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UNIVERSITY OF VETERINARY AND PHARMACEUTICAL SCIENCES BRNO FACULTY OF VETERINARY HYGIENE AND ECOLOGY Department of Biology and Wildlife Diseases HANDBOOK FOR BIOLOGY AND GENETICS PRACTICAL COURSES Eva Bártová Eva Roubalová Brno 2009 INTRODUCTION This handbook is addressed to the students of the English Master's degree study programme, at the University of Veterinary and Pharmaceutical Sciences in Brno. The book contains protocols for practical courses of “Biology and genetics I and II” that are focused on general biology and on the basis of genetics. We hope that this book will be helpful for you and that it will give you some feeling of the need for scientific knowledge, and how it can be implemented into practice. Protocols are divided into chapters (see “Content”) corresponding to particular lessons. Each chapter contains theoretical introduction, summary of important information, terms and definitions to the respective subject matter followed by individual tasks related to the respective chapter. This introduction is not an exhaustive review of the particular topic. To successfully pass each lesson, you are expected to comprehend the theoretical bases of the chapter by the study of both materials concerning corresponding lecture and additional materials before every lesson (for recommendations of suitable textbooks and study materials see the list at the end of this book). A great deal of the tasks includes microscopic examination. Therefore it is necessary to know how to work with the microscope, with its setting and with various microscopic techniques. You will also master the techniques of microscopic samples preparation (both native and permanent) and you will learn how to design, carry out and evaluate simple biological experiments. In one of the lessons you will learn the most important methods of molecular biology and you will see practical applications of these methods. You will also get the basic information about animal experiments, animal welfare and other related issues, including excursions to user facilities, where experimental animals are kept. Genetic section of the practicals will be based on exemplary genetic tasks solving, but you will also experience e.g. designing and interpretation of Drosophila melanogaster crossing and much more. Your success as a student in this course will require regular attendance, careful note taking (for style of protocol see chapter 1) and the mastery of each particular topic through careful study. You will spend 90 minutes every week in the laboratory practicing your acquired knowledge, therefore you have to study the relevant topics in advance. We strongly recommend you attending the lectures regularly, even if your presence on the lectures is not compulsory. The information provided there gives you the basis for your studies and the personal contact with your teacher gives you the opportunity to discuss the questionable topics, which will help you to make sense of the acquired information more easily. If questions or problems arise, do not hesitate to contact your teacher. We are always willing to talk with you and, if possible, assist you with your concerns. We wish you a successful and exciting first year at our university and all the best in all your academic pursuits. Authors 2 CONTENTS 1. Making records from the lessons ........................................................................................5 2. Methods used for obtaining information in biological sciences ...........................................6 3. Magnifying devices (magnifying glass, light microscope, electron microscope) .................7 3.1. Magnifying glass .........................................................................................................7 3.2. Light (optical) microscope ...........................................................................................8 3.2.1. History of light microscopes ..................................................................................8 3.2.2. Parts of a light microscope ....................................................................................8 3.2.3. Types of light microscopes .................................................................................. 14 3.3. Electron microscopes ................................................................................................. 17 3.3.1. Evolution of electron microscopes ....................................................................... 17 3.3.2. Types of electron microscopes............................................................................. 17 3.4. Microphotography ..................................................................................................... 19 4. Microscopic technique ..................................................................................................... 20 4.1. Dry objectives, centring the objects, iris diaphragm function ..................................... 20 4.2. Optical planes, measuring the size and thickness of microscopic objects .................... 23 4.3. Permanent preparations.............................................................................................. 25 4.4. Native preparation, phase contrast ............................................................................. 28 5. Prokaryotes and immersion microscopy ........................................................................... 31 5.1. Prokaryotes................................................................................................................ 31 5.1.1. Domain Bacteria ................................................................................................. 31 5.1.2. Domain Archaea ................................................................................................. 33 5.2. Observation using immersion objective ..................................................................... 34 6. Chemical composition of bioplasm .................................................................................. 37 6.1. Elements .................................................................................................................... 37 6.2. Chemical compounds ................................................................................................ 37 7. Non-cellular life ............................................................................................................... 42 8. Eukaryotes ....................................................................................................................... 46 8.1. Plant cell.................................................................................................................... 49 8.2. Animal and protozoan cells........................................................................................ 50 9. Research methods in biology............................................................................................ 