biological tools and techniques

Microscopy

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Microscopy

Content 3.1. Introduction 3.2. Basic Principle of Microscopy 1.2. Simple Microscope 1.3. Compound Microscope

1.4. Light Microscopy and its types 1.4.1. Sample Preparation of Light microscopy 1.5. Electron Microscopy 1.5.1. Transmission Electron Microscopy 1.5.2. Scanning Electron Microscopy

3.1. INTRODUCTION All living organisms are made up of one or more cells which are the basic structural and functional units of life that carries out all the biological processes. Microorganisms such as bacteria, amoeba and yeast are single celled organisms. Humans, on the other hand, are made up of approximately 30 trillion cells (1 trillion = 1012). These cells organize themselves to form tissues and organs. Cells have different shapes and sizes that range from 10 to 50 µm in diameter. Objects that are less than 100 µm in size are generally invisible to naked human eyes. Thus, the resolving power or distinguishing limit between two separate objects as separate entity is also very low. Resolving power can be defined as the ability to distinguish two closely spaced points as two separate points. Structural study of any kind of cells requires magnification of cell size so as to distinguish the various structural compositions present within the cell. Magnification may also be expressed as the act of increasing the size of an object.

3.2. BASIC PRINCIPLE OF MICROSCOPY Microscope is an important scientific instrument that can magnify an image or object. The science of studying microscope and its functions is called microscopy. The formation of image in a microscope depends upon two important parameters - magnification and resolution or resolving power. The property by how much large the object or specimen is enlarged compared to the real object is commonly known as magnification. It is expressed as the ratio of the size of the image to that of the real object. Magnification is a function of the number of lenses. Magnified or increased magnification of an object without increased resolution produces only large but blurred images. The magnification of a compound microscope is the product of the magnification of the objective and the eyepiece. Below the limit of resolution two objects will look like single object. The maximum magnification of a compound light microscope is usually 1500x and has a limit of resolution of about 0.25 μm.

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Biological Tools and Techniques

Resolving Power of Microscope The resolving power of a microscope lens is defined as the ability of the lens to distinguish two particles and render them visible as two different objects rather than one. Thus, resolution is the smallest distance between two objects that can approach one another and still be resolved as two distinct objects. The resolving power is inversely related to the limit of resolution. The limit of resolution is defined as the minimum distance between two points that allows for their discrimination as two separate points. Thus, the higher the resolving power, the smaller the limit of resolution. The limit of resolution of light microscope depends upon the three main factors: the wavelength (λ) of the light used to illuminate the specimen, the angular aperture (α) and the refractive index (n) of the medium surrounding the specimen. The effect of these three variables on the limit of resolution is described quantitatively by the following equation known as the Abbe equation:

Limit of resolution (R) = 0.61λ/n x Sin α The quantity ‘n × sinα’ is called the numerical aperture of the objective lens, abbreviated NA. The NA is the measure of the ability of a lens to collect light from the specimen. Numerical aperture is said to be the "light gathering capability" of a lens. Lenses with a low NA collect less light than those with a high NA. The refractive index is the measure of the extent to which light is slowed down by a given medium. The smaller the value of R the greater the resolving power.

Figure 3.1. Numerical aperture of objective lens.

3.3. TYPES OF MICROSCOPE Traditionally, microscopes can be classified into two types - Simple microscope and Compound microscope.

3.3.1. Simple Microscope This is the simplest form of microscope that consists of single glass lens system mounted on a metal frame that uses visible light as the source of illumination to magnify the image.

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It is also known as optical microscope. A simple microscope is used to obtain small magnifications. It is usually used for study of microscopic algae, fungi and biological specimen. Light from a light source (mirror) passes through a thin transparent object. A biconvex lens magnifies the size of the object to get an enlarged virtual image (Fig. 3.2). The image is viewed from the other side.

Component Parts of a Simple Microscope The component parts of a simple microscope can be divided into two – i) Mechanical part and ii) Optical part. 1. Mechanical parts These parts support the optical parts and help in their adjustment for focusing the object. They include the following components: I) Metal Stand: It has a heavy base plate and a vertical rod fitted to it, which provide support and stability to other parts of the microscope. II) Stage: It is a rectangular metal plate fitted to the vertical rod. It has a central hole for light to pass from below. Slide with specimen to be observed is kept on the stage, in such a way that, the specimen remains just on the central hole. Some microscopes have a pair of slanting wings projecting from the both the sides of the stage. They provide support to hand for manipulating the object.

2. Optical parts These parts are involved in passing the light through the object (specimen) and magnifying its size. The components of the optical parts are as follows I) Mirror: A Plano-convex mirror is fitted below the stage to the vertical rod by means of a frame. It focuses the surrounding light on the object to be observed. II) Lens: A biconvex lens is fitted above the stage, to the vertical rod, by means of a frame. It magnifies the size of the object and the enlarged virtual image formed is observed by keeping the eye above it. For proper focusing, the lens can be moved up and down by the frame.

Figure 3.2. Simple microscope and its magnification.

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3.3.2. Compound Microscope Compound microscopes are modern microscopes made up of combination of more than single lens. Like simple microscopes, compound microscope also uses visible light as the source of illumination. The major components of a compound microscope include a light source and three glass lens system. The light source may be sunlight or some other artificial lights. The three glass system includes the condenser lens, the objective lens and the eye piece lens (Fig. 3.3). Each of these lenses work together to magnify the images of an object. Condenser lens condenses the incident lights coming from light sources to the specimen. Transmitted light through the specimen are collected by objective lens and the image formed are magnified. Magnified images are finally focused at eyepiece lens that increases the size and resolution of the images by further magnification. Eyepiece lens is the lens that conveys the magnified images to the human eye. The image so formed can be viewed directly by eye in the eyepiece or it may be projected to a digital camera. The camera may sometimes be connected to computer system to display the images in computer screens or stored in digital format. Based on the position of light source light microscope may be upright or an inverted light microscope. In an upright microscope the light source lies below the condenser lens. Objective lens lies above the specimen stage. Inverted microscope is a slightly modified microscope where both the condenser and light source are above the specimen stage. Objective lens lies below the specimen stage. (A specimen stage is the component of a microscope where specimen can be kept and moved according to the requirement).

