User:Mjalalab/sandbox

Microscopy

There are materials, which are very small in size and can not seen by unaided eye. The use of device (i.e. a microscope) to view these materials is called as microscopy. The term microscope was introduced by members of the Italian Accademia de Lincei (Academy of The Lynx), whose most prominent member was G. GALILEI. HANS and ZACHARIAS JENSEN (father and son) from the Dutch town Middelburg invented the compound microscope at the beginning of the 17th century by placing one lens behind the other. They are regarded as the inventors of the telescope, too.

Most of the people often regard the microscope as no more than a magnifying device and do not really appreciate the vital property of resolution. Magnification achieved by microscope can be defined as the ration of the apparent size of the object to the actual size of the structure. In practice, this can be calculated by multiplying the primary magnification of the objective lens by that of the eyepiece. It is possible to increase the magnifying power of the instrument by increasing the power of the objective or eyepiece or both. Above a certain level, simple enlargement does not increase the amount of detail but only serves to increase the size of the image.

Resolving Power and limit of Resolution

The ability to distinguish neighboring points or objects as distinct and separate entities is called as resolving power. The resolving power of our naked (unaided) eye is 0.1 mm (equalent to 100 microns i.e. 01 is equal to 1/1000 mm). Thus any object smaller than 0.1 mm can not be visible to us or objects or points separated by a distance of less than 0.1 mm will be appear as one to the human unaided eye.

01 cm = 10 mm or 1/100 meter 01 mm = 1000  (micrometer) or 1/10 cm 01 m or micron () = 1000 m (milimicron) or 1/1000 mm 01 Nanometer (nm) or 01 milimicron (m) = 10Å or 1/1000  01 Angstrom (Å) = 0.1 m or 0.1 nm

The resolving power represents the capacity of magnification of the lens system or instrument used i.e. microscope. If the resolving power of a microscope is low, the images of two closely placed points will overlap and only a blurred single point will be visible, however microscope having high resolving power will differentiate these two points and will appear sharply distinct.

The resolving power of a microscope depends upon the wavelength of illuminating agent λ and the light gathering capacity of the objective lens, which is called as numerical aperture.

Numerical aperture represents width of cone of illumination.

Numerical aperture: NA = n sin θ where θ =	half the angular width of the cone of rays collected by the objective lens from a typical point on the specimen (since the maximum width is 180o, sin θ can have maximum value of 1) n=	refractive index of the medium (usually air or oil) separating the specimen from the objective and condenser lens. λ =	the wavelength of light used (for white light it is presumed to be 0.53 m)

Thus NA for light microscope is 01 and 1.4 for oil immersion. So, higher the numerical aperture, the greater is the resolution and brighter is the image.

Sin θ cannot exceed 1 and the refractive index of most optical material does not exceed 1.6.

The limit of resolution can be calculated by using the following formula: 0.61 λ Limit of resolution:	   R=				   NA

Thus with monochromatic light (violet light, wavelength λ –400) the limit of resolution cannot exceed 170nm (=0.17 m) and with white light (wavelength 600 nm) the resolving power of microscope can reach upto 250 nm (0.25 m). By using illuminating agents of different wavelength, the resolving power of microscopes have been increased and is now possible to see all cell structures as small as one billionth of a meter (Å). Depending upon the source of illumination, the microscopes are light microscope, electron microscope, X-ray microscope, ultraviolet and fluorescent microscope.

Limit of Resolution:

It is the smallest distance by which 2 objects can be separated and still be distinguishable as 2 separate objects.

The optical theory employed in designing of microscope was given by German physicist Ernst Abbe in 1876. He gave an equation called Abbe’s equation after his name as: λ  		  λ Lm =		= NA		n Sin θ

Where:	Lm = Limit of resolution λ    = Wave length of light to illuminate the object NA = Numerical aperture of the object

So as per the equation, if Lm becomes smaller, the resolution increases and thus finer details can be seen in specimen.

Thus resolving power of a microscope is inversely proportional to the limit of resolution.

There are two ways to reduce the Lm (1)	by decreasing the wavelength of visible light or (2)	by increasing the Numerical aperture (NA)

(1)	The range of wavelength of visible light is 400 nm to 700 nm. From Blue region to Red region less than 400 falls in ultraviolet light and more than 700 falls in infrared.

So in light microscopy the highest resolution can be obtained with light of shortest wavelength i.e. light at blue end spectrum. Therefore, a blue filter is generally used in microscope.