53 9.1. Cell and tissue cultures .............................................................................................. 53 9.2. Molecular biology techniques .................................................................................... 56 9.3. Care of laboratory animals and animal experiments ................................................... 64 10. Transport of substances, osmosis .................................................................................... 68 11. Cell growth and reproduction ......................................................................................... 71 11.1. Mitosis in plant cell ................................................................................................. 74 11.2. Mitosis in animal cell ............................................................................................... 75 12. Movement and irritation ................................................................................................. 77 12.1. Movement ............................................................................................................... 77 12.2. Irritation .................................................................................................................. 80 13. Reproduction and development ...................................................................................... 82 13.1. Development ........................................................................................................... 84 13.2. Meiosis .................................................................................................................... 86 13.2.1. Meiosis in humans ............................................................................................. 87 14. Influence of surroundings onto the bioplasm .................................................................. 92 14.1. Physical stress .......................................................................................................... 92 14.2. Chemical stress ........................................................................................................ 94 15. Genetics ......................................................................................................................... 96 15.1. Cytogenetics - study of chromosomes, karyotypes ................................................... 97 3 15.2. Model organism - Drosophila melanogaster .......................................................... 102 15.3. Monohybridism ..................................................................................................... 103 15.4. Dihybridism, polyhybridism and branching method ............................................... 106 15.5. Polymorphic genes................................................................................................. 108 15.6. Gene interactions ................................................................................................... 109 15.7. Inheritance and sex ................................................................................................ 112 15.8. Genetic linkage ...................................................................................................... 114 15.9. Population genetics ................................................................................................ 116 15.10. Quantitative genetics ............................................................................................ 118 16. Recommended literature............................................................................................... 120 4 1. Making records from the lessons You have to elaborate a protocol from each lesson onto an undersigned A4 sized paper or into notebook. Each protocol has to contain the date and topic of the lesson, followed by the individual tasks inscribed with number and title and containing drawings of observed objects. The drawings have to be done by a pencil (colored parts can be drawn by a crayons), they have to be large enough (two pictures on one page at the most) and have to contain a description (also by a pencil). The magnification used for the observation has to be pointed out alongside each drawing. In case you prepare the sample yourself, or if you perform an experiment, the protocol has to contain a description of the procedure. The outcome of an observation or the findings resulting from an experiment has to be summarized in “Conclusion”. The protocols have to be at university level including handwriting and overall appearance Submission of complete and accurate protocols at the end of each semester (along with regular attendance and with successful passing of specified tests) is one of the requirements for granting your credit. It is necessary to elaborate the protocol(s) even in case of your absence in the particular lesson. PROTOCOL: Date: Topic of the practical work: TASK 1: (the title of the task) Procedure: Drawing: (sufficiently large, with a pencil and also a crayon, description) Magnification (10/4, 10/10, 10/40 and 10/100 or 40x, 100x, 400x and 1000x): Conclusion: (result and evaluation of observation or experiment) TASK 2: ............. 5 2. Methods used for obtaining information in biological sciences Each scientific branch, including biology, makes prompt efforts to obtain new information, that can supplement or extend current level of knowledge, or that can be instrumental to the verification of scientific hypotheses. In general biology, the main method for obtaining new data is the observation. For observation we can use all our senses (above all the sight), efficiency of which can be multiplied using various instruments (e.g. magnifying glass or a microscope). In biological and medical sciences, more complicated methods, requiring special knowledge and techniques, can be used for obtaining information. These include clinical, biochemical, serological, haematological, immunological, microbiological (bacteriological, virological, parasitological), cytogenetic, histologic or pathoanatomic examinations. It is also possible to employ various procedures involving animals, bacterial or eukaryotic cell cultures, or distinct methods of molecular biology. Additional information can be acquired from medical records, statistics, interviews, and questionnaires, from scientific and professional journals and from various databases available on the internet. The most important biochemistry, virology, haematology, bacteriology, cytology, cytogenetic and molecular biology methods will be detailed in separate chapters. Another method used for obtaining data is an experiment, which is a research method used for the determination of consequences induced in an experimental object by alteration of one of the factors that influence the object. The experiment is performed in the context of solving a particular problem or question, to retain or disprove a hypothesis or research concerning phenomena. Well defined and stable conditions have to be kept during the whole experiment to