Figure 3.3. Compound Microscope and its image formation.

Microscope may also be classified fundamentally into two types based on the sources of light. Microscopes that use visible light as a source of illumination, and uses a series of glass lenses as the main organ of magnification is known as light microscope. A light microscope can magnify an object up to 1500 time its original size. The resolution power of light microscope is approximately 250 nm (1 µm = 1000 nm). In other higher levels of microscopes called electron microscope, beam of electrons are used as the source of illumination. An electron microscope is used to reveal the ultrastructural and surface

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topological details of a specimen (cell, cell organelles etc.) or object. Electron microscope is much more powerful than the light microscopes. Its resolution power is down to about 0.2 nm which is about 1000 times smaller than the light microscope.

3.4. LIGHT MICROSCOPE Light microscopes are used to observe the tissues or slices of organs which are generally colorless and transparent. Visualization of such structures requires staining of the samples with a dye or staining solutions. Different regions of a cell can be viewed by staining with different stains. The contrast of different structures can also be made by means of differential arrangement of microscope components. This can be done by introducing extra lenses or color filters that changes the pattern of light passing through the specimen. Light microscope may be of several types, such as bright field, dark field, phase contrast, fluorescence or confocal microscopes.

3.4.1. Bright field microscope It is the most commonly used laboratory microscope where a specimen or a tissue section is observed by colourful staining. The contrasting of the image therefore can be obtained by keeping the background white.

3.4.2. Dark field microscope The working principle of Dark Field microscope is similar to conventional compound microscope except that the viewing is done at dark background and the image formed is due to reflected rays. Delicate, brightly illuminated objects or living bodies are observed on a black background. Dark field microscopes are generally used to view the outline or boundary of an object in liquid media such as living spermatozoa, microorganism, or cells in culture. To achieve a dark field the condenser is designed in such a form that it forms a hallow cone of light. It is generally obtained by putting a dark field stop (Fig. 3.4) below the condenser. The light at the apex of the cone is focused at the plane of the specimen; as this light moves past the specimen plane it spreads again in the form of a hollow cone. The objective lens sits in the dark hallow of this cone and no light pass through the objective although the cone of light travel and pass around the lens. The entire area becomes dark if there is no sample and therefore, dark filed microscopy. If a sample placed on the stage the apex of the cone (light) strikes the sample. Once the light strikes at the sample some light will be scattered (reflected rays) by the sample and some lights will be transmitted (oblique rays). Thus, the object becomes brightly illuminated because of the reflected rays which pass through the objective lens of the microscope.

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Figure 3.4. Dark-field microscope image formation.

3.4.3. Phase contrast microscope Protoplasmic compositions of a living cell are generally undetectable in bright field microscopes because of its little contrast and low natural pigmentation. Generally these structures are made visible by staining processes that kill the specimen. Phase contrast microscope is an advanced form of optical microscope where a living cells or specimen can be studied. A microscope that is able to differentiate transparent protoplasmic structures without staining and killing them is the phase contrast microscope.

Working principle of phase contrast microscope The basic principle of phase contrast microscope is based on the property of phase shifting (amplitude) of direct and diffracted light rays. By shifting the amplitudes of light rays proper contrasting can be achieved. Most living biological objects are transparent and the incident light falling on the object emerges as either direct or diffracted rays. Those rays that pass straight through the object are called direct rays. They are unaltered in amplitude and phase. By contrast some rays are diffracted (bent) by the medium and object and emerge as diffracted rays. The diffracted rays are retarded (phase shift) 1/4 (-90°) wavelength compared to direct rays. Diffracted rays reduce the contrasting of an object in a microscopy study. In a phase contrast microscope the direct and diffracted rays can be brought into exact phase called coincidence that result in the increase of brightness because of summation of two rays. On the other hand, if two rays of equal amplitude are in reverse phase (1/2 wavelength off), their amplitudes cancel each other to produce a dark object. This phenomenon is called interference or reverse phase. The former produces bright background and therefore called bright phase microscope and the latter is known as dark phase microscope.

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Figure 3.5. Phase contrast microscope and the mechanism of image formation.

Structural Component of Phase Contrast Microscope All types of compound microscopes share almost similar structural components. Phase contrast microscope, however, differs from other conventional bright field microscope by having 1) different diaphragm and 2) a phase plate. The diaphragm consists of an annular stop that allows only a hollow cone of light rays to pass through the condenser to the object on the slide. The phase plate is a special optical disk located at the rear focal plane of the objective. It has a phase ring on it that advances or retards the direct light rays 1⁄4 wavelength (Fig. 3.5).

Operation of phase contrast microscope a. b.

c. d. e.

Adjust the alignment of annular ring concentrically with phase ring so that a good phase contrast image can be obtained. The brightness of the field in phase contrast microscope is controlled by adjusting the voltage supplied. Considerably more light is required than common bright field microscope since much light are blocked by annular stop. Use optically perfect slides and cover glasses. Use of completely wet mounts slides is better compared to hanging drop of preparations. Increase the intensities of the light slowly.

3.4.3. Fluorescence Microscope A fluorescence microscope is much the same as a conventional light microscope with added features to enhance its capabilities. It is currently the most widely used microscope.