(2)	The Numerical aperture can be increase again by two ways

(a)	The angle of cone of light is increased by using condensor lens. However, there is a limit to it. The objectives have an aperture angle, about 70o (i.e. angle of full cone = 140o ). Sin θ 70o = 0.94

(b)	Increasing refractive index of medium through which light travels.

If air is present between specimen and objective lens, than n=1 (in case of dry lenses).

If Immersion oil is present between objective lens and specimen than n=1.6 (In case of immersion oil objectives). Now if we calculate the Lm of good light microscope than we use the formula of Lm

i.e. 	  	  λ 	       	           400 nm	       400		400 Lm =			     =		           =		      = NA (i.e n sin θ)      1.6 x sin 70o	    1.6 x 0.94	   	1.5

= 266 nm or 0.26 m

So a best light microscope will have a resolution limit of approximately 200 nm or 0.2.. That means 2 adjacent points closer than 200 nm can not seen or recorded as 2 separate object by light microscope. We can see with the help of this microscope only those structures, which roughly have a diameter of 0.2 or more.

1.Light Microscope

The oldest and still the most widely used instrument for studying the structure of organisms and cell is the light microscope. Until 1940, most of the study regarding cell structure was done by using the compound light microscope. Even today, the most popularly used microscope is the compound light microscope. The first operational light microscope was constructed by Janssen and Hans in 1950. The light microscope, so called because it employs visible light to detect small objects, is probably the most well-known and well-used research tool in biology. Light microscopes can magnify objects up to 1,000 times, revealing microscopic details. Light-microscopy technology has evolved far beyond the first microscopes of Robert Hooke and Antoni van Leeuwenhoek.

Types of light microscopes The bright field microscope is best known to students and is most likely to be found in a classroom. Better equipped classrooms and labs may have dark field and/or phase contrast optics. Differential interference contrast, Nomarski, Hoffman modulation contrast and variations produce considerable depth of resolution and a three dimensional effect. Fluorescence and confocal microscopes are specialized instruments, used for research, clinical, and industrial applications.

The essential parts of present-day microscope includes:

(1) Glass lenses that magnify the image of object and focus light on retina of the observer’s eye. It consist of two lens system, one at each end of hollow tube:

(i) an objective lens system which is closer to object and form the initial image of the object and enlarges it. Generally the objective lenses are of various magnifying power which are mounted on a revolving device at lower end of the tube. These are of 10x, 40x, 45x or 100x

(ii)an ocular lens system or eye piece which is closer to eye and forms image of the image and thus further magnifies it. These are also of various magnifying power such as 2x, 5x, 10x, 15x or 20x.

(2) Condenser which focuses light rays on the specimen.

The object is supported on a glass slide under objective lens. It is illuminated by light beneath it. The light is reflected on the object by using mirror (plain or concave). In a good microscope there is a third lens called as condenser which focus the light on object. It is present between light source and object.

The source of illuminating the objects in compound light microscope is visible light. The wavelength of visible spectrum of light ranges from 4000 Å-8000 Å. Taking the average range as 6000 Å, the resolving power of a light microscope will be 3000 Å (3000 angstrom) or 0.3 micron. It means if visible spectrum of light is used for illumination, even the best objective lens cannot resolve structures smaller than 0.3 .

The progress of microscope building enabled a quick series of numerous discoveries in the subjects of histology, cytology and bacteriology. The progress was helped by the development and use of suitable methods of fixation, embedding and cutting, specific dyes and conservatives.

The structures are visualized as a result of differences in light absorption by different portions of the object. In untreated (unstained) cells, the differences in light absorption are less, therefore, fixation and staining are done to improve differentiation of structural components. Methods of sample preparation:

To study the structure of tissue or a cell, first it must be fixed in any medium. Then dehydrated, then embedded in any material to obtained the fine section. The section of these tissues or cell are mounted on glass slides and stained with any dye and put under microscope.

(1)	Fixing of cells: First cells are killed or fixed in any fixatives like alcohol, formaldehyde, picric acid etc. The process of fixation involves various events: (a)	proteins and other organic molecules are precipitated (b)	Intracellular hydrolytic enzymes are denatured. (C)	cross links are formed, making the cells more stable (d)	tissues become stiffer, making their sectioning easy. (e)	affinity to stains increased

2.	Dehydration: Dehydration is the gradual removal of water vapours from the tissue by using organic solvents like ethanol/methanol

3.	Embedding:The dehydrated materials are embedded i.e. infiltrated with paraffin wax which hardens upon cooling. Embedding also provides enough support to allow thin section cutting on a device called as microtome (5-10 micron thick).

