviewing. 2s well as Amy Johnson, Amie Hewish, Les Hopper. Clodagh Burke, Sharon ‘Thom for their tireless work and encouragement. In addition they ‘would like to thank the teams ar science and plants for schools (SAPS) and at the Wellcome Trust Sanger Institute for their valuable input to the project. and finally the help zeceived from Dr Jeremy Pritchard and Jennifer Collins. ‘Ann Fullick would like to thank her partner Tony for his support and amazing photographs, and al of the boys William, Thomas, ames, Edward and Chis for their expert advice and for making her ake time of Paul Rircher wold like to thank his wife, julie, and the rest of his family for their patience and support throughout the writing ofthis book. A special mention goes to his irrepressible grandchildren, Leo and Toby, who provided a welcome distraction, Their insanity has kept him sane. Jo Locke would like to thank her husband Dave forall his suppor, encouragement, and endless cups of tea, as well s her girs Emily and Hermione who had to wait patiently for Mummy "to just finish this paragraphAS/A Level course structure This book has been written to support students studying for OCR A Biology A. It covers all A level modules from the specification. The chapters within each module are shown in the contents list, Which also shows you the page numbers for the main topics within each chapter. There is also an index at the back to help you find what you are looking for. If you are studying for OCR AS Biology A, you will only need to know the content in the blue box. Year 1 content Year 2 content 1. Development of practical skills 5 Communication, homeostasis, in biology and energy 2. Foundations in biology 6 Genetics, evolution, and 3 Exchange and transport ecosystems 4 Biodiversity, evolution, and disease AS exam Level exams will cover content from Year 1 and Year 2 and will be at a higher demand. You will also carry out practical activities throughout {your course. Alevel examHow to use this book viii Kerboodle xi Module 1 Development of practical skills 5.4 Active transport 112 in Biology 2 5.5 Osmosis 114 Chapter 5 Practice questions 118 Module 2 Foundations in Biology 6 b " Chapter 6 Cell division Chapter 2 Basic components oflivingsystems 8 6.1 Celi cycle 120 2.4 Microscopy 8 62 Mitosis 124 2.2. Magnification and calibration 15 6.3. Meiosis 128 2.3. More microscopy 18 6.4. The organisation and specialisation 2.4 Eukaryotic cell sucture 26 of cells 133 2.5 The ultrastructure of plant cells, 33° 6.5 Stemcells 138 2.8 Prokaryotic and eukaryotic cells 35 Chapter 6 Practice questions 143 Chapter 2 Practice questions 38 Module 2 summary 146 Chapter 3 Biological Molecules 40 Module 2 practice questions 148 3.1 Biological elements 40 ie. 44 Module 3 Exchange andtransport = 152 3.3 Carbohydrates 46 Module 3 Introduction 152 3.4 Testing for carbohydrates 51 Chapter 7 Exchange surfaces and breathing 154 3.5 Lipids 54 24 Specialised exchange surfaces 154 3.6 The structure of proteins 5822 Mammalian gaseous exchange system 157 3.7 Types of proteins 84 23 Measuring the process 163 3.8 Nucleic acids 68 2.4 Ventilation and gas exchange in 3.9 DNAreplication and the genetic code 72 other organisms 165 3.10 Protein synthesis 76 Chapter 7 Practice questions 172 3.11 ATP 80 ican Chapter 3 Practice questions ee ee ina ees ersten aa 8.1 Transport systems in multicellular Chapter 4 Enzymes 84 animals 174 4.1 Enzyme action 84 8.2 Theblood vessels 178 4.2 Factors effecting enzyme activity 88 3 Blood, tissue fluid, and lymph 182 4.3 Enzyme inhibitors 94 8.4 Transport of oxygen and carbon 4.4 Cofactors, coenzymes, and dioxide in the blood 185 prosthetic groups 9? 95 theheart 189 Chapter 4 Practice questions ai Chapter 8 Practice questions 196 Chapter 5 Plasma membranes 102 Chapter 9 Transport in plants 198 5.1 Thestructure and function of membranes 102 9.4 Transport systems in dicotyledonous 5.2 Factors affecting membrane structure 106 plants 198 5.3 Diffusion 108 9.2 Watertransportin multicellular plants 2029.3. Transpiration 9.4 Translocation 9.5 Plant adaptations to water availability Chapter 9 Practice questions Module 3 summary Module 3 practice questions Module 4 Biodiversity, evolution and disease Module 4 introduction Chapter 10 Classification and evolution 10.1 Classification 10.2. The five kingdoms 10.3 Phylogeny 10.4. Evidence for evolution 10.5 Types of variation 10.6 Representing variation graphically 10.2. Adaptations 10.8 Changing population characteristics Chapter 10 practice questions Chapter 14 Biodiversity 11.4 Biodiversity 11.2 Sampling 11.3 Sampling techniques 11.4 Calculating biodiversity 11.5 Calculating genetic biodiversity 11.6 Factors affecting biodiversity 11. Reasons for maintaining biodiversity 11.8 Methods of maintaining biodiversity Chapter 11 practice questions Chapter 12 Communicable diseases 12.4. Animal and plant pathogens 12.2. Animal and plant diseases 12.3. The transmission of communicable diseases 12.4 Plant defences against pathogens 12.5. Non-specific animal defences against pathogens 12.6 The specific immune system 205 2a. 214 219 222 224 228 228 230 230 235 240 242 247 254 259 264 268 270 270 272 274 279 282 286 291 295 300 302 302 305 341 314 316 320 12.7 Preventing and treating disease Chapter 12 practice questions Module 4 summary Module 4 practice questions Module 5 Communcation, homeostasis, and energy Chapter 13 Neuronal communication 13.1 Coordination 13.2. Neurones 13.3. Sensory receptors 13.4 Nervous transmission 43.5. Synapses 13.6 Organisation of the nervous system 13.7 Structure and function of the brain 13.8 Reflexes 13.9. Voluntary and involuntary muscles 13.10 Sliding filament model Chapter 13 Practice questions Chapter 14 Hormonal communication 44.1. Hormonal communication 325 333 334 336 340 342 342 344, 347 349 355 360 362 365 369 374 380 382 382 44.2. Structure and function of the pancreas 386 14.3. Regulation of blood glucose concentration 14.4 Diabetes and its control 44.5 Coordinated responses 14.6 Controlling heart rate Chapter 14 Practice questions Chapter 15 Homeostasis 45.4 The principles of homeostasis 15.2. Thermoregulation in ectotherms 45.3. Thermoregulation in endotherms 15.4. Excretion, homeostasis, and the liver 45.5 The structure and function of the mammalian kidney 45.6 The kidney and osmoregulation 15.7 Urine and diagnosis 15.8 Kidney failure Chapter 15 Practice questions 389 393 397 400 404 406 406 408 411 415 420 428 431 433 438Chapter 16 Plant responses 440 Chapter 21 Manipulating genomes 550 16.1 Planthormones and growth inplants 440 24.1. DNAprofiling 550 16.2. Plant responses to abiotic stress 445 21.2 DNAsequencing and analysis 555 16.3. Plant response to herbivory 448 241.3. Using DNAsequencing 559 16.4 Tropismsin plants 451 21.4 Genetic engineering 564 16.5 The commercial use of planthormones 456 24.5. Gene technology and ethics 569 Chapter 16 Practice questions 458 Chapter 21 Practice questions 574 Chapter 17 Energy for biological processes 460 Chapter 22 Cloning and biotechnology 576 12.1 Energy cycles 480 22.1. Natural loningin plants 576 47.2 ATP synthesis 463 22.2 Artificial cloningin plants 578 42.3. Photosynthesis 486 22.3 Cloningin animals 581 17.4 Factors affecting photosynthesis 474 22.4 Microorganisms and biotechnology 586 Chapter 17 Practice questions 428 22.5. Microorganisms, medicines, and Chapter 18 Respiration 480 lore mediation am 181 Glycolysis 4gq 228. Culturing microorganisms in the 18.2. The link reaction 482 eo we 183 Krebs cycle 4g4 22? Culturing microorganisms on an 18.4 Oxidative phosphorylation 486 indllstraliscale ae8 BE resphctorusubsirates 49g 22.8 Usingimmobilised enzymes 599 IRE Acioek Fesuiation eet Chapter 22 Practice