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The various applications of these microscopes include: i) Imaging structural components of small specimens, such as cells, ii) Conducting viability studies on cell populations (are they alive or dead?), iii). Imaging the genetic material within a cell (DNA and RNA), and iv) Viewing specific cell within a large populations with the help of a technique such as FISH. A fluorescence microscope differs from an ordinary bright field microscope in several respects such as: (1) It uses much more powerful light source such as laser light, (2) A dark field condenser is used in place of bright field condenser, (3) It employs three sets of filters to alter the light that passes up through the instrument to the eye and (4) It uses fluorescence dye called fluorochrome to stain the specimen.

Fluorochromes Fluorochromes are a group of fluorescent chemical compounds that can emit light upon absorbance to light. Chemically, fluorochromes contain a large number of aromatic groups or cyclic molecules with several π-bonds. The fluorophore absorbs light energy of a specific wavelength and re-emits light at a longer wavelength. Excitation energies range from ultraviolet through the visible spectrum, and emission energies may continue from visible light into the near infrared region. Examples - fluorescein, rhodamine, Hoechst33342, DAPI, ethidium bromide etc.

Principle of fluorescence Whenever a molecule is illuminated with high energy light changes occurs in the physical state of the molecule. The energy from the light source are absorbed by the molecule which causes the electrons of lower orbital to jump to the higher orbital which is called the excited state of the molecule. Since molecules cannot stay in the excited state for long time, electrons come back to the lower orbital and the molecule attains its normal state called ground state of the molecule. During the time of movement of electrons from excited state to ground state, electrons releases some amount of energy in the form of light, also called emission light. The process of emission of such light by energized molecule is also known as photoluminescence. In photoluminescence there is always a certain time lapse between the absorption and emission of light. If the times lag is greater than 1/10,000 of a second it is generally called phosphorescence. On the other hand, if the time lapse is less than 1/10,000 of a second, it is known as fluorescence. During fluorescence the wavelength of emission light is longer than the wavelength of absorption light. This is because energy loss occurs in the process so that the emitting light has to be of a longer wavelength.

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Figure 3.6. Fluorescence microscope and image formation.

Components of a fluorescence microscope The essential components of a fluorescence microscope are the light source, excitation filter, condenser (dichromatic mirror), and emission filter (Fig. 3.6). Light Source: Fluorescence microscope requires high energy light source. A specific wavelength is used to excite the fluorescent molecules. For an example, the commonly used laboratory fluorophore is excited at 488 nm, and emits maximally at 518 nm. The high-pressure mercury or xenon vapor lamp, and more recently lasers and LED are also used as light sources. Excitation Filters: Different light rays coming from the light source passes through a glass filter called excitation filter. This filter blocks all the other light rays but allows only the required light ray (Example - 448 nm). Condenser or Dichromatic mirror: To achieve the best contrast of a fluorescent object in the microscopic field, a dark field condenser is used. Condenser condenses the light rays to the specimen and therefore excites the molecule.

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Why dichromatic? Fluorescence microscopes use dichroic mirrors, particularly because these mirrors have coatings with high reflectivity for shorter wavelengths, and high transmission for longer wavelengths. Most of the time scientists use them in conjunction with barrier filters and exciter filters to create filter cubes for their microscopes. The orientation of these dichromatic mirrors is generally at a 45-degree angle to the path of the excitation light entering the cube through the reflected light fluorescence illuminator. Their function is to direct the selected wavelengths through the objective and onto the specimen. They also have the additional functions of passing longer wavelength light to the barrier filter and reflecting any scattered excitation light back in the direction of the lamp house. Using dichroic mirrors has many advantages. These mirrors cleanly separate the light spectrum between transmission and reflection while absorbing very little light; this low absorption rate also minimizes the risk of thermal stress and keeps the mirror working clearly and efficiently. They have spectral stability regardless of operating temperatures or humidity levels. Most are also scratch and mechanical resistant.

Barrier or Emission Filter: This filter is situated between objective and the eyepiece. It allows only the emitted light rays to fall to the eyepiece but remove all the remnants of other lights. The fluorescence emitted from the specimen is often too low to be detected by the human eye or it may be out of the wavelength range of detection of the eye. A sensitive digital camera easily detects such signals.

3.4.4. Confocal microscopy Confocal microscopy is a powerful and modern microscopic tool invented by Marvin Minsky in 1955; it produces sharp images of relatively thicker specimens compared to conventional microscope. It is particularly useful for examining fluorescent stained specimens. The image generated by conventional light microscopes do not produce sharp and contrasting image because – (i) If the specimen is thicker than the depth of focus of the objective lens, light coming from structures above or below the plane of focus will also enter the detector (eye or camera), (ii) In fluorescent microscope, any fluorescent molecule present in the specimen above or below the plane of the focal point will also be stimulated and fluorescence light enter the detector (eye), and (iii) Light coming from the focal point will be mixed with the out-of-focal length light rays giving low contrasting blur image in the detector.

Principle of Confocal Microscope Confocal microscopy is a form of light microscopy in which the illuminating light and the light-collecting optics are focused on the same diffraction-limited spot in the specimen. Unlike the conventional microscope, the confocal instrument images only the single spot onto the detector rather than the entire field of view of the objective lens. The image in a confocal microscope is achieved by scanning one or more focused beams of light, usually from a laser or arc-discharge source, across the specimen. It works by increasing the optical resolution and contrast of a micrograph by means of adding a spatial pinhole placed at the confocal plane of the lens to eliminate out-of-focus light. It enables the

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reconstruction of three-dimensional structures from the obtained images by collecting sets of images at different depths within a thick object which is also known as optical sectioning.