4.	Mounting: After section cutting, the ribbon is mounted on a glass slide using egg albumin as adhesive. These sections are deparaffinized in xylene for staining.

5.	Staining:These sections are stained with different chemicals (stains) which can selectively attach to particular molecules of particular cellular structures thereby giving different colours to different cellular structures. These chemicals (stains) are broadly of two types (i) acidic stains and (ii) Basic stains

(i) Acidic stains: These stains bind with basic molecules like proteins of the fixed cells, e.g. Eosine, Aniline blue etc. The cellular structures which stains with acidic dyes are called as acidophillic.

(ii) Basic stains: These stain combine with nucleic acids and other acid molecules and give a particular colour. These stains are methylene blue, crystal violet, haematoxylin etc. Those structures, which stain with basic dyes, are called as basophilic.

Beside these two types of stains, there are certain specific dyes known as cytochemical stains which selectively bind with some specific macromolecules or micromolecules e.g. Millon reaction is used for protein localisation Feulgen reaction for DNA presence Periodic Acid Schiff’s (PAS) reaction for demonstration of polysaccharide materials such as starch, cellulose, hemicellulose etc. Fat-soluble dyes such as Sudan Red and Sudan Black B are used for presence of lipids.

After staining the tissue is again dehydrated in ethanol series, cleared in xylen and mounted for viewing in DPX or Canada balsam.

The picture shows the way the fan of rays of an object that is collected by a single lens is imaged.

If you want to magnify an object even more, two lenses in tandem have to be used (objective and eyepiece or ocular). The resulting construct is a simple microscope. The objective magnifies the object or specimen (O) and a turned up real image (O´) is formed in front of the focal plane of the second lens. The eyepiece than forms an enlarged virtual image (O''), that can be seen as a turned up image at a distance of 250 mm. The magnification of a microscope is thus a product of Vobjective x Vocular ________________________________________

Light path in the microscope: F = Focal plane, O = Object (Specimen), Ob = Objective, Oc = ocular (eyepiece) Dark field and Phase contrast

Phase-Contrast Microscopy

A main obstacle in the microscopy of biological objects is their poor contrast. Only where a contrast exists or where it can be achieved by contrast-enhancing dyes, structures can be made visible. Light-absorbing parts of a preparation weaken the amplitude of the light waves that pass through them. It is thus also spoken of amplitude preparations. The change of stronger and feebler light is perceived by the eye as a difference in brightness. The invisible parts of the preparation are went through by the light without a change of amplitude, but the phase of the light may be changed depending on the consistency of the material. This change is due to the altered speed of the lightwaves. Differences in phase can be perceived neither by the eye nor by a photographical film.

The Dutch physicist F. ZERNIKE succeeded in 1935 to convert phase to amplitude differences. He was awarded the Nobel price for this achievement in 1953. Today his method is known as phase-contrast microscopy and it is by now an integral part of nearly all research and many teaching microscopes. Its eminent advantage is that it allows the examination of living objects and thus to follow the processes within cells. It was the phase-contrast microscope that made it possible to make mitosis visible and even to film it (K. MICHEL, Company CARL ZEISS, 1943).

For the procedure itself a special condenser with a ring-shaped mask and an additional "phase-ring" that is fixed within the back focal plane of the objective is needed. The "phase-ring" has two important tasks:

It has to achieve an alignment of the brightness of refracted and unrefracted light as the rays that pass through the preparation are weakened in their intensity. In contrast to a conventional image gained with the help of a light microscope, the background of a phase-contrast image is thus dark.

The phase difference of most biological preparations is one-quarter of a wavelength or less (lambda / 4). The phase-ring is constructed so as to achieve an additional difference of lambda / 4. The complete phase difference is thus lambda / 2 and crest and trough of refracted and unrefracted rays extinguish each other. A disadvantage of this method is the appearance of light halos around some objects ("halo-effect").

To the left: Arrangement of the ring-shaped mask below the objective and of the phase-ring within the objective. Only the direct light beams are influenced by the phase-ring (according to a works photography of CARL ZEISS).