enable its reproducibility. Usually several replicate samples (duplicates, triplicates…) and both a positive and a negative control are included in an experiment. The results from replicate samples are often averaged, or if one of the replicates is obviously inconsistent with the others, it can be discarded as being the result of an experimental error. A positive control is a procedure that is very similar to the actual experimental test but which is known from a previous experience to give a positive result. The positive control confirms that the basic conditions of the experiment were able to produce a positive result, even if none of the actual experimental samples produce a positive result. The negative control demonstrates the base-line result obtained when a test does not produce a measurable positive result. The value of the negative control is often considered as a "background" value and can be subtracted from the test samples results. Various statistic methods are used to evaluate the results of an experiment. Since one of the main goals of Practice in Biology and Genetics is to learn to observe and classify some nature's phenomena at the microscopic level, the first chapters are applied to the microscopic technique. 6 3. Magnifying devices (magnifying glass, light microscope, electron microscope) The studies of microscopic structures and their functions require the use of various magnifying devices enabling observation of details that are normally below the resolution limits of the human eye (0.2 mm). Optical magnifying devices e.g. magnifying glass, light microscope can enlarge the image of an object 2-2000× depending on the optical system used and on the number and type of lenses employed. The resolution limit is up to 0.2 μm, enabling observation of the eukaryotic and prokaryotic cell. LENSES are simple optical devices with axial symmetry which transmit and refract light, concentrating or diverging the light beams. Lenses are usually made of glass or other suitable transparent material, such as plastic, fluorite (CaF ), synthetic resin, etc. Lenses 2 are usually spherical, which means that their two surfaces are parts of the surfaces of spheres. Each surface can be convex (bulging outwards from the lens), concave (depressed into the lens), or planar (flat). If the lens is biconvex or plano-convex, a collimated or parallel beam of light passing through the lens in parallel with its axis and passing through the lens will be converged (focused) to a spot on the axis, at a certain distance behind the lens (known as the focal length); the lens is thus called a positive or converging (connecting) lens. Such lenses enlarge the image of the observed object. If the lens is biconcave or plano-concave, a collimated beam of light passing through the lens is diverged (spread). In this case, the lens is called a negative or diverging (dispersing) lens. Negative lenses make the image of the observed object smaller. Using an electron microscope we can achieve magnification of about 2 000 000× with the resolution limit up to 2-20 nm, enabling observation of e.g. viruses, that have the average size of about 50 nm. Electron microscopes use electrons to illuminate a specimen and electrostatic and electromagnetic lenses to focus the electron beam, thus creating an enlarged image of the observed object. Electron microscopes have much greater resolving power than light microscopes due to the wavelength of an electron, which is much smaller than that of a photon. The main disadvantages of the electron microscopes are that they are expensive to build and maintain and that they are very sensitive to external influence e.g. vibrations or magnetic fields and that the sample preparation is quite complicated and samples have to be viewed in a vacuum which disables observation of living objects. 3.1. Magnifying glass The magnifying glass is the simplest optical device giving only a small magnification, thus serving for observation of e.g. flower parts, small insects or plankton. Magnifying glasses consist of one or more positive (converging) lenses, magnifying 2-30×. The image of the observed object is acquired using a magnifying glass that is direct (not inverted) and magnified. 7 3.2. Light (optical) microscope 3.2.1. History of light microscopes On September 17, 1683, Dutch amateur microscope inventor Antonie van Leeuwenhoek (1632-1723) wrote to the Royal Society in London about his observations of the samples he prepared from the teeth plaque. Using his simple microscope he found "…an unbelievably great company of living animalcules, swimming more nimbly than any I had ever seen up to this time. The biggest sort bent their body into curves in going forwards. Moreover, the other animalcules were in such enormous numbers, that all the water seemed to be alive". The optical part of this microscope contained only one lens, the sample was placed on a tip in front of the lens and the microscope was held in a hand, close to the eye. This type of microscope magnified up to 270×, reached the resolution of up to 1.35 μm. But Leeuwenhoek was not the first microscope inventor. Around 1595 another Dutch, Zacharias Janssen invented a microscope formed by two lenses placed on the opposite ends of a movable tube. Nevertheless, this microscope magnified only 9× and showed serious optical defects. Microscopes invented by Englishman Robert Hook (1635-1703) reached higher magnifications, however the image quality was low. Optical defects of microscopes th were corrected as late as in the 19 century, when Carl Zeiss Company began to manufacture microscopes using glass with improved features. Early microscopes were monocular, with an external light source (daylight, lamp) and th a mirror. The principle of the optical microscope has not changed since the 19 century, when it had already reached its limits given by physical laws: magnification up to 2000× and resolution up to 0.2 µm. The light microscope is an optical device consisting usually of two systems of lenses: the eyepiece and objective. The light microscope can be monocular (for observation with one eye) or binocular (for two eyes). The latter one is used more often nowadays. In our lessons we will use transmission microscopy which means that the light passes through thin (transparent) objects. Therefore we usually observe small objects in thin layers of medium, thin sections of tissues, smears, compressed objects, etc. 3.2.2. Parts of a light microscope 1. Optical part – eyepiece (ocular) and objective 2. Illumination part – light source, condenser, condenser iris diaphragm, filters and mirror 3. Mechanical part (frame) – base, arm, tube, revolving nosepiece, stage with specimen holder, coarse and fine focus adjustment knobs OPTICAL PART The optical part of microscope includes a system of lenses (or by single lens) that form the eyepieces (lens close to eye) and objectives (the lens close to the object). The objective produces an enlarged, reversed and real image of the observed object which