Components of confocal microscope A conventional confocal microscopy consists of the following parts such as Light source (laser system), Filters, Scanner, Detector and Pinhole (Fig. 3.7). 1. Light Source: The most commonly used source of light in a confocal microscope is the LASER light. LASER stand for Light Amplification by Stimulated Emission of Radiation which can be defined as the materials with altered distribution of atoms is such that there are more excited atoms, ready to emit energy. The most commonly used light source of a microscope such as mercury and xenon lights are found to be too week for confocal microscope. 2. Filtering Devices: In any fluorescence microscope, like confocal microscope which uses fluorescent stained sample, a filtering device is needed to separate the light beams on the basis of their wavelength. Four different types of filters are used to selectively transmit or block a desired range of wavelengths. a. Short pass filters - they cut off wavelengths longer than a certain wavelength e.g. heat filters are used to exclude infra-red light to reduce specimen heating by illumination. b. Long pass filters - e.g. fluorescent filters that transmit light of longer than a certain wavelength. c. Band pass filter- that transmit light only between a cut-on and cut-off wavelength, especially useful when one is trying to image signals from more than one fluorochrome simultaneously. d. Dichromatic mirrors – that separates the emitted light from the excited light. 3. Scanner: Confocal imaging relies upon the sequential collection of light from spatially filtered individual specimen points, followed by electronic signal processing and eventually the visual display as corresponding image points. The point-by-point signal collection process requires a mechanism for scanning the focused illuminating beam through the specimen volume under observation which is achieved by scanning the stage or the beam. 4. Detectors: In confocal microscopy fluorescence emission is directed through a pinhole aperture positioned near the image plane to exclude light from fluorescent structures located away from the objective focal plane, thus reducing the amount of light available for image formation. As a result, the exceedingly low light levels most often encountered in confocal microscopy necessitate the use of highly sensitive photon detectors that respond very quickly with a high level of sensitivity to a continuous flux of varying light intensity. Thus in confocal microscopy the collection and measurement of secondary emission gathered by the objective is accomplished by several classes of photosensitive detectors.

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5. Pinhole: The optical sectioning capability of a confocal microscope depends on pinhole and its capability to reject out-of-focus light rays i.e., the strength of the optical sectioning depends strongly on the size of the pinhole. Thus, one can assume that making the pinhole as small (approximately 0.25 AU) as possible is the best way to enhance optical sectioning. However, as the pinhole size is reduced a large number of photons that arrive at the detector from the specimen are blocked. This may lead to a reduced signal-to-noise ratio.

Figure 3.7. Confocal microscope and mechanism of image formation.

Types of Confocal Microscopes Four types of confocal microscopes are commercially available 1. 2. 3. 4.

onfocal laser scanning microscopes Spinning-disk (Nipkow disk) confocal microscopes Microlens Enhancer or Duel Spinning Disk Confocal Microscopes. Programmable array microscopes (PAM)

Applications of Confocal Microscope The broad range of applications available to laser scanning confocal microscopy includes a wide variety of studies in neuroanatomy and neurophysiology, as well as morphological studies of a wide spectrum of cells and tissues. In addition, the growing use of new fluorescent proteins is rapidly expanding the number of original research reports coupling these useful tools to modern microscopic investigations. Other applications include resonance energy transfer, stem cell research, photobleaching studies, lifetime imaging, time-lapse imaging, multiphoton microscopy, total internal reflection, DNA

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hybridization, membrane and ion probes, bioluminescent proteins, and epitope tagging. Many of these powerful techniques are described in these reviews.

3.5. SPECIMEN PREPARATION FOR LIGHT MICROSCOPY Preparation of specimen for light microscope depends on the nature of the specimen and the information to be obtained. Cells are generally transparent and optically homogenous. The observation of a biological sample requires preparation of a particular cell or tissue for better contrasting and viewing. The basic steps for the preparation of sample for light microscopy includes – fixation of the sample tissue or organ, dehydration, embedding, sectioning, staining, mounting and finally viewing using light microscopes. Fixation: Cells are delicate and fragile in nature. Fixation allows a cells or tissue to become hard and also preserve them from decay. Many chemicals such as alcohols, formaldehyde, mercuric chloride, picric acid, acetic acid and mixture of these are used as fixatives. Fixation prevents any postmortem changes in the cell and preserves the cell to its natural state by many ways – a. It inactivates the proteolytic enzymes which otherwise digest the cellular content. b. Proteins and other macromolecules are precipitated. c. Fixatives create cross-links between proteins and cytoskeletons leading to the increase of rigidity of the cell. d. Fixation alters the properties of fixed tissue making less palatable to opportunistic micro-organisms. e. Fixation increases the staining affinity of tissue. Dehydration: After fixation the tissue must be dehydrated with series of increasing concentration of alcohols. The purpose of dehydration is to remove the presence of water in the tissue. During dehydration the water molecules from tissue are replaced by alcohol which is followed by washing with xylene which removes the alcohols from the tissue. Infiltration and Embedding: Biological tissue must be supported by hard matrix such as paraffin. After washing with xylene the dehydrated tissue is infiltrated with molten paraffin wax which replaces the xylene. Upon cooling the infiltrated waxes harden the tissue and the tissue can be sectioned into thin slices using a machine called microtome. Sectioning: After the tissue is embedded into the paraffin wax, the tissues must be made into thin slices of 5 - 10 µm. The process of cutting thin slices of tissue using a microtome is called sectioning. Staining: Transparent biological tissue slices are colored by incubating with a specific staining solution which will give the color of its choice. Hematoxylin and eosin is the most commonly used light microscopical stain in histology. Hematoxylin is a basic dye and it stains nucleus give blue color to it. Eosin on the other side is an acidic dye which stains the cytoplasm and gives pink color. Certain specific stains called Cytochemical stains bind

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selectively to some specific groups of cellular macromolecules such as proteins, nucleic acids, polysaccharides and lipids. For example, Millons reagent is used to stain proteins, Feulgen reaction (using Schiff’s reagent) is used for DNA, periodic acid-Schiff (PAS) reaction is used for the demonstration of polysaccharide materials such as starch, cellulose, hemicellulose, and pectin in the plant cells and mucoproteins (glycoproteins), hyaluronic acid and chitin in animal cells; and fat soluble dyes such as Sudan Red and Sudan Black B are used for the lipids. The Sudan Black B is a specific stain for phospholipids and is also use to stain Golgi apparatus.