To the right: The path of light rays within a phase-contrast microscope. 1. ring-shaped mask, 2. condenser, 3. specimen, 4. objective, 5. phase plate, 6. focal plane of the objective. The wave character of the light is indicated by the change of light and dark areas (according to a works photography of CARL ZEISS)

Dark Field Microscopy: This method uses a special condenser with an aperture that big that the light beams that go through it pass by the objective. Only if the object is brought into the center of the light, the light is diffracted, collected by the objective, and used for image formation. Shiny structures are seen in front of a dark background. This method has no very important role in biology, but is impressive with crystals.

Fluorescence Microscopy

This method is of critical importance in the modern life sciences, as it can be extremely sensitive, allowing the detection of single molecules.

Fluorescence microscopy is based on the fact that some molecules emit part of the light absorbed by them as longer waves. A well-known example is the red fluorescence of chlorophyll. It is known that a number of so-called fluorochromes exist, with which microscopic preparations can be stained so that they emit fluorescence indirectly. These vital dyes are used in low concentrations to mark specific parts of living cells or of tissues.

Fluorescence microscopy is a rapid expanding technique, both in the medical and biological sciences. The technique has made it possible to identify cells and cellular components with a high degree of specificity. For example, certain antibodies and disease conditions or impurities in inorganic material can be studied with the fluorescence microscopy.

In fluorescence microscopy, the sample you want to study is itself the light source. The technique is used to study specimens, which can be made to fluoresce. The fluorescence microscope is based on the phenomenon that certain material emits energy detectable as visible light when irradiated with the light of a specific wavelength. The sample can either be fluorescing in its natural form like chlorophyll and some minerals, or treated with fluorescing chemicals.

The basic task of the fluorescence microscope is to let excitation light radiate the specimen and then sort out the much weaker emitted light to make up the image. First, the microscope has a filter that only lets through radiation with the desired wavelength that matches your fluorescing material. The radiation collides with the atoms in your specimen and electrons are excited to a higher energy level. When they relax to a lower level, they emit light.

To become visible, the emitted light is separated from the much brighter excitation light in a second filter. Here, the fact that the emitted light is of lower energy and has a longer wavelength is used. The fluorescing areas can be observed in the microscope and shine out against a dark background with high contrast.

Since fluorescence emission differs in wavelength (color) from the excitation light, a fluorescent image ideally only shows the structure of interest that was labelled with the fluorescent dye. This high specificity led to the widespread use of fluorescence light microscopy in biomedical research. Different fluorescent dyes can be used to stain different biological structures, which can then be detected simultaneously, while still being specific due to the individual color of the dye.

There are two ways in which fluorescence microscopes can be constructed: as epifluorescence- and as transmission microscopes. The second is the older way of construction. Three components are needed:

A strong source of light, that emits mainly short lightwaves. Mercury high pressure lights have proven useful.

The first barrier filter: This filter helps to shut off all radiation other than the one that activates the specific dye. It is placed behind the light source within the light cone. Besides it is advantageous to work with a dark field condenser.

Second barrier filter: This filter is brought into the light cone between objective and eyepiece. It lets through only long wavelengths, that are caused by emission of the preparation (so-called "secondary radiation").

To block the excitation light from reaching the observer or the detector, filter sets of high quality are needed. These typically consist of an excitation filter selecting the range of excitation wavelengths, a dichroic mirror, and an emission filter blocking the excitation light. Most fluorescence microscopes are operated in the Epi-illumination mode (illumination and detection from one side of the sample) to further decrease the amount of excitation light entering the detector.

In the last years the epifluorescence microscope has replaced the transmission fluorescence microscope more and more. But the transmission fluorescence microscope is still better suited to only weakly magnifying objectives (2.5 x, 6.3 x). With the epifluorescence the objective is also a condenser and the stronger it is the more intensive radiation can be used. The heart of epifluorescence is a construction within the light cone between objective and eyepiece that serves to feed activating radiation into the system and is constructed of first barrier filter, beam-splitting mirror and second barrier filter.

Most fluorescence microscopes in use are epifluorescence microscopes (i.e. excitation and observation of the fluorescence are from above (epi) the specimen). These microscopes have become an important part in the field of biology, opening the doors for more advanced microscope designs, such as the confocal laser scanning microscope and the total internal reflection fluorescence microscope (TIRF).

Fluorophores lose their ability to fluoresce as they are illuminated in a process called photobleaching. Special care must be taken to prevent photobleaching through the use of more robust fluorophores or by minimizing illumination.