questions 604 Chapter 18 Practice questions 496 Chapter 23 Ecosystems 606 Module 5 summary 498 23.1. Ecosystems 606 Module 5 questions 500 23.2 Biomass transfer through an ecosystem 608 Module 6 Genetics, evolution, 23.3. Recycling within ecosystems 614 and ecosystems 504 = 23.4 Succession 619 Chapter 19 Genetics of living systems sog 23.5. Measuring distribution and 19.1. Mutations and variation 506 abundehice cr OreeriSmse oe 19.2 Control of gene expression 510 Chapter 23 Practice questions 628 19.3 Body plans 514 Chapter 24 Populations and sustainability 630 Chapter 19 Practice questions 518 24.1 Population size 630 Chapter 20 Patterns of inheritance exe! Competition G3) oe 520 24.3 Predator—prey relationships 635 0d Vajistionand iaherence: 520 24-4 Conservation and preservation 637 20.2 Monogenetic inheritance s2q, 24S Susteinabitigy ea 20.3. Dinybrid inheritance B29, “PAB: Ecosystem management ’— 20.4. Phenotypic ratios 531 Masa! mara ee 20.5 Evolution 53g 24.2. Ecosystem management ~ Terai 20.6: Speciation and artificial selection 543 region of Nepal BAS Chapter 20 Practiosapaeionis 54g 248 Ecosystemmanagement—Peat bogs 64724.9. Environmentally sensitive ecosystems 649 Chapter 24 Practice questions Module 6 summary Module 6 questions Synoptic concepts ‘Synoptic concepts questions Glossary Answer OR page Index Appendix (Statistics data tables) Acknowledgements, 654 656 658 662 667 672 692 693 703 705How to use this book Learning outcomes > Atthe beginning of each topic, there isa list of learning outcomes. > These are matched tothe specification and allow youto monitor your progress. > Aspecitication reference is also included. Specification reference: 2.1.3 Stee ‘Study tips contain prompts to help you with your understanding and revision, Btn These highlight the key areas where topics relate to each other ‘As you go through your course, knowing how to link different areas of biology together becomes increasingly important. Many exam questions, particularly at ALevel, wll require you to bring ‘together your knowledge from. different areas. air This book contains many different features. Each feature is designed to support and develop the skills you will need for your examinations, as well as foster and stimulate your interest in biology. Terms that you will need to be able to define and understand are highlighted by bold text. hae +—— WOM Application features These features contain important and interesting applications of biology in order to emphasise how scientists and engineers have used their scientific knowledge and understanding to develop new applications and Technologies. There are also practical application features, with the icon 10 suppor futher development of your practical skis. There are also application features with the icon @fo which help support development of your understanding of scientific issues and their impact in society, 4 All application features have a question to link to material covered with the concept from the specification. a Extension features These features contain material that is beyond the specification. They are designed to stretch and provide you with a broader knowledge and Understanding and lead the way into the types of thinking and areas you might study in further education. As such, neither the detail nor the depth of questioning will be required for the examinations. But this book is about more than getting through the examinations. 4 Extension features also contain questions that link the off-specification material back to your course. Summary Questions 1 These are short questions at the end of each topic. 2 They test your understanding of the topic and allow you to apply the knowledge and skills you have acquired. 3. The questions are ramped in order of difficulty. The icon gf indicates where a question relates to scientific issues in society.MODULE 6 Introduction at the beginning ms of each module summarises teri sl yrmcmrinins Sriranicaen GO eect irene intent Fee eens ey ora eeesencnaeriannimenasitars ene O Sietatersinalenennprnernssnntinnrpinal lal 0) eerarngete etme Achecklist helps you assess your knowledge from KS4, before starting work on the module. There is also.a maths skills checklist to demonstrate Wanna the skills you will learn in that module, some of the key concepts of ‘that module interlink with other modules, across the entire A Level course. Application task brings together some of the key concepts of the module in »| anew context, v Extension task bring together some key concepts of the module and develop them further, leading you a towards greater understanding and further study.Practice questions at the end of each chapter and the end ane of each module, including : ‘questions that cover practical and math skills. Tara ad Adedicated Synoptic Concepts section at the end of the book, with help and advice on answering ‘synoptic questions that cover multiple different topics. This section also contains further practice questions.Kerboodle This book is supported by next generation Kerboodle, offering unrivalled digital support for independent study, differentiation, assessment, and the new practical endorsement If your school subscribes to Kerboodle, you will also find a wealth of additional resources to help you with your studies and with revision. study guides Maths skills boosters and calculation worksheets On your marks activities to help you achieve your best Practicals and follow up activities to support the practical endorsement Interactive objective tests that give question-by-question feedback Animations and revision podcasts Self-assessment checklists Test your knowledge with the progress quizzes, and learn from your mistakes with the detailed explanations given for each answer. For teachers, Kerboodle also has plenty of further assessment resources, answers to the questions in the book, and a digital markbook along with full teacher support for practicals and the worksheets, which include suggestions on how to support and stretch students, All ofthe resources are pulled together into teacher guides that suggest 2 route through each chapter.MODULE 1 Development of practical skills in biology Developing your practical skills isa fundamental part of a complete education in science. A good grounding of practical skills will help your understanding of Biology and help to prepare you for studying beyond Alevel. You will carry out a number of practicals. throughout the A level course. Practical skills knowledge will account for 15% of the marks in your written exams. There are no assessed practical components in the AS qualification, however, if you choose to study for the full Alevel qualification then these practicals will count towards the practical endorsementin your second year of study. Practical coverage throughout this book Itis a good idea to keep a record of your practical work during your course, this will be very useful when you come to revise for your exams, and you can use it later as part of your practical endorsement. You can find more details of the practical 1.1.1 Planning Experimental design Identification of variables Evaluation of experimental method endorsement from your teacher or from the specification. In this book and its supporting materials, practical skills are covered in a number of ways — look out for the conical flask symbol. By studying Application boxes and practice questions in this student book you will have many opportunities to learn about scientific method and how to carry out practical activities. There are also more resources available on Kerboodle to help you practise your skills. (kerboodle 1.1 Practical skills assessed in written examinations There are four key