3.6. ELECTRON MICROSCOPY An electron microscope is a modern and sophisticated microscope that uses highly energetic ‘beam of electron’ as source of illumination and the magnification in obtained by ‘electromagnetic lenses (coils)’ unlike light microscopes where light waves are used to produce image and magnification obtained by optical lenses. Electron microscopes are generally used to study the fine details of the surface topography or ultrastructural details of a cell. The resolution of an image depends upon the wavelength of the light used to illuminate the specimen. The wavelength of an electron can be up to 100,000 times shorter than that of visible light photons and therefore the electron microscope has a higher resolving power. A transmission electron microscope can achieve better than 50 picometer resolution (1 pm = 10-12 m) and magnifications of up to about 10,000,000x whereas most light microscopes are limited by diffraction to about 250 nm resolution and useful magnifications below 1000x. The Electron: An atom is made up of three kinds of particles – protons, neutrons, and electrons. The positively charged protons and neutral neutrons are held tightly together in a central nucleus. Negatively charged electrons surround the nucleus. Normally, the number of protons equals the number of electrons so that the atom as a whole is neutral. When an atom deviates from this normal configuration by losing or gaining electrons, it acquires a net positive or negative charge and is referred to as an ion. The electrons, which are about 1800 times lighter than the nuclear particles, occupy distinct orbits, each of which can accommodate a fixed maximum number of electrons. When electrons are liberated from the atom, however, they behave in a manner analogous to light. It is this behavior which is used in the electron microscope. 1 µm = 10-6 m, (also known as micron); 1 Å = 10-10 m; 1 nm = 10-9 m

Types of electron microscope There are two types of electron microscopes a. b.

Transmission Electron Microscope Scanning Electron Microscope

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3.6.1. Transmission Electron Microscope The Transmission Electron Microscope (TEM) was the first type of Electron Microscope to be developed and is patterned exactly on the Light Transmission Microscope except that a focused beam of electrons is used instead of light to "see through" the specimen. It was developed by Max Knoll and Ernst Ruska of Germany in 1931. The transmission electron microscope can be compared with a slide projector (Fig. 3.8). In a slide projector light from a light source is made into a parallel beam by the condenser lens; this passes through the slide (object) and is then focused as an enlarged image onto the screen by the objective lens. Similarly, in electron microscope, the light source is replaced by an electron source, the glass lenses are replaced by electromagnetic lenses, and the projection screen is replaced by a fluorescent screen, which emits light when struck by electrons, or, more frequently in modern instruments, an electronic imaging device such as a CCD (charge-coupled device) camera. The whole trajectory from source to screen is under vacuum and the specimen (object) has to be very thin to allow the electrons to travel through it.

Figure 3.8. Comparison of image formation in projector and fluorescence microscope.

Working Principle of TEM Transmission electron microscopy uses high energy electrons (up to 300 kV) emitted from a tungsten filament (cathode) which are accelerated to nearly the speed of light from the top of a cylindrical column of about 2 m high. The electron beam behaves like a wave

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with wavelength about a million times shorter than light waves. When an electron beam passes through a thin-section of the specimen electrons are transmitted and scattered. The transmitted and scattered electron beams are focused and magnified by series of electromagnetic lenses until it is recorded by hitting a fluorescent screen, photographic plate or light sensitive sensors like CCD (Charge-Coupled Device) camera. The electromagnetic coils present along the specific intervals of the microscope column act as condenser lens system which focuses the electron beam.

Figure 3.9. Image formation in transmission electron microscope

Component parts of TEM There are four main components or parts for a transmission electron microscope 1. 2. 3. 4.

An electron source (Electron gun) Electromagnetic lens system Sample holder Imaging system

1. Electron Guns: The first and basic part of the microscopes is the source of electrons. It is usually a V-shaped filament made of tungsten filament, also known as the cathode because of high negative potential (Fig. 3.10). Three main types of electron sources are used in electron microscope: tungsten filament, lanthanum hexaboride (LaB6) or field emission gun. Due to negative potential of the electrode, the electrons are emitted from a small area of the filament (point source). A point source is important because it emits monochromatic electrons (with similar energy). The function of an electron gun is to emit an intense beam of electrons into the vacuum which accelerates between the cathode and the anode. There are two main types of electron gun: thermionic electron gun and field emission gun. The metals contain free electrons. The valences are free electrons, which are loosely bound in the nucleus. Those electrons cannot escape from the metal surface. The

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positively charged nucleus will try to pull back the free electrons when they try to escape from the surface. Hence, the electrons have to overcome the potential barrier in order to escape from the surface of the metals. The energy required to overcome this potential barrier is called work function.

Figure 3.10. Electron gun in transmission electron microscope.