Epifluorescence microscopy

Epifluorescence microscopy is a method of fluorescence microscopy that is widely used in life sciences. The excitatory light is passed from above (or, for inverted microscopes, from below), through the objective and then onto the specimen instead of passing it first through the specimen. (In the latter case the transmitted exitatory light reaches the objective together with light emitted from the specimen). The fluorescence in the specimen gives rise to emitted light which is focused to the detector by the same objective that is used for the excitation. A filter between the objective and the detector filters out the excitation light from fluorescent light. Since most of the excitatory light is transmitted through the specimen, only reflected excitatory light reaches the objective together with the emitted light and this method therefore gives an improved signal to noise ratio. A common use in biology is to apply fluorescent or fluorochrome stains to the specimen to provide an estimated count.

‎This image uses epifluorescence to image three components of a dividing human cancer cell. DNA is stained blue, a protein called INCENP is green and the microtubules are red. Each fluorophore is imaged separately using a different combination of excitation and emission filters. The images are captured sequentially using a digital CCD camera, then overlaid to give a complete image.

Epifluorescence microscopy can be used to find routine direct total counts of bacteria in water samples.

Excitation and fluorescence with chromatic beam splitters. Similar to the interference filters these are specially coated mirrors used under 45° to the illuminating beam. They reflect certain spectral ranges, while others are completely transmitted. The separating line between reflection and transmission may be set at any point of the spectrum. 1. Exciting radiation, 2. Fluorescence emission. (Redrawn from diagram by CARL ZEISS).

Principle of Fluorescence 1. Energy is absorbed by the atom which becomes excited. 2. The electron jumps to a higher energy level. 3. Soon, the electron drops back to the ground state, emitting a photon (or a packet of light) - the atom is fluorescing

So, a fluorescence microscope is a light microscope used to study properties of organic or inorganic substances 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 labeled with a fluorescent molecule called a fluorophore (such as green fluorescent protein (GFP), fluorescein or DyLight 488). The specimen is illuminated with light of a specific wavelength (or wavelengths) which is absorbed by the fluorophores, causing them 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 beamsplitter), and the emission filter (see figure below). The filters and the dichroic are chosen to match the spectral excitation and emission characteristics of the fluorophore used to label the specimen. In this manner, a single fluorophore (color) is imaged at a time. Multi-color images of several fluorophores must be composed by combining several single-color images.

X-ray Diffraction microscopy

This technique is used to analyse three dimentional structure or tertiary structure of several large molecules like DNA molecules, proteins such as myoglobin, haemoglobin, collagen, myelin sheath of nerve cells, myofibrils of striated muscles etc.

In x-ray microscopy, x-rays are used as source of illumination which possess extremely short wavelength but greater penetration power. Therefore, (i) these rays can pass through comparatively thick biological preparations surrounded by water vapours or gas. (ii) These bears no electric charge and can be brought to focus by reflecting mirrors. (iii) Because of short wavelength, these allow resolution of much finer detail.

Principle: In x-rays diffraction, x-rays of narrow beam are passed through the specimen and which are scattered or diffratcted by atoms of substance. These scattered or diffracted beams are recorded by a photographic plate which is placed at a short distance from the object. If the material through which x-ray are passed has an ordered crystalline atomic structure, the x-ray diffracted pattern is also ordered. The x-ray diffraction pattern reflects 3-dimensional arrangement of atoms in crystal. The diffraction pattern appears as concentric rings or spots which help in calculating the distance between molecules in the specimen.

Uses: With x-ray diffraction, (i) quantitative determination of dry matter as well as the analysis of crystalline structure is possible. (ii) By x-ray diffraction, the arrangement of individual atoms in the molecules can be determined which is not possible with even highest resolution electron microscope e.g. Bernal and Crowfoot presented first X-ray diffraction pattern of pepsin crystal; Astbury (1941) obtained first X-ray pattern of DNA molecule and Watson and Crick proposed double helical structure of DNA based on X-ray diffraction pattern obtained by Franklin and Wilkins.

Electron Microscopy

What are Electron Microscopes?

Electron Microscopes are scientific instruments that use a beam of highly energetic electrons to examine objects on a very fine scale. This examination can yield the following information:

Topography The surface features of an object or "how it looks", its texture; direct relation between these features and materials properties (hardness, reflectivity...etc.) Morphology The shape and size of the particles making up the object; direct relation between these structures and materials properties (ductility, strength, reactivity...etc.) Composition The elements and compounds that the object is composed of and the relative amounts of them; direct relationship between composition and materials properties (melting point, reactivity, hardness...etc.) Crystallographic Information How the atoms are arranged in the object; direct relation between these arrangements and materials properties (conductivity, electrical properties, strength...etc.) ________________________________________ Where did Electron Microscopes Come From?