is then seen through the eyepiece and an even more enlarged, reversed, but virtual image is obtained. OBJECTIVES are optic systems composed of several lenses placed in a metal cover. Characteristic features of objectives: Focal length (f) indicates the distance between the lens and the focus. It ranges usually from 1.5 mm (the most magnifying objectives) to 20 mm. Magnification (M) = 250/f, where 250 is conventional working distance of the human eye in mm. It is the ratio between the apparent size of an object and its true size. It is 8 a dimensionless number. The magnification of an objective depends upon its focal length. The shorter is the focal length, the bigger magnification (and the opposite). Free working distance is the distance between the front lens of the objective and the observed object. The greater the magnification of the objective, the shorter the free working distance. Size of the field of view (visual field) decreases with increasing magnification. The greater the magnification, the smaller the field of view. Numerical aperture (NA) = n.sinα, where n is the refractive index of the substance between the objective and the sample and α is the entrance angle of the objective (see Fig. 1). NA is also related to the resolution (the higher NA, the better resolution). NA is an important technical/optical characteristic of the objective and its value is recorded on each objective. Aperture is the ability of the objective to accept (catch) as many light beams as possible. Aperture literally means “the opening“. s´ Immersed Dry objective objective Immersion r´ α oil Cover glass Slide glass A B r s Fig. 1: A – (α) is half the maximal angle under which the objective lens collect light from the object (entrance angle). B – The effect of refractive index of the environment between the objective lens and the cover glass of the sample (comparison of dry and immersion objectives) on the numerical aperture of the objective. In case of dry objective, the transversal ray (r) refracts at the glass/air interface and thus the light does not enter the objective (r´). In case of an immersion objective, the analogical light ray (s) goes straight through slide and cover glass and through immersion oil (lens glass, specimen glass and immersion oil have very similar refractive indexes, thus light rays do not refract) and enters the objective (s´). Resolution (D) is the ability of the objective to distinguish two closely situated points as separate points. D = NA/λ, where NA is a numerical aperture and λ is wave length of used light. This formula can also be written in a different way, depending on how you define D. If you express D in length units (nm, µm), you must use reversed version: D = λ/NA. The resolution of the light microscope is generally limited by the wave length of visible light, which is about 0.2 µm. Lens speed (lens opening) describes the ability of a lens to retain the light rays. It is a qualitative concept related to the relative aperture diameter. A lens may be referred to as "fast" or "slow" depending on its maximum aperture compared to a lens of similar focal length. Lens speed is given by the minimum relative aperture. A lens with a larger maximum aperture is a fast lens because it delivers more light intensity (illuminance) to the focal plane. Lens speed depends on the numerical aperture and on the size of the cone angle (2xα). The amount of light that enters the objective is also dependent on the refraction index (n) of the substance as light rays go through. In case the light passes through slide and cover glass of the sample (n = 1.5) and subsequently through air (n = 1), the light rays refract and some of them miss the objective and are lost. Lens speed can be increased by homogenizing the optical features (refractive indices) of the substance as the light rays go through, e.g. by the use of immersion oil (see Fig. 1). Among the substances that are used for immersion microscopy are e.g. cedar tree oil (n = 1.516), 9 Canada balsam (n = 1.515 – 1.530) or synthetic immersion oils, that are more commonly used. Refraction index of water is n = 1.33. Penetration ability (called depth of field in photographic objectives) is the ability of an objective to render a sharp image of several optical planes of the object at the same time. It is inversely related to NA and thus high magnification objectives have small penetration ability and lower magnification means greater penetration ability. The more you open the iris diaphragm (wider opening = greater aperture), the greater the penetration ability. It is therefore necessary to start the search for inconspicuous objects (such as scattered transparent epithelial cells, or pollen grains) with a damped (dim) light and to add more light when shifting to a greater magnification. Aberration of objectives: Chromatic aberration (or defect) is the phenomenon of different color focusing at different distances from a lens. It causes that an image in white light tends to have colored edges. Spherical aberration (or defect) is a deviation resulting in an image imperfection that occurs due to the increased refraction of light rays when rays strike a lens near its edge, in comparison with those that strike nearer the centre. It makes focusing of lenses less than ideal due to their spherical shape. Types of objectives: 1. According to the substance used between the front lens of the objective and the observed object: a) Dry – objectives with smaller magnification (usually 2-60×), that have air (n = 1) between objective and sample. b) Immersion – more magnifying objectives (usually 90-100×), that are immersed into the medium (medium is between objective and sample). The medium must be removed from both the objective and the permanent sample after finishing the observation using a cloth soaked with ethanol or xylen. Immersion objectives usually have spring-loaded tips that protect them from damage and are marked with a black ring. 2. According to optical features (degree of correction of various aberrations of lenses): a) Achromatic objectives are designed to limit the effects of chromatic aberration. They are corrected to bring two wavelengths (typically yellow and green) into focus in the same plane. Achromatic objectives belong to the most commonly used objectives. b) Planachromatic objectives specified as "plan", will show visual field that is focused both in the centre and at its periphery. Planachromatic objectives have been corrected for both achromatic and spherical aberrations. These objectives belong to the best and the most expensive and are used for microphotography. c) Apochromatic objectives are corrected for both chromatic and spherical aberrations. The chromatic aberration is usually corrected for green, yellow, blue and red colors, while spherical aberration is corrected for two colors. They are used for color microphotography. d) Monochromatic objectives are designed for observation in monochromatic light (light of a single wavelength). They can be used e.g. for microscopy using ultraviolet (UV) radiation. 