A thermionic (tungsten) gun comprises of a filament, a Wehnelt cylinder, and an anode. These three together form a triode gun, which is a very stable source of electrons. The tungsten filament is hairpin-shaped and is heated to about 2700°C. At such high temperature tungsten filament emits electrons into the surrounding vacuum by the process known as thermionic emission. Raising the temperature of the cathode causes the nuclei of its atoms to vibrate with increased amplitude. By applying a high positive potential difference between the filament and the anode, thermally excited electrons are extracted from the electron cloud near the filament and accelerated towards the anode. The anode has a hole in it so that an electron beam, in which the electrons may travel faster than two thousand kilometers per second, emerges and is directed down the column. The Wehnelt cylinder, which is held at a variable potential slightly negative to the filament, directs the electrons through a narrow cross-over to improve the current density and brightness of the beam. Like tungsten, LaB6 guns depend on thermionic emission of electrons from a heated source, a lanthanum hexaboride crystal. LaB6 sources can provide up to 10x more brightness than tungsten and have significantly longer lifetimes, but require higher vacuum levels, which increases the microscope’s cost. The emitting area of LaB6 is smaller than tungsten, increasing brightness but reducing total beam current capability. Field emission guns, in which the electrons are extracted from a very sharply pointed tungsten tip by an extremely high electric field, are the most expensive type of source, but generally provide the highest imaging and analytical performance. High

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resolution TEM, based on phase contrast, requires the high spatial coherence of a field emission source. The higher brightness and greater current density provided by these sources produce smaller beams with higher currents for better spatial resolution and faster, more precise X-ray analysis. 2. Electromagnetic lens system: Like conventional compound microscopes, the TEM requires a series of lenses called an electromagnetic coil that focuses the beam of electrons emitted from the electron gun to the sample. The transmitted electron beams are finally projected and magnified onto a fluorescent screen through a series of electromagnetic coils that act as objective lens. 3. Sample holder: The sample holder consists of a mechanical arm which holds the specimen. It is a standard sized grid on which the self-supporting standard sized sample section is placed. The grids are made up of copper, molybdenum, gold or platinum. A standard TEM grid sizes about 3 mm diameter ring, with a thickness and mesh size ranging from a few to 100 μm. 4. Imaging system: At the bottom part of the column a fluorescent screen is place that emits light when impacted with transmitted electrons. The screen was under vacuum in the projection chamber, but could be observed through a window, using a binocular magnifier if needed. The fluorescent screen usually hinged up to allow the image to be projected on the film below. Modern instruments rely primarily on solid-state imaging devices, such as a CCD (charge-coupled device) camera, for image capture.

What happens in the specimen during the electron bombardment? Contrary to what might be expected, most specimens are not adversely affected by the electron bombardment as long as beam conditions are controlled judiciously. When electrons impinge on the specimen, they can cause any of the following

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Some of the electrons are absorbed as a function of the thickness and composition of the specimen; these cause what is called amplitude (or mass thickness) contrast in the image. Other electrons are scattered over small angles, depending on the composition and structure of the specimen; these cause what is called phase contrast in the image. In crystalline specimens, the electrons are scattered in very distinct directions that are a function of the crystal structure; these cause what is called diffraction contrast in the image. Some of the impinging electrons are deflected through large angles or reflected (back scattered) by sample nuclei. The impinging electrons can knock electrons from sample atoms which escape as low energy secondary electrons. The impinging electrons may cause specimen atoms to emit X-rays whose energy and wavelength are related to the specimen’s elemental composition; these are called characteristic X-rays. The impinging electrons cause the specimen to emit photons (or light); this is called cathodoluminescence.

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Finally, transmitted beam electrons can be counted and sorted by an energy loss spectrometer according to the amount of energy they have lost in interactions with the specimen. The energy loss carries information about the elemental, chemical, and electronic states of the sample atoms.

In a standard TEM, mass thickness is the primary contrast mechanism for non-crystalline (biological) specimens, while phase contrast and diffraction contrast are the most important factors in image formation for crystalline specimens (most non-biological materials).

3.6.2. Sample Preparation for TEM Sample preparation is important for electron microscopy. There are three main steps for sample preparation: processing, embedding and polymerization. 1. Processing: It includes fixation, rinsing, post fixation, dehydration and infiltration. a) Fixation: This is done to preserve the sample and to prevent further deterioration so that it appears as closely as possible to the living state, although it is dead now. It stabilizes the cell structure. There is minimum alteration to cell morphology and volume. Glutaraldehyde is often used as the fixative in TEM. As a result of glutaraldehyde fixation, the protein molecules are covalently cross linked to their neighbors. b) Rinsing: The samples should be washed with a buffer to maintain the pH. For this purpose, sodium cacodylate buffer is often used which has an effective buffering range of pH 5.1 - 7.4. The sodium cacodylate buffer thus prevents excess acidity which may result from tissue fixation. c) Post fixation: A secondary fixation with osmium tetroxide (OsO4), which is to increase the stability and contrast of fine structure. OsO4 binds phospholipid head regions, which creating contrast with the neighboring protoplasm (cytoplasm). OsO4 helps in the stabilization of many proteins by transforming them into gels without destroying the structural features. Tissue proteins are stabilized by OsO4 and do not coagulate by alcohols during dehydration. For imaging electrons scattering, heavy metals like uranium and lead are used and thus, it gives contrast between different structures. Thus, adding more electron density to the internal structures. d) Dehydration: The water content in the tissue sample should be replaced with an organic solvent since the epoxy resin used in infiltration and embedding step is not miscible with water. e) Infiltration: Epoxy resin is used to infiltrate the cells. It penetrates the cells and fills the space to give hard plastic material which will tolerate the pressure of cutting. 2. Embedding: After processing the next step is embedding. This is done using flat molds. 3. Polymerization: Next is polymerization step in which the resin is allowed to set overnight at a temperature of 60°C in an oven.

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4. Sectioning: The specimen must be cut into very thin sections for electron microscopy so that the electrons are semitransparent to electrons. These sections are cut on an ultramicrotome which is a device with a glass or diamond knife. For best resolution the sections must be 30 to 60 nm. The resin block can be made ready for the sectioning by trimming it at the tip with a razor blade or black trimmer so that the smallest cutting face is available. Fix the block to a microtome and cut the sections. Sections float onto a surface of liquid held in trough and remain together in a form of ribbon. Freshly distilled water is generally used to fill the trough. These sections are then collected onto a copper grid and viewed under the microscope.