components of practical skill assessments. These are laid out here with a skills checklist to help you as you study.1.1.2 Implementing Use of practical apparatus Appropriate units for measurement Presenting observations and data 1.1.3 Analysis Processing, analysing, and interpreting results Appropriate mathematical skills for data analysis Use of appropriate number of significant figures Plotting and interpreting A Evaluate results to draw conclusions Identify anomalies Explain limitations in method Precision and accuracy of measurements Uncertainties and errors Suggest improvements to help improve experimental designMaths skills and How Science Works across A level Biology Maths is a vital tool for scientists, and throughout this course you will become familiar with maths techniques and equations that support the development of your science knowledge. Each module opener in this book has an overview of the maths skills that relate to the theory in the chapter. There are also questions using maths skills throughout the book that will help you practice. How Science Works are skills that will help you to apply your knowledge in a wider context, and the relevance of what you have learnt in the real world. This includes developing your critical and creative skills to help you solve problems in many different contexts. How Science Works is embedded throughout this book, particularly in application boxes and practice questions. 1.2 Practical skills assessed in practical endorsement You are required to carry out 12 assessed practical activities over both years of your Alevel studies. The practical endorsement does not count towards your final A level grade but is reported alongside it as a pass ora fail. Universities and employers will look for this as evidence that you have a good level of practical competence. These practicals will be assessed by your teacher and will help to develop your skills and confidence. The practicals you carry out should be recorded in a lab book or practical portfolio where the hypothesis, method, results, and conclusion are clearly displayed. The information here details the types of skills and equipment you should become familiar with. 1.2.1 Practical skills Independent thinking * Investigating and analysing the methods used in practicals in order to solve problems Use and application of scientific methods and practices * using practical equipment correctly and safely * following written instructions, recording observations, taking measurements, and presenting data scientifically Using appropriate software and technology throughout. Research and referencing Using information available from a variety of different sources including websites, scientific journals, and textbooks to help provide context and background for the practical. Itis important ‘to use many sources of information as you can and to cite these correctly. Instruments and equipment Correct and appropriate use of a wide range of equipment, instruments, and techniques.1.2.2 Use of apparatus and techniques Apparatus for quantitative measuring, Use of glassware apparatus Use ofa light microscope at high and low power and the use of stage graticule Producing clear and well labelled scientific drawings Use of qualitative reagents Experience of carrying out electrophoresis or chromatography Ethical and safe use of organisms Microbial aseptic techniques Use of sampling techniques for fieldwork Use of IT to collect and process data. Alevel PAG overview and Application features The practical activity requirements (PAGs) for the OCR A Biology practical endorsement are listed in the table. You should take all opportunities to develop you practical skills and techniques in your first year of study to help build a greater understanding. The table below shows the practical activity requirements, and which topics these are covered in. Bieber PAG1 Microscopy Btls 2.1, 2.2, 2.3, 13.9, 13.10, 14.2, 15.4 PAG2 Dissection 7.4, 8.2, 8.5, 15.5, 22.1 || PAG3 Sampling techniques 11.3, 23.5 PAG4 Enzyme controlled reactions 4.2,18.6 PAGS Colorimeter or potometer 3.4 PAGG Chromatography or electrophoresis 3.6, 17.3, 21.1 PAG? Microbial technique 22.6, 22.7 PAG8 Transport in and out of cells 5.2, 5.3, 5.5 PAGS Qualitative testing PAG10 Investigation using a data logger or computer modelling 3.4, 15.2 17.4, 18.5 PAG11 Investigation into the measurement of plant or animal responses 9.3, 12, 13.8, 13.10, 14.6, 16.1, 16.4, 18.6 PAG12 Research skills Throughout courseMODULE 2 Foundations in Biology Introduction Erna ed aes CTU ea Sue nt eae Ree ee CSch RUC OCT Rte mu eso) SO Cc ers MRO Lal) Since Robert Hooke coined the phrase ‘cells’ in 1665, careful observation using microscopes has revealed details of cell Rei Meee ei razr) evidence to support hypotheses regarding Reg Seung are Basic components of living systems PCE st ak kee Se) microscopy techniques. An understanding Oe ont eee ne mus E I routes for biologists. Cree Sant cd cule nec ag ee COU Cmca ee curity POEM uber sme id key biological disciplines, such as medicine er eee ec ce TRO Oaue ME ie Pees ee Reta oat hn Tad they are structured and how they function. De Cee eeu ete CEU Rte au) Poet ed Rue er for all areas of biology involving cellular ees Perea eee uce neces sct eee eS a PiU cue ee escort rad oon aE nua tn coc Pe ee nce ee aad ee en ate TeasKnowledge and understanding checklist From your Key Stage 4 study you should be able to answer the following questions. Work through each point, using your Key Stage 4 notes and other resources. There is also support available on Kerboodle. Describe a cell as the basic structural unit of all organisms. Describe the main sub-cellular structures of eukaryotic and prokaryotic cells. Relate sub-cellular structures to their functions. Describe the cell cycle, Describe cell differentiation. Relate the adaptation of specialised cells to their function. Explain the mechanism of enzyme action. Recall the difference between intracellular enzymes and extracellular enzymes, Describe some anabolic and catabolic processes in living organisms including the importance of sugars, amino acids, fatty acids, and glycerol in the synthesis and breakdown of carbohydrates, lipids, and proteins. Maths skills checklist All biologists need to use maths in their studies and field of work. In this module, you will need to use the following maths skills. Changing the subject of an equation. You will need to be able to do this. when working with microscopy. Converting units. You will need to be able to do this when working with microscopy Working with negative numbers. You will need to be able to do this when calculating water potential when studying osmosis. Working in standard form. You will need to be able to do this throughout this chi f this chapter. @ MyMaths.ouPMO tee LIVING SYSTEMS 2.1 Cd cetetetey Specification reference: 2.1.1 Learning outcomes Demonstrate knowledge, understanding, and application of: > the use of light microscopy > the preparation of microscope slides for use in light microscopy > the use of taining in light microscopy > the representation of cell structure seen under light microscope using scientific annotated drawings. Before the invention of microscopes, we knew nothing of bacteria, cells, sperm, pollen grains, chromosomes the list is endless ‘Microscopes have given us the power to understand disease, see how a new life is formed, watch the dance of the chromosomes as cells divide and manipulate the processes of life itself Seeing is believing A microscope is an instrument which enables you to magnify an object hundreds, thousands and even hundreds of thousands of times. We can see many large organisms with the naked eye, but