Electron Microscopes were developed due to the limitations of Light Microscopes which are limited by the physics of light to 500x or 1000x magnification and a resolution of 0.2 micrometers. In the early 1930's this theoretical limit had been reached and there was a scientific desire to see the fine details of the interior structures of organic cells (nucleus, mitochondria...etc.). This required 10,000x plus magnification which was just not possible using Light Microscopes. 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 in Germany in 1931.

The first Scanning Electron Microscope (SEM) debuted in 1942 with the first commercial instruments around 1965. Its late development was due to the electronics involved in "scanning" the beam of electrons across the sample. An excellent article was just published in Scanning detailing the history of SEMs and I would encourage those interested to read it. ________________________________________ How do Electron Microscopes Work?

Electron Microscopes(EMs) function exactly as their optical counterparts except that they use a focused beam of electrons instead of light to "image" the specimen and gain information as to its structure and composition. The basic steps involved in all EMs: A stream of electrons is formed (by the Electron Source) and accelerated toward the specimen using a positive electrical potential This stream is confined and focused using metal apertures and magnetic lenses into a thin, focused, monochromatic beam. This beam is focused onto the sample using a magnetic lens Interactions occur inside the irradiated sample, affecting the electron beam These interactions and effects are detected and transformed into an image The above steps are carried out in all EMs regardless of type. A more specific treatment of the workings of two different types of EMs are described in more detail: A TEM works much like a slide projector. A projector shines a beam of light through (transmits) the slide, as the light passes through it is affected by the structures and objects on the slide. These effects result in only certain parts of the light beam being transmitted through certain parts of the slide. This transmitted beam is then projected onto the viewing screen, forming an enlarged image of the slide. TEMs work the same way except that they shine a beam of electrons (like the light) through the specimen(like the slide). Whatever part is transmitted is projected onto a phosphor screen for the user to see. A more technical explanation of a typical TEMs workings is as follows (refer to the diagram below): The "Virtual Source" at the top represents the electron gun, producing a stream of monochromatic electrons. This stream is focused to a small, thin, coherent beam by the use of condenser lenses 1 and 2. The first lens(usually controlled by the "spot size knob") largely determines the "spot size"; the general size range of the final spot that strikes the sample. The second lens(usually controlled by the "intensity or brightness knob" actually changes the size of the spot on the sample; changing it from a wide dispersed spot to a pinpoint beam. The beam is restricted by the condenser aperture (usually user selectable), knocking out high angle electrons (those far from the optic axis, the dotted line down the center) The beam strikes the specimen and parts of it are transmitted This transmitted portion is focused by the objective lens into an image Optional Objective and Selected Area metal apertures can restrict the beam; the Objective aperture enhancing contrast by blocking out high-angle diffracted electrons, the Selected Area aperture enabling the user to examine the periodic diffraction of electrons by ordered arrangements of atoms in the sample The image is passed down the column through the intermediate and projector lenses, being enlarged all the way The image strikes the phosphor image screen and light is generated, allowing the user to see the image. The darker areas of the image represent those areas of the sample that fewer electrons were transmitted through (they are thicker or denser). The lighter areas of the image represent those areas of the sample that more electrons were transmitted through (they are thinner or less dense) Scanning Electron Microscope (SEM) SEMs are patterned after Reflecting Light Microscopes and yield similar information: Topography The surface features of an object or "how it looks", its texture; detectable features limited to a few manometers Morphology The shape, size and arrangement of the particles making up the object that are lying on the surface of the sample or have been exposed by grinding or chemical etching; detectable features limited to a few manometers Composition The elements and compounds the sample is composed of and their relative ratios, in areas ~ 1 micrometer in diameter Crystallographic Information The arrangement of atoms in the specimen and their degree of order; only useful on single-crystal particles >20 micrometers A detailed explanation of how a typical SEM functions follows (refer to the diagram below): ________________________________________