3.6.3. Scanning Electron Microscope Scanning electron microscope (SEM) is another important tool for biological sciences. It was invented soon after the TEM but took longer to be developed into a practical tool for scientific research by Manfred von Ardenne in 1937. Unlike, TEM, SEM do not produce ultrastructural details of a specimen but produces the fine surface topography or external morphology of a specimen. A focused beam of electrons interact with the sample and produces various signals that contain the information about sample’s surface morphology and composition. In a SEM, areas ranging from approximately 1 cm to 5 microns in width can be imaged in a scanning mode using conventional SEM techniques.

Working Principle of SEM Beam of electrons emitted by the electron guns fitted with tungsten filament cathode passes through the condenser lenses down to the specimen. The electron beam with energy of 0.2 kV to 40 kV is focused by one or two condenser lenses to a spot of about 0.4 nm - 5 nm in diameter. When the primary beam of electrons interacts with the sample, the electrons lose energy by repeated random scattering and absorption within a very small volume of the specimen known as interaction volume. This causes the release of secondary electrons and other types of radiations from the sample. The intensities of the secondary electrons depend upon the shape and the chemical compositions of the irradiated samples. The electrons are collected by electron detectors generating electronic signals. These signals are scanned in the manner of a television system to produce an image. Various electronic amplifiers amplify the signals which are finally displayed with various brightness on a computer monitor.

Microscopy

Figure 3.11. Scanning electron microscope image formation and the instrument (right).

Resolution of SEM The resolution of a SEM is determined by the size of the region (specimen) from which the signal originates. Certainly this will not be smaller than the extent of the spot illuminated by the beam on the sample surface. In conventional SEM, it is easier to form a smaller spot at higher beam energies because the degrading effects of chromatic aberration are relatively less significant. However, at higher beam energies, the electron beam penetrates deeply and scatters widely within the sample, contributing signal from locations well outside the spot and thus degrading image resolution. When beam energy is reduced in a conventional SEM, spot size increases as the fixed energy spread among electrons in the beam becomes larger relative to the nominal.

Magnification in SEM Magnification in an SEM can be controlled over a range of about 6 orders of magnitude from about 10X to 30,000X times. Unlike optical and transmission electron microscopes, image magnification in a SEM is not a function of the power of the objective lens. SEM may have condenser and objective lenses, but their function is to focus the beam on to the specimen as small spot, and not to magnify the image of the specimen. Provided the electron gun can generate a beam with sufficiently small diameter, a SEM could in principle work entirely without condenser or objective lenses, although it might not be very versatile or achieve very high resolution. In a SEM, as in scanning probe microscopy, magnification results from the ratio of the dimensions of the raster on the specimen and the raster on the display device. Assuming that the display screen has a fixed size, higher magnification can be obtained by reducing the size of the raster on the specimen and viceversa. Magnification is therefore controlled by the current supplied to the x, y scanning coils, or the voltage supplied to the x, y deflector plates, and not by objective lens power.

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Component Parts of a SEM A scanning electron microscope, like a TEM, consists of an electron source (gun), a vacuum system, condensers, detectors and monitoring software (Fig. 3.11). The column is considerably shorter because the only lenses needed are those above the specimen used to focus the electrons into a fine spot on the specimen surface. 1. Electron Source: Electron beams in EM are produce by thermionic heating. The electrons are then accelerated to a voltage between 1 – 40 kV. SEM also uses 3 common types of electron sources – tungsten filament, solid state crystal (LaB 6) or field emission gun. 2. Condenser electromagnetic lenses: A series of electromagnetic lenses (2 to 3 condenser lens) focuses the electron beam as it moves from the electron source down the column to the specimen. The main role of lens is to control the size of the beam and for a given objective aperture size, determines the number of electrons in the bean which finally heat the sample. Unlike TEM, SEM does not possess lenses below the specimen. 3. Sample Chamber: Sample chamber is the chamber where researchers place their sample or specimen for observation. The sample must be keep extremely still to produce clear image and therefore the sample chamber must be very sturdy and insulated from the vibrations. The specimen chamber is larger in EM compared to TEM because the SEM technique does not impose any restriction on specimen size other than that set by the size of the specimen chamber. Images can also be manipulated by rotating the angles of the specimens. 4. Detectors: The electrons coming out from the specimen are detected by several detectors in SEM. In SEM, two types of electrons are typically used for imaging: secondary electrons and backscattered electrons. Secondary electrons are low energy electrons produced when electrons are ejected from k-orbital of the sample atoms by the focused electron beam. The most commonly used secondary electron detector is the EverhartThornley detector. Backscattered electrons are higher energy electrons that are elastically backscattered by the atoms of the specimen. Atoms with higher atomic numbers backscatter more efficiently and therefore these detectors can give compositional information about the specimen. These detectors can be either scintillators or semiconductors.