microscopes open up a whole world of unicellular organisms. By making visible the individual cells which make up multicellular organisms, microscopes allow us to discover how details of their structures relate to their functions. The first types of microscopes to be developed were light microscopes in the 16th to 17th century. Since then they have continued to be developed and improved. By the mid-19th century, scientists, for the first time, had access to microscopes with a high enough level of magnification to allow them to see individual cells Il theory was developed. It states that © both plant and animal tissue is composed of cells © cells are the basic unit of all life © cells only develop from existing cells Light microscopy continues to be important ~ it is easily available. relatively cheap and can be used out in the field, and it can be used to observe living organisms as well as dead, prepared specimens OF History of the light microscope and the development of cell theory Late in the Roman Empire the Romans began to develop _ who invented the telescope when experimenting with and experiment with glass. They noted how objects looked multiple glass lenses in a tube in the late 15th century. bigger when viewed through pieces of glass that were Others claim it was Galileo Galilei in 1609 who developed thicker in the middle than at the edges. There was little further development of glass lenses until ‘around the 13th century and the invention of spectacles or eye glasses. the first true ar compound microscope (Table 1). Galileo's instrument was the first tobe given the name ‘microscope’ Cell theory The credit forthe invention of the light microscope is much The development of cell theory is a good example of haw disputed, Some accredit it to two Dutch spectacle makers _ Scientific theories change over time as new evidencePe Li Lee ae ko ee is gained and as knowledge increases. Theories are is the case with cell theory - as microscopes with higher proposed, accepted and can then be later disproved as magnification and resolution were developed, cells could new evidence comes to light. New evidence can arise ina___be observed for the first time. Table 1 summarises some ‘number of ways, including as technology develops. This __of the developments in cell theory. V Table 1. Cell theory timeline Cell first observed Robert Hooke, an English scientist, observed the structure of thinly sliced cork using an early ight microscope. He described the compartments he saw as ‘cells’ — coining the term we still use today. As this was dead plant tissue he was observing only cell walls. 1674-1683 First living cells observed Anton van Leeuwenhoek, a Dutch biologist, developed a technique for creating powerful glass lenses and Used his handcrafted microscopes to examine samples of pond water. He was the first person to observe bacteria and protactista and described them as ‘little animals’ or ‘animalcules’ — today we call them microorganisms, He went on to observe red blood cells, sperm cells, and muscle fibres forthe first time. Evidence for the origin of new plant cells Barthélemy Dumorter, a Belgian botanist, was the first to observe cell division in plants providing evidence against the theories of the time, that new cells arise from within old cells or that cells formed spontaneously from non-cellular material, However it was several more years until cell division as the origin ofall new cells became the accepted theory, Nucleus first observed Robert Brown, an English botanist, was the first to describe the nucleus of a plant cell 1837-1838 1844 (1855) ‘The birth of a universal cell theory Matthias Schleiden, a German botanist, proposed that all plant tissues are composed of cells. Jan Purkyné, a Czech scientist, was the first to use a microtome to make ultra-thin slices of tissue for microscopic examination, Based on his observations, he proposed that not only are animals composed of cells but also that the ‘basic cellular tissue is clearly analogous to that of plants” Not long after this, and independently, Theodor Schwann, a German physiologist, made a similar observation and declared that “all living things are composed of cells and cell products”. He is the scientist credited with the ‘birth’ of cell theory, Evidence for the origin of new animal cells Robert Remak, a Poish/German biologist, was the first to observe cel division in animal cells, disproving the existing theory that new cells originate from within old cells, He was not believed at the time however, and Rudolf Virchow, a German biologist, published these findings as his own a decade later in 15S. Spontaneous generation disproved Louis Pasteur disproved the theory of spontaneous generation of cells by demonstrating that bacteria would only grow in a sterile nutrient broth after it had been exposed to the air 41 Outline the importance of microscopes in the study of living organisms. 2 Suggest, with reasons, why cell theory was not fully developed before the mid-19th century,2.1 Microscopy ‘A Figure 1. Drawing of cork rom 1663 seen under an early micrascope. Rabert Hooke described the pores as cells, thus coining the term, He prepared the specimen by making thin slices with a razor blade, inventing the technique of sectioning A Figure 2. Robert Hooke’s drawing of his own compound microscope, which he used to see the ‘cells’ in a sample of cork How a light microscope works SOc na A compound light microscope has two lenses ~ the objective lens, which is placed near to the specimen, and an eyepiece lens, through which the specimen is viewed. The objective lens produces a magnified image, which is magnified again by the eyepiece lens. This objective/eyepiece lens configuration allows for much higher magnification and reduced chromatic aberration than that in a simple References to clarity or cleamess of images are not relevant when discussing microscopy. Itis important to explain that objects have become visibly distinct with increased resolution, in other words more detail can be seen. Ulumination is usually provided by a light underneath the sample Opaque specimens can be illuminated from above with some light microscope microscopes, image viewed here ccoarse-ocusing knob fine focusing knot light from oF ght bull A Figure 3 Acompound light micrascopePe LiLo ae ko ee Sample preparation There are a number of different ways in which samples ‘and specimens can be prepared for examination by light microscopy. The method chosen will depend on the nature of the specimen and the resolution that is desired. ‘© Dry mount — Solid specimens are viewed whole or cut into very thin slices with a sharp blade, this is called sectioning. The specimens placed on the centre of the slide and a cover slip is placed over the sample. For example hair, pollen, dust and insect parts can be viewed whole in this way, and muscle tissue or plants can be sectioned and viewed in this way. coverslip BAN slide Wet mount — Specimens are suspended in aliquid such as water or an immersion oil. A cover slip is placed on from an angle, as shown. For example, aquatic samples and other living organisms can be viewed this way. cover slip droplet ‘Squash slides — A wet mount is first prepared, then a lens tissue is used to gently press down the cover slip. Depending on the material, potential damage to a cover slip can be avoided by squashing the sample between two microscope slides. Using squash slides is a good technique for soft samples. Care needs to be taken that the cover slip is not broken when being pressed. For example, root tip squashes are used to look at cell division, © Smear slides ~ The edge of a slide is used to smear the sample, creating a thin, even