10 Color markings of objectives: Objectives are usually marked by color stripes. The microscopes in the biology classrooms are equipped by the objectives that are typed in bold: Magnification 1 2 4 10 20 40 50 60 100 black brown green light blue cobalt blue Color red yellow blue white Description of an objective contains (see Fig. 2): Type of an objective (e.g. “A” - meaning that this objective is apochromatic with corrected chromatic aberration, PL - objective for phase contrast microscopy) Magnification (e.g. 40 ) Numerical aperture (0.65) Observation tube length (160 mm) Recommended thickness of the cover glass in mm (0.17 mm) Producer Type of objective (A - achromatic) PL - objective for phase contrast microscopy Numerical aperture Magnification Recommended thickness of the cover glas in mm Observation tube length in mm Fig. 2: Description of an objective. EYEPIECE (OCULAR) is a cylinder containing two or more lenses to bring the image into focus for the eye. The eyepiece is inserted into the top end of the tube. Eyepieces are interchangeable and many different eyepieces can be inserted with different degrees of magnification. Conventional eyepieces magnify 10×. An eyepiece can be equipped with eyepiece micrometer that enables the measuring of observed objects. Eyepiece can also have dioptric adjustment mechanism that allows users who wear eye-glasses to adjust their diopters so they don't need glasses for microscopic observation. The tube also enables the interpupillary distance adjustment that allows to change the distance between the two eyepieces to fit it for user’s individual distance between the centres of his/her pupils. TOTAL MAGNIFICATION (M) = M x M , usually given as a fraction, e. g. objective eyepiece 10/40. This means that a specimen viewed by the 40 magnifying objective is actually enlarged 400 . The magnification of light microscopes used in practicals ranges from 40× to 1000×. (Note: Every drawing of a microscopic object must be completed with these data) Magnification Ocular 10× 10× 10× 10× Objective 4× 10× 40× 100× 40× 100× 400× 1000× Total magnification 11 ILLUMINATION PART Illumination part provides the light to illuminate the sample and to regulate and homogenize light rays. LIGHT SOURCE - both daylight and artificial light (electric bulb) can be used for observation. In most of the modern microscopes, the light source is incorporated in the base of the microscope. CONDENSER is a system of lenses that concentrates the light in the form of a cone on the object (specimen) so that the observed area is equally illuminated. Its distance from the object should be the same as the distance between the object and the objective. However, in some microscopes the condenser is in the fixed highest position. IRIS DIAPHRAGM is a mechanism with an opening (aperture) at its centre, that is used to close and open the hole the light passes through from the light source to the condenser. Iris diaphragm has an adjustable opening (like the iris of human eye), that is shaped in a near- round fashion by a number of movable blades that can change the diameter of the opening, thus regulating the light intensity. MIRROR is important to direct and concentrate the light into the condenser. Microscopes with light source incorporated into the base usually have flat metal mirror incorporated into the base. FILTERS modify the light from the light source to make it suitable for direct observation or for microphotography. Protective filters (yellow or orange) protect eyes from harmful UV radiation, the yellow-green filter is used in phase contrast microscopy to monochromatize the light, polarization filters are used to polarize the light, blue filter made of cobalt glass is used to absorb the yellow component of the artificial light. MECHANICAL PART Mechanical part forms the frame of the microscope and carry the optical and illumination parts. BASE forms the foot of the microscope and harbours the light source, mirror and filter holder. ARM holds the revolving nosepiece, the tube with the eyepieces, the movable stage with specimen holder, and a condenser that is located below the stage. TUBE is connected to revolving nosepiece (on its bottom) and it carries eyepiece(s) on its top. Tubes can be monocular, binocular or even trinocular (one output can be used for redirecting the light into the camera, CCD or other recording device). REVOLVING NOSEPIECE (TURRET) is a part of the microscope that holds two or more different objectives. It is located on the bottom of the tube and it can be rotated to easily change the resolving power. STAGE is a platform below the objective which supports the specimen being viewed. In the centre of the stage, there is a hole enabling the passage of light from illumination parts, through the sample and further into the optical system. Stage is equipped with the movable specimen holder that facilitates searching for desired objects. SPECIMEN HOLDER enables moving the sample in two different directions (left-right and back-forward). It is usually scaled, which allows localization of appropriate region of the sample. COARSE AND FINE FOCUS ADJUSTMENT KNOBS are mounted on both sides of the arm and control the focusing by moving the stage up and down. The larger knurled wheel is to adjust coarse focus, while the smaller knurled wheel controls fine and accurate focusing. The fine focus adjustment wheel is equipped with a graded scale (with divisions at 2.5 µm each), that enables the measuring of the thickness of the observed objects. 12 Eyepiece/ocular Diopter adjustment ring Tube Observation Revolving nosepiece tube securing knob Objective Arm Vertical and horizontal feed Specimen holder knob Stage Coarse focus adjustment knob Condenser iris diaphragm dial Condenser Window lens Base Fine focus adjustment knob Main switch Brightness adjustment knob Fig. 3: Parts of a light microscope. 13 3.2.3. Types of light microscopes 1. According to the number of eyepieces: a) Monocular microscopes b) Binocular microscopes 2. According to the light course: a) Transmitted light microscopes (including inverted microscopes) b) Incident light microscopes (reflected light microscopes) c) Dissecting microscopes 3. According to the light source/illumination type: a) Phase contrast microscopy b) Polarized light microscopy c) Differential interference contrast microscopy d) Dark field microscopy e) Fluorescent microscopy f) Confocal microscopy Monocular microscopes can be used for observation by one eye, with the possibility to use the other eye for drawing. Binocular microscopes enable observation by both eyes. The light collected by the objective is divided by reflecting prisms (and/or a beam splitter) located in the tube equally into both eyepieces. Transmitted light microscopy – the light from the light source is reflected by the mirror into the condenser, where it is condensed into small area. Subsequently it passes through the sample into the objective. Note: microscopes