3.6.4. Sample Preparation for SEM Conventional SEM operates at very high vacuum conditions to avoid gas molecules interfering with both the primary electron beam and the secondary or backscattered electrons emitted from the sample. This means that everything going into the SEM must be completely dry and free of any organic contaminants that may potentially outgas in a high vacuum environment. This poses a problem when dealing with biological specimens which are largely composed of water, necessitating additional preparatory steps to ensure the native structure of the organism is retained. The steps necessary depend on sample type and the purpose of the study. Some types of biological tissues or specimens, including

Microscopy

insects like beetles with hard exoskeletons, will require less stringent processing to preserve their structure. Other more delicate types of samples will take time and care to prepare to avoid the introduction of drying artifacts, such as shrinkage and collapse that can be seen in this micrograph of poorly preserved fungal spores. In order to produce a good image in a SEM, a biological sample needs to be processed as described below. 1. Fixation: Fixation is the first and most important step for optimum preservation of biological samples. Transferring the samples immediately into fixative prevents any cellular changes. Most commonly used fixative for biological samples include 2.5 - 3% glutaraldehyde in either phosphate or cacodylate buffer (about pH 6.8 - 7.4). Another popular fixative is paraformaldehyde, which is usually mixed with phosphate buffer (Davidson’s fixative), or with a low concentration of glutaraldehyde together with phosphate buffer. For some specimen primary fixation may be followed by secondary fixation in 1% osmium tetraoxide. However, the post-fixation step is often bypassed if the specimen has been fixed for long enough in the aldehydes. 2. Washing and dehydrating procedures: Post-fixation, samples need to be washed and dehydrated with graded series of ethanol or acetone (for example 50%, 60%, 70%, 80%, 90% and 100% ethanol/acetone). Dehydration Procedure a. b. c. d.

Rinse your samples with fresh buffer (no fixative added) - repeat three times. Replace buffer solution with the lowest concentration ethanol or acetone solution in your dehydration series – e.g. 50%, and leave for 10 - 20 minutes. Continue with this process until your samples are in 100% ethanol/acetone, then repeat the 100% ethanol step. After the samples are dehydrated, they are now ready for drying.

3. Drying All the specimens placed in the SEM are examined under vacuum and therefore they must first be thoroughly dried. Direct air drying can result in considerable distortion of specimen shape due to the adverse effects of surface tension forces. Any deformation that occurs may then be mistakenly identified as a native feature of the sample, or as an effect of a particular treatment applied. Therefore, biological samples are dried via ‘Critical Point Drying’ (CPD) to avoid these effects. The basic principle of CPD lies in the fact that a liquid CO2 held in the closed chamber will simultaneously expand and evaporate when subjected to an increase in temperature. As the kinetic energy of molecules in the liquid phase increases, more of them enter the gas phase resulting in a progressive decrease in density of the liquid and consequent increase in density of the gas. At a certain combination of temperature and pressure, the "critical point", the densities of both phases are equal and the boundary between them disappears, thus reducing surface tension to zero. The 100% ethanol/acetone dehydrated specimen is placed in the chamber of a CPD apparatus. The chamber is then sealed and cooled, as valves are opened to let liquid

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CO2 in and vent ethanol out, until liquid CO2 has completely replaced the ethanol. The chamber is then sealed again and slowly heated. When the chamber pressure exceeds 1072 psi and the temperature exceeds 31°C, a critical point is achieved whereby the liquid and gas phases of CO2 are in equilibrium. The CO2 is then slowly drained from the chamber and the sample as a gas, thereby avoiding the effects of surface tension that occur as water changes from liquid to gas. Q. Why is liquid CO2 used during critical point drying? Liquid CO2 is used as a transition element during critical point drying because it has low critical point than water. Since CO2 is miscible in ethanol, not in water, samples are dehydrated with ethanol before drying. By adding liquid CO2 to the chamber, the dehydrating solvents are vented off. Once the temperature and pressure is increased to slightly above 31ºC about 72.9 atm CO2 obtain its critical point and both the liquid and gas phase of CO 2 become equilibrium reducing the surface tension to nearly zero. Although other solvents can also be used as transitional element, it is readily available, inexpensive, environmentally friendly, and has a reasonable critical temperature and pressure (31.1°C and 72.9 atm). Q. Can we use specimen fixed for light microscopy? It is also possible to process specimens that have been previously preserved for light microscopy since most of the common fixatives used, i.e. AFA (acetic acid, formaldehyde and alcohol), Bouin's, etc., preserve structure well enough for SEM examination. Some specimens can be adequately preserved by simply immersing them directly into boiling 95% ethanol.

4. Gold Coating The gold splutter coater is a machine that we use to coat the mounted specimens in gold before they go into the SEM. The reason why we gold coat the specimens is because the SEM uses an electron beam instead of a light globe to illuminate the specimen. There are two detectors in the SEM chamber that are used to detect the two types of electrons that are bouncing of the gold metal specimen. It is these electrons - secondary and backscatter that go to make up an image of the specimen. This image of the electrons is what we are seeing, not the reflected light image we see in a light microscope. If the specimen is not finely covered with a metal like gold we will get a very poor signal thus the image derived will be very dark and perhaps not even there.

Advantages and Disadvantages of SEM Advantages of SEM 1. Electrons in scanning electron microscopy penetrate into the sample within a small depth, so that it is suitable for surface topology, for every kind of samples (metals, ceramics, glass, dust, hair, teeth, bones, minerals, wood, paper, plastics, polymers, etc.). 2. It can also be used for chemical composition of the sample’s surface since the brightness of the image formed by backscattered electrons is increased with the atomic number of the elements. This means that regions of the sample consisting of

Microscopy

light elements (low atomic numbers) appear dark on the screen and heavy elements appear bright. 3. Backscattered are used to form diffraction images, called EBSD, that describe the crystallographic structure of the sample. 4. In SEM, X-rays are collected to contribute in Energy Dispersive X-Ray Analysis, which is used to the topography of the chemical composition of the sample. 5. The quality of the image in a SEM depends on the orientation and distance of the specimen from the detectors and the final lens. The specimen stage allows the specimen to be moved in a horizontal plane (X and Y directions), up and down (Z direction), rotated, and tilted as required. These movements are generally motorized and controlled by a computer using a joystick or mouse.

Disadvantages of SEM Unlike TEM, SEM is only used for surface topography study and both resolution and crystallographic information are limited (because they’re only referred to the surface). Other constraints are firstly that the samples must be conductive, so non-conductive materials are carbon-coated and secondly, that materials with atomic number smaller than the carbon are not detected with SEM.

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