coating on another slide. A cover slip is then placed over the sample. An ‘example of 9 smear slide ic a sample of blood. This is 9 ‘good way to view the cells in the blood. ‘Suggest reasons for the following, with reference to slide preparation: a Specimens must be thin. b When preparing a wet mount the refractive index (ability to bend light) of the medium should be roughly the same as glass, © Acover slip must be placed onto a wet mount at anangle. On OS, Using staining In basic light microscopy the samples illuminated from below with white light and observed from above {brightfield microscopy). The whole sample is illuminated at once (wide-field microscopy). The images tend to have low contrast as most cells do not absorb alot of light, Resolution is limited by the wavelength of light and diffraction of light as it passes through the sample Diffraction is the bending of light as it passes close to the edge of an object. The cytosol (aqueous interior) of cells and other cell structures are often transparent. Stains increase contrast as, different components within a cell take up stains to different degrees. The increase in contrast allows components to become visible so they can be identified (Figure 4) Toprepare a sample for staining its first placed on a slide and allowed to air dry, Thisis then heat fixed by passing through a flame. The specimen will adhere to the microscope slide and will then take up stains,2.4 Microscopy anhOnAaa red blood cell (erythrocyte) white blood cell (leucocyte) o@ me ‘A Figure 4 Light micrograph and annotated diagram ofa blood sample that has been stained using Wright's stain (0 mixturo of eosin red and methylene blue dyes). The nuclei of the white blood colls are stained purplo Paya ese rege Crystal violet or methylene blue are positively charged dyes, which are attracted to negatively charged materials in cytoplasm leading to staining of cell components. Dyes such as nigrosin or Congo red are negatively charged and are repelled by the negatively charged cytosol. These dyes stay outside cells, leaving the cells unstained, which, ‘then stand out against the stained background, This is @ nogative stain technique. Differential staining can distinguish between two types of organisms that would otherwise be hard to identify. It can also differentiate between different organelles of single organism within a tissue sample. © Gram stain technique is used to separate bacteria into ‘two groups, Gram-positive bacteria and Gram-negative bacteria (Figure 5]. Crystal violets first applied to a bacterial specimen on a side, then iodine, which fixes the dye. The slide is then washed with alcohol. The Gram-positive bacteria retain the crystal violet stain and will appear blue or purple under a microscope. Gram- negative bacteria have thinner cell walls and therefore Jose the stain. They are then stained with safranin dye, which is called a counterstain, These bacteria will then appear red. Gram-positive bacteria are susceptible to the antibiotic penicillin, which inhibits the formation of Cell walls, Gram-negative bacteria have much thinner cell walls that are not susceptible to peni Acid-fast technique is used to differentiate species of Mycobacterium from other bacteria. A lipid solvent is used to carry carbolfuchsin dye into the cells being studied, The cells are then washed with a dilute acid- alcohol solution. Mycobacterium are not affected by the acid-alcohol and retain the carbolfuchsin stain, at o ayy S) ‘A Figure S Gram stain of Streptococcus pneumoniae, a Grom positive bacteria which cause pneumonia (200 ‘magnification). Gram-positive bacteria retain a crystal violet dye during the Gram stain process and appear blue or violet ‘under a microscope a . te set ese ‘A Figure 6 Yersinia pestis, the Gram-negative bacteria which cetise tha boi plgue fect humans annals («1450 magnification). Gram-negative bacteria appear red or pec sagePe Li Lee ae ko ee which is bright red. Other bacteria lose the stain and are exposed toa methylene blue stain, which is blue. You will often look at slides that have been pre-prepared, Anumber of stages are involved in the production of these slides: © Fixing — chemicals like formaldehyde are used to preserve specimens in as near-natural a state as possible. Sectioning — specimens are dehydrated with alcohols and then placed in a mould with wax orresin to form a hard block. This can then be sliced thinly with a knife called a microtome, Staining ~ specimens are often treated with multiple stains to show different structures, Mounting — the specimens are then secured to a microscope slide and a cover slip placed on top. Risk management Many of the stains used in the preparation of slides are toxic or irritants. A risk assessment must carried out before any practical is started to identify any procedures involved that may result in harm. CLEAPSS (Consortium of Local Education Authorities for the Provision of Science Services) is the organisation that, provides support for the practical work carried Lessis more @ out in schools. One of the main areas covered is health and safety, including risk assessment. Advice and support is provided to all types of educational establishments and their employees about all aspects of practical work such as the use of chemicals or living organisms, laboratory design, and even where to obtain the right equipment. CLEAPSS provide student safety sheets that identify specific risks, advise on the measures to be taken to reduce these risks and the action to be taken in any emergency, In fact, in schools many of the microscopy slides that are used are bought in ready-prepared and pre-stained, not only because of the harmful nature of the stains but also because of the long complex process needed to produce high quality sections. 4 Use your knowledge of the staining technique used to distinguish Gram-positive from Gram-negative bacteria and information from the paragraph above to answer the following question: Suggest why Gram-negative infections are more difficult to treat than Gram-positive infections. Crystal violet and potassium iodide are chemicals classed asiritants. Crystal violet is also toxic. Describe the precautions you should take when using these chemicals. Scientific drawings are line drawings not pictures. They are used to highlight particular features and should not include unnecessary detail. The focus can be changed to help draw celected features. The followingis a list of rules for producing good scientific drawings: © include a ttle state magnification use a sharp pencil for drawings and labels, use white, unlined paper use as much of the paper as possible for the drawing draw smooth, continuous lines draw clearly defined structures ensure proportions are carrect label lines should not cross and should not have arrow heads . . . . . © donot shade . . . ° label lines should be parallel to the top of the page and drawn with a ruler2] 2.1 Microscopy The light micrograph (Figure 7) shows a layer of onion cells as seen under a light microscope. Next tothe micrograph is an example of a good scientific drawing of this image. x 18 magnification A Figure 7 Top: Light micrograph of a layer of onion cuticle, showing the bands of large, rectangular cells. he dark spot in the centre of each cell sits ‘nucleus. x 18 magnification. Bottom: A scientific drawing from the micrograph Below is an example of a poor scientific drawing: nucleus 4 Describe how this diagram is incorrect as a scientific drawing. BITC toca 1 Outline the basic concepts of cell theory. (3marks) 4 @ Calculate the low and high power magnifications of ‘a microscope with the following lenses: 2 Explain why staining is used in microscopy. (2 