used in the biology classrooms are binocular light (optical) microscopes for observation in transmitted light. Inverted (inversion) microscope – optical system of inversion microscope is „upside-down“, i.e. the light source and condenser are located above the sample, while objectives are below the stage. The inversion microscope is mainly used for observation of living cells in the cell or tissue cultures at the bottom of cultivation vessels (bottles, flasks). Incident light microscopy (reflected light microscopy) – this technique is used for the observation of opaque (non-transparent) objects (e.g. minerals). Because light is unable to pass through these specimens, it must be directed onto the surface and eventually returned to the microscope objective by either specular or diffused reflection. Dissecting microscopes are configured to allow low magnification of larger objects (objects larger or thicker than the compound microscope can accommodate). Dissecting microscopes are binocular and allow seeing objects in three dimensions (3D) i.e. in stereo. Dissecting microscopes utilize both incident light (direct illumination) and transmitted light, enabling observation of opaque objects. Microscopes can be equipped with additional accessories for special microscopic techniques (e.g. phase contrast microscopy, dark field microscopy, UV or polarized light microscopy). Phase contrast microscopy differs from normal transmitted light microscopy in an illumination technique in which a small phase shift in the light passing through transparent specimens is converted into amplitude or contrast changes in the image. This method does not require staining to view the objects, thus enabling to study structures and processes in living cells (e.g. the cell cycle). Phase contrast microscopy proved to be such advancement in microscopy that Dutch physicist Frits Zernike, who discovered this technique, was awarded the Nobel Prize in physics in 1953. In optical microscopy, many objects (e.g. cell parts in protozoan, bacteria or sperm flagella) are essentially fully transparent unless stained (and 14 therefore killed). The difference in densities and composition within these objects, however, often give rise to changes in the phase of light passing through them. The use of phase contrast technique makes these structures visible even in native preparations. Equipment for phase-contrast microscopy consists of diaphragm (a phase ring) located in the phase objective and a correspondent diaphragm, which is located in the phase condenser. Phase contrast illumination can be established only through correctly centring the two diaphragms. A phase microscope (also called an auxiliary microscope) that temporarily replaces one of the oculars is used to centre the objective and condenser diaphragms. When aligned properly, light waves emitted from the illumination source arrive at the eye 1/2 wavelength out of phase. The phase contrast microscope uses the fact that the some of the light passing through a specimen is diffracted (and/or refracted) and due to this is shifted compared to the uninfluenced light. This phase shift is not visible to the human eye. However, the change in the phase can be increased to half a wavelength using the phase condenser and the phase objective, that are inserted into the optical path of the microscope, causing a difference in brightness. This makes the transparent object shine out in contrast to its surroundings. In the positive phase contrast optics, the phase condenser reduces the amplitude of all light rays travelling through the phase annulus by 70 to 90 % and advances the phase by yet another 90° (λ/4). The recombination of these waves with waves that were not shifted results in a significant amplitude change at all locations where there is interference due to 180° (λ/2) phase shifted waves. The net phase shift of 180° (λ/2) results directly from the 90° (λ/4) retardation of the wave due to the phase objective and the 90° (λ/4) phase advancement of the wave due to the phase condenser. π B A C Fig. 4: A – wave length of light ( ) and π = /2, B – quarter a wavelength phase shift (1/4 , i.e. +π/2), C – three quarters a wavelength phase shift (3/4 , i.e. -π/2). B A Fig. 5: Interference: A – light beams in the same phase (sum), B – light beams in reverse phase (difference). Polarized light microscopy (polarizing microscopy) can distinguish between isotropic and anisotropic materials. Isotropic materials which include gases or liquids, demonstrate the same optical properties in all directions. They have only one refractive index and no restriction on the vibration direction of light passing through them. Anisotropic materials which include 90 % of all solid substances in contrast have optical properties that vary with the orientation of incident light with the crystallographic axes. They demonstrate 15 a range of refractive indices depending both on the propagation direction of light through the substance and on the vibrational plane coordinates. More importantly, anisotropic materials act as beam splitters and divide light rays into two parts. Polarizing microscopy exploits the interference of the split light rays, as they are re-united along the same optical path to extract information about these materials. Differential interference contrast microscopy (DIC) or Nomarski Interference Contrast (NIC) is used to enhance the contrast in unstained, transparent samples. It works by separating a polarized light into two beams which take slightly different paths through the sample. Where the lengths of each optical path differ, the beams interfere when they are recombined. This gives the appearance of a 3D physical relief corresponding to the variation of optical density of the sample, emphasizing lines and edges. Image produced using DIC helps to visualize otherwise invisible features. It is similar to image obtained by phase contrast microscopy but without the bright diffraction halo. Dark field microscopy (dark ground microscopy) describes microscopy methods (in both light and electron microscopy), which exclude the unscattered beam from the image. As a result, the field around the specimen (i.e. where there is no specimen to scatter the beam) is generally dark. This microscopy technique requires special illumination parts 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 to form part of the image. Light enters the microscope for illumination of the sample. In dark field microscopy, specially sized disc (the patch stop) blocks some light from the light source, leaving an outer ring of illumination. The condenser lens focuses the light towards the samples; after the light enters the sample, most of it is directly transmitted, while some is scattered from the sample. The scattered light enters the objective lens, while the directly transmitted light simply misses the lens and is not collected due to a direct illumination block. Only the scattered light goes on to produce the image, while the directly transmitted light is