The "Virtual Source" at the top represents the electron gun, producing a stream of monochromatic electrons. The stream is condensed by the first condenser lens (usually controlled by the "coarse probe current knob"). This lens is used to both form the beam and limit the amount of current in the beam. It works in conjunction with the condenser aperture to eliminate the high-angle electrons from the beam The beam is then constricted by the condenser aperture (usually not user selectable), eliminating some high-angle electrons The second condenser lens forms the electrons into a thin, tight, coherent beam and is usually controlled by the "fine probe current knob" A user selectable objective aperture further eliminates high-angle electrons from the beam A set of coils then "scan" or "sweep" the beam in a grid fashion (like a television), dwelling on points for a period of time determined by the scan speed (usually in the microsecond range) The final lens, the Objective, focuses the scanning beam onto the part of the specimen desired. When the beam strikes the sample (and dwells for a few microseconds) interactions occur inside the sample and are detected with various instruments Before the beam moves to its next dwell point these instruments count the number of interactions and display a pixel on a CRT whose intensity is determined by this number (the more reactions the brighter the pixel). This process is repeated until the grid scan is finished and then repeated, the entire pattern can be scanned 30 times per second. Electron Source (GUN) All Electron Microscopes utilize an electron source of some kind with the majority using a Themionic Gun as shown below: A Thermionic Electron Gun functions in the following manner An positive electrical potential is applied to the anode The filament (cathode) is heated until a stream of electrons is produced The electrons are then accelerated by the positive potential down the column A negative electrical potential (~500 V) is applied to the Whenelt Cap As the electrons move toward the anode any ones emitted from the filament's side are repelled by the Whenelt Cap toward the optic axis (horizontal center) A collection of electrons occurs in the space between the filament tip and Whenelt Cap. This collection is called a space charge Those electrons at the bottom of the space charge (nearest to the anode) can exit the gun area through the small (<1 mm) hole in the Whenelt Cap These electrons then move down the column to be later used in imaging This process insures several things: That the electrons later used for imaging will be emitted from a nearly perfect point source (the space charge) The electrons later used for imaging will all have similar energies (monchromatic) Only electrons nearly parallel to the optic axis will be allowed out of the gun area

In 1924 L. de BROGLIE discovered the wave-character of electron rays thus giving the prerequisite for the construction of the electron microscope. The prototype was built by M. KNOLL and E. RUSKA (Technische Universität Berlin, 1932). One of the first biological objects depicted was the tobacco mosaic virus (TMV). The first picture of a cell was published in 1945 by K. R. PORTER, A. CLAUDE and E. F. FULLAM (Rockefeller Institute, New York). ________________________________________ The Transmission Electron Microscope (TEM)

The conventional electron microscopy is nowadays called TEM (transmission electron microscopy). We will therefore start with its construction. The ray of electrons is produced by a pin-shaped cathode heated up by current. The electrons are vacuumed up by a high voltage at the anode. The acceleration voltage is between 50 and 150 kV. The higher it is, the shorter are the electron waves and the higher is the power of resolution. But this factor is hardly ever limiting. The power of resolution of electron microscopy is usually restrained by the quality of the lens-systems and especially by the technique with which the preparation has been achieved. Modern gadgets have powers of resolution that range from 0,2 - 0,3 nm. The useful resolution is therefore around 300,000 x.

The accelerated ray of electrons passes a drill-hole at the bottom of the anode. Its following way is analogous to that of a ray of light in a light microscope. The lens-systems consist of electronic coils generating an electromagnetic field. The ray is first focused by a condenser. It then passes through the object, where it is partially deflected. The degree of deflection depends on the electron density of the object. The greater the mass of the atoms, the greater is the degree of deflection. Biological objects have only weak contrasts since they consist mainly of atoms with low atomic numbers (C, H, N, O). Consequently it is necessary to treat the preparations with special contrast enhancing chemicals (heavy metals) to get at least some contrast. Additionally they are not to be thicker than 100 nm, because the temperature is raising due to electron absorption. This again can lead to destruction of the preparation. It is generally impossible to examine living objects.

After passing the object the scattered electrons are collected by an objective. Thereby an image is formed, that is subsequently enlarged by an additional lens-system (called projective with electron microscopes). The thus formed image is made visible on a fluorescent screen or it is documented on photographic material. Photos taken with electron microscopes are always black and white. The degree of darkness corresponds to the electron density (= differences in atom masses) of the candled preparation. ________________________________________ The Scanning electron microscope (SEM)

The path of the electron beam within the scanning electron microscope differs from that of the TEM. The technology used is based on television techniques. The method is suitable for the depiction of preparations with conductive surfaces. Biological objects have thus to be made conductive by coating with a thin layer of heavy metal (usually gold is taken). The power of resolution is normally smaller than in transmission electron microscopes, but the depth of focus is several orders of magnitude greater. Scanning electron microscopy is therefore also well-suited for very low magnifications. Numerous examples will be given in the following.