marks) atderiacelens 10, 3 Compound microscopes led to new discoveries li Objective lenses x10 and «40, (2marks) essential for cell theory to be fully explained. b Youare observing a specimen of squamous tissue Explain the benefit of having two lenses in under high power. The individual cells have an amicroscope. (4 marks) average diameter of 60m and the diameter of the field of view of the objective lens is 2 mm. Calculate the maximum number of whole cells that are visible inthe field of view. (3 marks)2.2 Magnification and calibration Specification reference: 2.1.1 Magnification, resolution, and the magnification formula Magnification is how many times larger the image is than the actual size of the object being viewed. Interchangeable objective lenses on a compound light microscope allow a user to adjust the magnification Simply magnifying an object does not increase the amount of detail that can be seen. The resolution also needs to be increased. The resolution of a microscope determines the amount of detail that can be seen ~ the higher the resolution the more details are visible, Resolution is the ability to see individual objects as separate entit Imagine a car coming towards you at night with its headlights on. When it is a long way off you will only see one light but as the car gets dloser you eventually see that there are, in fact, two headlights ~ they have been resolved. Resolution is limited by the diffraction of light as it passes through samples (and lenses). Diffraction is the tendency of light waves to spread as they pass close to physical structures such as those present in the specimens being studied. The structures present in the specimens are very close to each other and the light reflected from individual structures can overlap due to diffraction. This means the structures are no longer seen as separate entities and detail is lost. In optical microscopy structures that are closer than half the wavelength of light cannot be seen separately (resolved). Resolution can be increased by using beams of electrons which have a wavelength thousands of times shorter than light (Topic 2.3, More microscopy). Electron beams are still diffracted but the shorter wavelength means that individual beams can be much closer before they overlap. This means objects which are much smaller and closer together can be seen separately diffraction blurring the image. Calculation for magnification The magnification of an object can be calculated using the magnification formula: size of image actual size of object magnification In practice, the size of the image refers to the length of the image as measured, for example with a ruler. You may need to change the units of measurement to that of the actual size of the object. ‘The magnification formula, like all mathematical equations, can be rearranged to find any of the unknowns, where the remaining values are known. To help you, you can imagine this three-part formula in a standard formula triangle (Figure 2) Learning outcomes Demonstrate knowledge, understanding, and application of. > slide preparation and examination > the magnification formula t > thedifference between ‘magnifcation and resolution. ‘A Figure 2 Formula triangle for ‘magnification calculations2.2 Magnification and calibration 7 So, if the actual size of the object isn’t known but the magnification is Sane know: When solving magnification to give: problems, carry out your calculations with all measurements having the actual size of object ssame units. the actual size can be calculated by rearra iging the formula size of image magnification Or, the size of the image can be calculated by rearrang as below: Measure the image size in mm 1g the formula and convert it tothe smallest unit present in the problem, usually : actual size of object ete size of image = "EE magnification 1000 nanometres [nm] = Tinian [am Worked example: Magnification calculation 1000 micrometres (im) ‘rilietre (rom) Calculate the magnification of the image of the nuclear pore shown here. UU milimetres (mm) = Ametre (m) To calculate the magnification we first convert all figures to the smallest unit, in this case nm Magnification itself does not have units of measurement. 24 millimetres is equal to (24 x 1000 x1000) nanometres or 24000000 nanometres. Students should also practice rearranging the magnification formula. size of image actual size of object _ 24000000nm 120nm = 2000000 or 2 million times. ‘Magnification = OF Using a graticule to calibrate a light microscope @fo @ To measure the size of a sample under a microscope you The scale marked on the micrometer slide is usually use an eyepiece graticule. The true magnification of the 100 divisions = 1 mm, so 1 division = 10 um. different lenses of a microscope can vary slightly from the magnification stated so every microscope, and every lens, has to be calibrated individually using an eyepiece graticule anda slide micrometer. You calibrate the eyepiece graticule scale for each objective lens separately. Once all three lenses are calibrated, if you measure the same cell using the ‘three different lenses you should get the same actual + An eyepiece graticule is a glass disc marked with measurement each time. afine scale of 1 to 100. The scale has no units and For example: remains unchanged whichever objective lens is in r place, The relative size of the divisions, however, Calibrating a x4 objective lens increases with each increase in magnification. 4. Put the stage micrometer in place and the eyepiece You need to know what the divisions represent at raticule in the eyepiece. the different magnifications so you can measure specimens. The scale on the graticule at each magnification is calibrated using a stage micrometer. Align the micrometer scale with the scale in the eyepiece. Take @ reading from the two scales ~ see Get the scale on the micrometer slide in clear focus. Astage micrometer is a microscope slide with a very next page: ‘accurate scale in micrometres (um) engraved on it biaPe Li Lee ae ko ee 30 divisions on the eyepiece graticule = 10 divisions on the stage micrometer Use these readings to calculate the calibration factor for the x4 objective lens. 100 micrometer divisions = 1 mm So each small division is 1/100 mm = 0.01 mm or 10.0 um 30 graticule divisions = 10 micrometer divisions 10 micrometer divisions = 10 x 10 = 100 um number of eyepiece divisions number of micrometres 30 graticule divisions = 100 um so 1 graticule division = 1NN/3N= 3.33 pn 1 graticule division = The magnification factor is 3.33 Tose this magnification factor remove the stage micrometer and place a prepared slide on the stage. Measure the size of an abject in graticule units. To find the actual size multiply the number of graticule units measured by the magnification factor to give you the length in ym graticule divisions x magnification factor = measurement. (um) xg, the diameter of a cell seen using the x4 objective lens measures 10 graticule divisions. Each graticule division = 3.33 um so the cell diameter= 10 x 3.33 = 33.3ym, eyepiece graticule scale ° 10 Py lilaililititilililititititilil PEPeprpry ed Calibration example Astudent was asked to calibrate the x10 lens of alight ‘microscope and then to determine the diameter of a pollen grain from a sample slide provided 1. The student placed a scale on the stage of the microscope and facused on it with the x10 lens. 100 small divisions of the scale are 1 mm long, $01 division is 10 um, The student then aligned the scale in the eyepiece with the scale on the microscope stage and calibrated it 3. The student replaced the micrometer slide with pollen sample slides and used the calibrated scale in the eyepiece