omitted. Fluorescent microscopy is a light microscopy technique that is using the phenomena of fluorescence and phosphorescence instead of (or in addition to) reflection and absorption. In most cases, a component of interest in the specimen is specifically labelled with a fluorescent molecule called a fluorophore (or fluorochrome) e.g. GFP (green fluorescent protein) or fluorescein. The specimen is illuminated with light of a specific wavelength(s) that is absorbed by fluorophore, causing it to emit longer wavelengths of light of a different color than the absorbed light. The illumination light is separated from the much weaker emitted fluorescence through the use of an emission filter. Typical components of a fluorescence microscope are the light source (xenon arc lamp or mercury- vapor lamp), the excitation filter, the dichroic mirror (or dichromatic beam splitter), and the emission filter. Also, many biological molecules (e.g. chlorophyll, haemoglobin or some vitamins) are naturally fluorescent, thus emitting light of visible wavelengths after induction by UV light. Confocal microscopy is an optical technique used to increase image contrast and/or to reconstruct 3D image by using a spatial pinhole to eliminate out-of-focus light or flare in specimens that are thicker than the focal plane. Confocal microscopes also use fluorescence phenomenon and they use laser (Light Amplification by Stimulated Emission of Radiation) as a light source. They use “point illumination” and a pinhole in an optically conjugate plane in front of the detector to eliminate out-of-focus information. Only the light within the focal plane can be detected, so the image quality is much better than that of “wide-field” images. As only one point is illuminated at a time, 2D or 3D imaging requires scanning over a regular raster (i.e. a rectangular pattern of parallel scanning lines) in the specimen. The thickness of the focal plane is defined mostly by the square of the numerical aperture of the objective lens, and also by the optical properties of the 16 specimen and the ambient index of refraction. This microscopic technique enables dynamic observations, such as live cell imaging. 3.3. Electron microscopes 3.3.1. Evolution of electron microscopes Some objects, e.g. viruses, are too small to be observed even by the most powerful microscopes equipped with the best glass lenses. This was discovered by a German physicist th Ernst Abbe in the 19 century. The resolving power of optical microscopes is limited by the wavelength of visible light that ranges from 380 to 750 nm. Thus even the best light microscopes are limited to magnifications of approximately 2000 times. Electron microscopes can magnify objects up to two million times due to the wavelength of an electron that is much smaller than that of a light photon, enabling to visualize individual atoms. The first prototypes of electron microscopes (EM) were constructed in the thirties th of the 20 century and exceeded the resolution possible with optical microscopes. Construction of EM was enabled by technological progress in general and interconnected several discoveries of various investigators. One of them was the discovery of the electron by English physicist and Nobel laureate Sir Joseph John Thomson in 1897. The next step leading to the use of electrons to visualize atoms, molecules and other very small objects was the finding published by the French physicist Louis de Broglie in 1925. He introduced the theory of electron waves that included wave - particle duality theory of matter, based on the work of Albert Einstein and Max Planck. He stated that any moving particle or object (including electrons) had an associated wave and thus created a new field in physics - wave mechanics, uniting the physics of light and matter. The electron, carrying a negative electrical charge, is attracted by everything that is charged positively, which can be used to impart it certain velocity that corresponds to particular wavelength. Moreover, the trajectory of moving electron can be influenced by a strong electromagnetic field in like manner the light is influenced during its passage through optical lenses. These features predestined electrons to be used as the new “light” suitable for “microcosms” investigation. Subsequently, two types of electron microscopes (transmission and scanning) were constructed, both of them using beams of accelerated electrons. The first Transmission Electron Microscope was constructed in 1931 by German scientists Max Knoll and Ernst Ruska. In 1986, Ernst Ruska was awarded a Nobel Prize in Physics for his fundamental work in electron optics, and for the design of the first electron microscope. Electron microscopes have advanced the resolving power of up to tenths of nanometres, i.e. the dimensions of atoms. 3.3.2. Types of electron microscopes TRANSMISSION ELECTRON MICROSCOPE (TEM) involves a beam of electrons emitted by an electron gun (usually wolfram cathode). Electrons are accelerated by an anode, focused by electrostatic and electromagnetic lenses and transmitted through a specimen that is partially transparent to electrons and partially scatters them out of the beam. When electrons come through the specimen, they carry information about the structure of the specimen (the "image") that is magnified (about 50×) by the objective lens system of the microscope and by other lenses placed below the objective. The electron image formed by electrons that passes through the specimen is then recorded and projected onto a viewing screen coated with a small amount of fluorescent material (e.g. phosphorus). The image can be photographically recorded by exposing a photographic film or plate directly to the electron beam, or by a CCD (charge-coupled device) camera. 17 The image detected by the CCD may be displayed on a monitor or computer. The Image acquired by TEM is black and white. The operating temperature of an electron gun of TEM is about 2500°C. At such a temperature, interactions of electrons with matter would be very strong. Thus gas particles must be absent in the area where electrons proceed. The required high vacuum is maintained by a vacuum system consisting of various pumps (pre-vacuum pump, diffusion pump, ion getter pump). High Resolution TEMs (HRTEMs) allow the production of images with sufficient resolution to show e.g. carbon atoms in diamond separated by only 89 picometers at magnifications of 50 million times. Preparation of samples for observation by TEM is quite sophisticated and includes various procedures, such as chemical fixation, dehydration (e.g. by freeze drying or by replacement of water with organic solvents, such as ethanol or acetone) that prevents water evaporation; embedding (in resin); sectioning by ultramicrotome with a glass or diamond knife that produces ultra thin (about 90 nm thick) slices of specimen semitransparent to electrons and staining (treatment of samples by contrasting agents, such as lead citrate, uranyl acetate or other compounds containing