The surface of the object is scanned with the electron beam point by point whereby secondary electrons are set free. The intensity of this secondary radiation is dependent on the angle of inclination of the object's surface. The secondary electrons are collected by a detector that sits at an angle at the side above the object. The signal is then enhanced electronically. The magnification can be chosen smoothly (depending on the model) and the image appears a little later on a viewing screen. ________________________________________

________________________________________ The properties of the light microscope as opposed to that of transmission and scanning electron microscopes are collected in a table.

Finally some outlines of new and further developments are given. The high voltage electron microscope: it operates with an accelerating voltage of 700 - 3000 kV. Its power of resolution is greater, the preparation can be thicker, the strain on the preparation is smaller. But the enormous technical expenditure is disadvantageous. Only few gadgets exist. New results concerning botany have not been gained.

The scanning transmission electron microscope (STEM): In this development of the SEM do the electrons pass through the preparation and the secondary radiation thus generated is used for image formation. Here, too, the expenditure is large, but it is still worthwhile, since large molecules like nucleic acids or proteins or molecular complexes like viruses can be depicted much better and gentler than with the TEM. No news for botany, though.

The interpretation of images gained with electron microscopy is increasingly done with computerized interpretation programs. But they are usually only suitable for the reconstruction of regularly recurring patterns and these, again, are found more often on a molecular level than on a cellular one.

The Transmission Electron Microscope (TEM)

The Scanning electron microscope (SEM) Block diagram of a typical SEM (Redrawn from J. W. S. HEARLE, J. T. SPARROW, P. M. CROSS, 1972)

Preparation Techniques

Biological objects can only be examined with the electron microscope after long and careful preparations. The danger of artefacts is readily given. D. W. FAWCETT wrote in 1964:

"It has to be agreed without reservations that we have no objective criteria to judge the good maintenance of the examined structures. Maybe it is more a belief than a proven fact among morphologists that an image that is clear, continuous, well-ordered, detailed and generally aesthetic is more likely to represent reality than one that is rough, unordered and blurred. But to choose any other criteria as a basic principle would mean encouragement of less carefulness and technical bungling." Years of research and results achieved with different methods finally guaranteed the reliability of the methods and results. Biological preparations - even single cells - are usually too big and too thick to be used as a whole. Normally cross-sections have to be prepared. Sections require the following steps: Fixation of the material, usually with glutaraldehyde (covalent cross-linking of protein molecules) and osmium tetroxide (binds to and stabilizes membranes). Dehydration of the specimen.

Permeation with a monomeric resin that polymerizes to form a solid block of plastic. Without the thus strengthened structures the specimen would collapse in the vacuum of the microscope.

Cutting of the specimen: needed is an ultramicrotome that can produce sections of about 15 - 100 nm thickness (about 1/200 of the thickness of a cell). Ultramicrotomes do normally have fine glass knifes. Edges of break of glass are sharper than metal knifes, but they do not last very long. Diamond knifes are an alternative. They live longer, but are also much more expensive.

The thin sections are placed on a small circular metal grid that is coated with a coal-strengthened plastic (formvar) for viewing in the microscope.

Two different methods for contrast enhancement exist: coating and impregnation with heavy metal ions. Coating is achieved by placing the specimen in a vacuum and exposing it to a cloud of metal dust (platinium, platinium/coal, gold, vanadium, chrome, lead, etc.). The cloud is produced by the heating of a metal filament that is placed at a defined distance and angle from the specimen. The relief-like surface of the specimen helps the formation of irregular structured metal coats on the specimen (relief contrast). The imprint of a specimen can also be taken. Therefore slightly thicker metal coats are prepared that are taken off the specimen before viewing them in the microscope.

When contrast is achieved with heavy metal ions, then the preparation is impregnated with uranyl acetate- or lead citrate solutions. The salts are absorbed by the specimen with different strength, so that differently labeled structures are viewed in the microscope later on. It is spoken of positive staining, if a special structure has absorbed the ions and of negative staining, if the metal ions (phosphoric tungsten acid, uranyl acetate, uranyl formiate and others) accumulate around the actual structure. Negative staining is normally used to make macromolecules and molecular complexes (ribosomes, viruses) visible. Usually a special chemical has to be added that prevents the molecules from getting lumpy.

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