to measure the diameter of the pollen grains. Results The student decided to use the «10 objective lens. Result 1[2 Diameter of pollen grain/divisions | 11 | 16 4 State the correct names of the scale used on the stage of the microscope and the scale in the eyepiece. Calculate the calibration factor of the «10 objective lens. ‘a. Fillinthe missing reading in the table for the pollen grain shown in the artwork above, Using the table calculate the diameter of the different pollen grains using the calibration factor you have calculated in question 2 Calculate the mean diameter of the pollen grains. Suggest why itis important to calibrate ‘the lens that is going to be used to view the pollen grains.DD 2.2 Magnification and calibration 1. Suggest why you should put all measurements into the ‘same units before carrying out calculations. (2marks) 2 Calculate how many nanometres are present in 3846 centimetres. Give your answer in standard form. 3. Explain the difference between contrast and resolution. (2 marks) Calculate the magnification of the micrograph showing human cheek cells (Figure 3). ‘A Figure 3. Light micrograph of squamous epithelium cells fiom the inside ofa human cheek The average diameter of a cheek cellis 60 um. (2 marks) 5 Explain how diffraction limits resolution. (5marks) & Explain why eyepiece graticules do not have units. (2 marks)2.3 More microscopy BS) fella (er- Ula i(k 1a ear ae Learning outcomes Demonstrate knowledge, understanding, and application of > theuse offaserscanning ‘confocal microscopy > theuse of electron microscopy. Light microscopy started the science of cell biology, but it has limitations. In the middle of the 20th century a new invention, the electron microscope, revolutionised the study of cells and enabled biologists to see deep inside structures that were invisible under a light microscope. Electron microscopy In light microscopy, increased magnification can be achieved easily using the appropriate lenses, but if the image is blurred no more detail will be seen, Resolution is the limiting factor. In electron microscopy, a beam of electrons with a wavelength of less than 1 nm is used to illuminate the specimen. More detail of cell ultrastructure can be seen because electrons have a much smaller wavelength than light waves. They can produce images with magnifications of up to x500000 and still have clear resolution. Electron microscopes have changed the way we understand cells, but there are some disadvantages to this technique. They are very expensive pieces of equipment and can only be used inside a carefully controlled environment in a dedicated space. Specimens can also be damaged by the electron beam and because the preparation process is very complex, there is a problem with artefacts (structures that ‘A Figure 1 Coloured transmission are produced due to the preparation process). However, as techniques electron micrograph of a lymphocyte prove a lot of theses artefacts can be eliminated ee eal aecrefacon ato There are two types of electron microscope: © Ina transmission electron microscope (TEM) a beam of electrons is transmitted through a specimen and focused to produce an image. This is similar to light microscopy. This has the best resolution with a resolving power of 0.51nm (Figure 1). © Ina scanning electron microscope (SEM) a beam of electrons iy sent across the surface of a specinn are collected. The resolving power is from 3-10nm, so the resolution is not as good as with transmission electron microscopy but stunning three-dimensional images of surfaces are produced, giving us valuable information about the appearance of different organisms (see Figure 2). and the reflected electrons ‘A Figure 2 Coloured scanning electron ‘micrograph of a lymphocyte (white blood cel]. Magnification x 2000 Eo} Sample preparation for electron microscopes oe The inside of an electron microscope is a vacuum toensure fractured to expose the inside and will then need to be the electron beams travel in straight lines, Because of this, coated with heavy metals. samples need to be processed in a specific way. 4. Suggest reasons for the following steps in the preparation of samples for electron microscopy: + fixation * embedding in resin Specimen preparation involves fixation using chemicals or freezing, staining with heavy metals and dehydration with solvents. Samples for a TEM will then be set in resin and may be stained again. Samples fora SEM may be + dehydration staining with heavy metals.2.3. More microscopy Table 1 summarises the differences between light and electron microscopy. Y Table 1 A comparison of light and electron microscopy ere inexpensive to buy and operate | expensive to buy and operate ‘small and portable large and needs to be installed simple sample preparation complex sample preparation sample preparation does not | sample preparation often distorts material usually lead to distortion vacuum is not required vacuum is required natural colour of sample is seen | black and white images produced (but can (or stains are used) be coloured digitally) Up to «2000 magnification ‘over 500 000 magnification resolving poweris 2001nm resolving power of transmission electron microscope is 0.51nm and a scanning electron microscope is 3-10nm specimens can be living or dead_| specimens are dead Ae) GON scientific drawings from electron micrographs @ Electron microscopes produce * images with much greater resolution , than ight microscopes and teri $2 much more detail can be seen, \ Producing good scientific drawings K oO chloroplasts from electron micrographs takes cot wat nucleus practice. The same rules used in producing drawings from light micrographs must stil be observed (Topic 2.1, Microscopy). “4 Figure 3 Transmission electron ‘micrograph ofa section through two leaf cells at their junction, Their cell walls run {from top centre to lower lft. Anucleus is Seer it kavernghtShach grovmiles 1 Study the two drawings above and state, with {pale ovais) can be seen in the chloroplast (dark grey, upper left ond right). «18 700 ‘mognification reasons, which of them represents the best ‘scientific drawing. Creation of artefacts An artefact is a visible structural detail caused by processing the specimen and not a feature of the specimen. Artefacts appear in both light and electron microscopy. The bubbles that get trapped under the cover slip as you prepare a slide for light microscopy are artefacts. When preparing specimens for electron microscopy, changes inPe Li Lee ae ko ee the ultrastructure of cells are inevitable during the processing that the samples must undergo. They are seen as the loss of continuity in membranes, distortion of organelles and empty spaces in the cytoplasm of cells Experience enables scientists to distinguish between an artefact and a tue structure. Identifying artefacts Identifying artefacts in microscopy preparation can cause much discussion and controversy, ‘Mesosome’ was the name given to invaginations (inward foldings) of cell membranes that were observed using electron microscopes after bacterial specimens had been chemically fixed. They were thought to be a normal structure, or organelle, found within prokaryotes. The large surface area of the folded membrane was considered to be an important site for the process of oxidative phosphorylation. However, when specimens were fixed by the more recently developed, non-chemical technique called cryofixation, the mesosomes were no longer visible. itis now widely thought that the majority of mesosomes. observed are actually artefacts produced by the chemicals used in the fixation processin electron microscopy preparation, which damage bacterial cell membranes. However, there are still