heavy metals) to scatter imaging electrons and thus giving contrast to different structures (e.g. cell organelles), since biological materials are often too transparent to electrons and thus insufficiently contrastive. Electron gun Eye Eyepiece Condenser Sample Objective Objective Projector Sample Condenser CCD camera Light source (photographic film) Fig. 6: Principle of light microscope. Fig. 7: Principle of transmission electron microscope. SCANNING ELECTRON MICROSCOPE (SEM) is designed for direct studying of the surfaces of objects. By scanning with an electron beam that has been generated by heating of a metallic filament and focused by electromagnetic lenses, an image is formed in similar way as in TV. The electron beam is rastered across the sample and secondary electrons are emitted from the surface of the specimen due to excitation by the primary electrons. Detectors collect these secondary (or backscattered) electrons, mapping the detected signals with a beam position, and convert this information to a signal, thus building up an image. The most important step in sample preparation for observation by SEM is the conductive coating. Since SEM use electrons to produce an image, electrically conductive samples are required. The most widely used procedure for SEM sample preparation is the use of gold atoms to cover the sample surface. Biological samples are placed in a small vacuum chamber, inside of which an electric field is used to remove electrons from argon gas atoms to make positively charged ions that are attracted to 18 a negatively charged piece of gold foil. The argon ions are knocking gold atoms from the surface of the foil. These gold atoms settle onto the surface of the sample, producing a gold coating. The SEM resolution is about an order of magnitude smaller than resolution of TEM, however, the advantages of SEM are the relatively easy sample preparation, the ability to image bulky samples and much greater depth of view (compared to both TEM and optical microscope), producing attractive images that represent the 3D structure even of rugged samples, such as insects. Both light and electron microscopes have their advantages and disadvantages: Light (optical) microscopes use (visible) light to visualize the image. Light rays pass through the optical system of the microscope, usually formed by the glass lenses. The resolution (resolving power) of light microscope is up to 0.2 µm, with magnification up to 2000×. The advantage of light microscope is that it enables observation of native preparations without complicated sample preparation. Images acquired by optical microscope can be colored. Electron microscopes use electrons to visualize the image. Electron beams are controlled and focused by electrostatic and electromagnetic lenses. The average resolution of an electron microscope is about 2-20 nm, with the magnification approx. 2 000 000×. Among the disadvantages of the use of electron microscopes, we can mention their high purchase price, complicated and time-consuming sample preparation and the impossibility to observe living objects. Images acquired by EM are black and white. Favourable is the possibility to obtain 3D image by SEM. Electron gun Scanning generator Condenser Deflector Objective Electron Screen s Detector Sample orek Fig. 8: Principle of scanning electron microscope. 3.4. Microphotography Microphotography (photographic record of a microscopic image) can be divided into: a) Microphotography utilizing classical photographic materials processed in chemical way is employed mainly by users who want to obtain large format photographs. b) Digital microphotography processes digital record of an image. It is preferred by users who want to edit, analyze or archive their images using computer. 19 4. Microscopic technique 4.1. Dry objectives, centring the objects, iris diaphragm function EQUIPMENT FOR MICROSCOPIC OBSERVATION Laboratory glassware: slides (76 × 26 × 1-1.2 mm with or without cut edges), cover glasses or cover slips (square-shaped or rectangular, 18 × 18, 22 × 22, 30 × 40 mm), beakers, test tubes, Petri dishes, wash bottles, graduated cylinders, funnels. Instruments: microscope, water bath, magnifying glass, burner, scissors, scalpel, tweezers, inoculation loop, teasing needle and droppers. Chemicals: dyes and stains, acids, alkalies and their salts, immersion oil, glycerol. MICROSCOPIC OBSERVATION STEP BY STEP: 1. Remove the dust cover from your microscope. 2. Place the microscope to a position that enables comfortable sitting. 3. Check the completeness of the microscope and switch on the microscope using the main switch located on the base. Note: don’t switch off the microscope in between individual observations, because repeated switching shortens the lifetime of the bulb in the light source. 4. Look at the specimen by the naked eye, before placing it on the stage. Check if it is not upside down and look for the position of the object (in some specimens, the approximate position of the object is marked by a circle). 5. Set the revolving nosepiece to the least magnifying objective (4×). 6. Put the specimen on the stage, fasten it by the specimen holder and place the desired object it into the path of the light beam (above the hole in the middle of the stage). 7. Adjust the distance of eyepieces to fit it for the distance between the centres of your pupils. You should obtain one image (by fusion of images from both eyepieces) that you can observe by both eyes in the same time. Sometimes it needs a bit effort to train you to keep both eyes opened when looking through the oculars. Observation with only one eye leads to eye fatigue, headache or if practiced for a long time, it can cause eye disorders. 8. Adjust the diopter adjustment on the eyepiece, if necessary. 9. To arrange the object into the visual field, change simultaneously the focus (left hand, coarse focus knob) and position of the specimen (right hand, specimen holder movement knobs). 10. Adjust the amount of light by rotating the iris diaphragm and/or by rotating the brightness adjustment knob on the base of the microscope. 11. Look into the eyepieces and find the object first by using the coarse focus knob and then focus it by using the fine focus knob. 12. Observe the object first by using less magnifying objectives and then stepwise switch to more magnifying objectives (for adjusting use only fine adjusting knob). Note: place the object into the centre of the visual field, focus it and adjust the brightness both before and after switching to a more magnifying objective. When you lose the object, you must usually return to a smaller magnification or start again from the beginning. 13. Use a meandering (zigzag) movement and go through the whole space under the cover glass systematically. Start in one corner of cover glass and realize that the image is reversed, so moving the specimen to the left leads actually to moving the image to the right and vice versa. 20

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