a number of scientists who believe Laser scanning confocal microscopy Light microscopy has also continued to develop. Some images that are very different from electron micrographs but are just as useful. of the latest technology produce Conventional optical microscopes use visible light to illuminate spe is used to illuminate a specimen that has been treated with a fluorescent chemical (a fluorescent ‘dye’) Fluorescence is the absorption and re-radiation of light, Light of a longer wavelength and lower energy is emitted and used to produce a magnified image A laser scanning confocal microscope moves a single spot of focused light across a specimen {point illumination). This causes fluor with a ‘dye’, The emitted light from the specimen \ce from the components labelled is filtered through a pinhole aperture. Only light radiated from very close to the focal plane (the distance that gives the sharpest focus) is detected (Figure 4). imens and a lens to produce a magnified image. In fluorescent microscopes a higher light intensity specimen ‘that some species of bacteria do have mesosomes as part of their normal structure, but this is not the general consensus. This isa good example of haw the scientific community accepted an idea based on the evidence available at the time and as techniques improved and more evirlence became available, the collective knowledge and Understanding developed and changed, New evidence can either provide further support fora theory or disprove an earlier theory. Scientitic knowledge is constantly developing. 41 Structures that look similar to mesosomes have recently been observed in bacteria after treatment with certain types of antibiotics. ‘Suggest, with reasons, whether this information is evidence to support the current theory that ‘mesosomes are artefacts or the theory that they are, in fact, organelles. detector confocal pinhole —“ ‘mination pinhole dichroic abjective lens — excitation lignt == out of focus light — infocus emission focal plane light A Figure 4 The light rays from the laser and the fluorescing sample follow the same path and have the same focal plane2.3. More microscopy You willlearn more about antibodies in Topic 12.6, The ‘specific immune system. You will learn about genetic. engineering in Topic 21.4, Genetic engineering. Light emitted from other parts of the specimen would reduce the resolution and cause blurring. This unwanted radiation does not pass through the pinhole and is not detected. A laser is used instead of light to get higher intensities, which improves the illumination. As very thin sections of specimen are examined and light from elsewhere is removed, very high resolution images can be obtained. The spot illuminating the specimen is moved across the specimen and a two dimensional image is produced. A three dimensional image can be produced by creating images at different focal planes. Laser scanning confocal microscopy is non-invasive and is currently used in the diagnosis of diseases of the eye and is also being developed for use in endoscopic procedures, The fact that it can be used to see the distribution of molecules within cells means it is also used in the development of new drugs. The future uses for advanced optical microscopy inchude virtual biopsies, particularly in cases of suspected skin cancer. The beamsplitter is a dichroic mirror, which only reflects one wavelength (from the laser) but allows other wavelengths (produced by the sample) to pass through. The positions of the two pinholes n eans the light waves from the laser (illuminating the sample) follow the same path as the light waves radiated when the sample fluoresces. This means they will both have the same focal plane, hence the term confocal. Fluorescent tags By using antibodies with fluorescent ‘tags’, specific features can be targeted and therefore studied by confocal microscopy with much more precision than when using staining and light microscopy, Green fluorescent protein (GFP) is produced by the jellyfish Aequorea Victoria. The protein emits bright green light when illuminated by ultraviolet light. GFP molecules have been engineered to fluoresce different colours, meaning different components of a specimen can be studied at the same time. The gene for this protein has been isolated and can be attached, by genetic engineering, to genes coding for proteins under investigation. The fluorescence indicates that a protein is being made and is used to see ‘where it goes within the cell or organism. Bacterial, fungal, plant, and human cells have all been modified to express this gene and fluoresce. The use of these fluorescing proteins provides a non-invasive technique to study the production and distribution of proteins in cells and organisms. ‘a. Define the term resolution with reference to microscopy, b Suggest whether fluorescent microscopy has a higher resolution ‘than normal ight microscopy. Expiain your answer.Pe Li Lee ae ko ee + Atomic force microscopy The atomic force microscope (AFM) gathers information about a specimen by ‘feeling’ its surface with a mechanical probe. These are scanning microscopes that generate three-dimensional images of surfaces. An AFM consists of a sharp tip (probe) on a cantilever {alever supported at one end] that is used to scan the surface of a specimen, When this is brought very close toa surface, forces between the tip and the specimen cause deflections of the cantilever. These deflections are measured using a laser beam reflected from the top of the cantilever into a detector. Fixation and staining are not required and specimens can be viewed in almost normal cll conditions without the damage caused during the preparation of specimens for electron microscopy. Living systems can even be examined. The resolution of AFM is very high, in the order of O.1 nm Information can be gained at the atomic level, even about the bonds within molecules. The pharmaceutical industry in particular uses AFM to identify potential drug targets on cellular proteins and DNA. These microscopes can lead to a better understanding of how drugs interact with their target molecule or cell, AFM is also being emplayed to identify new drugs. Finding and identifying new chemical compounds from the natural world, which may have medical applications, takes a long time, and is expensive. The molecular structures need to be understood before their potential use in medicine is known. Atomic force microscopes can speed up this process, saving money and, potentially, lives. The case study below is a good example of the importance of AFM. Case Study: Deep sea molecules In 2010, scientists working on a species of bacterium froma mud sample taken from the Mariana Trench — the deepest place on the planet located nearly 11.000 metres beneath the Pacific Ocean, found that the bacteria produced an unknown chemical ‘compound. The chemical composition (the number and type of atoms present] was easily determined. However, detector laser beam cantilever lip atoms force surface atoms of specimen ‘A Figure 5 Top: The principle of atomic force microscopy. Bottom: Anuclear pore as shown by atomic force microscopy the molecular structure, the way in which the atoms were joined together, was not so easy to work out and would have taken months using conventional techniques. Using atomic force microscopy the scientists were able to image the molecules at very high, atomic level resolution within one week, giving them the molecular structure they needed This was the first time this method had been used in this way. This new approach could lead to much faster identification of unknown compounds and ultimately ‘speed up the process of the development of new medicines.