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Electron Microscopy (MM-535) MUHAMMAD SAJID ALI ASGHAR (Lecturer) 2 Department of Materials Engineering NED University of Engineering and Technology Karachi
Microscopy with Light and Electrons A microscope is an optical / visual system which transforms an object into an image. We are usually interested in making the image much larger than the object, that is magnifying it, and there are many ways in which this can be done. 1. Light Microscope 2. Electron Microscope 3
Methods of image formation There are three basic ways in which an image can be formed. Perhaps the simplest to imagine is the projection image, of which the commonest example is the formation of shadows when an object is placed in front of a point source of illumination, as shown in Figure 1.1. Figure 1.1 The formation of a projection (or shadow) image. Each point in the object is projected directly at the equivalent point in the image. 4
Methods of image formation The second type of image is formed by conventional lens systems, as for example in Figure 1.2, and we shall call this an optical image. 5 Figure 1.2 a single convex lens or 'magnifying glass'. The ray diagram for this is shown in Figure.
Magnifying glass A magnifying glass, is a single convex lens which is used to produce a magnified image of an object. The lens is usually mounted in a frame with a handle. The magnification of a magnifying glass is typically up to 10X 6
Methods of image formation Both projection and optical images are formed in parallel, that is all parts of the image are formed essentially simultaneously. However the third type of image we need to consider is the scanning image, in which each point of the picture is presented serially. The best-known example of this type of image is a television picture, in which several thousand picture points are displayed consecutively, but the process is repeated with such a high frequency that the image appears to the eye in its totality. 7
Pixels The smallest piece of information about the image is contained in one of these picture points. They are generally called pixels. Which is short for picture element. A single domestic TV picture therefore consists of more than 200 000 pixels, each of which can be of a different intensity or colour. The smallest detail which can possibly be shown in the image is a single pixel in size. 8
Pixels the images produced by electron microscopes are stored in computer memory and need to be in a digital form, that is each pixel is coded Such images are often composed of a number of pixels which is a power of two, and common image sizes are 256 x 256 (= 2 8 x 2 8 ) pixels or 1024 x 1024 (= 2 10 X 2 10 ) pixels. Large amounts of computer memory are then needed to store such images. 9
The Light-Optical Microscope The first lens, the objective, provides an inverted image at B with a magnification (V1 f1)if1 and the second lens, the projector, gives a final upright image at a further magnification of (V2 - f2)//2. The image is viewed on a screen or recorded on a photographic plate at C with a total magnification of Figure 1.3 The ray diagram of a simple two-stage projection microscrope. The object 10 is at 0 and the final image at C, with an intermediate image at B.
The Light-Optical Microscope we have a division into two classes of optical microscope: The biologist who needs to look at very thin sections of tissue uses a transmission arrangement such as that shown in Figure 1.4(a), Figure 1.4 The optical systems for the common types of projection microscope. (a) 11 Transmission illumination
The Light-Optical Microscope While the materials scientist or geologist who needs to examine the surface structure of a solid specimen uses a reflection arrangement as shown in Figure 1.4(b). Figure 1.4 The optical systems for the common types of projection microscope. (b) Reflected illumination. 12
Magnification Magnification is the process of enlarging something only in appearance, not in physical size. Magnification is also a number describing by which factor an object was magnified. (X) 13
14 Magnification
Simple Magnification A typical magnifying glass consists of a single thin convex lens that produces a magnification in the range of 1.5x to 30x, with the most common being about 2-4x for reading or studying rocks, stamps, coins, insects, and leaves. Magnifying glasses produce a virtual image that is magnified The unaided human eye can easily detect detail only 0 2 mm in size. 15
Empty Magnification Empty magnification: increasing magnification without increasing the resolving power. Thus any magnification greater than 1000 x only makes the details bigger. We cannot make finer details visible by magnifying the image an extra ten times. This is called as the 'empty magnification', is shown in Figure 1.6. 16
Empty Magnification It is not necessary to build a light microscope with three or more stages of magnification, since this will not improve the resolution but will rather degrade it by introducing extra aberrations 17
18 Effective Magnification
Resolution In order to compare the electron microscope with the light microscope we need to know what factors control the resolution (often called resolving power) Which we will define as the closest spacing of two points which can clearly be seen through the microscope to be separate entities. 19
Microscope Resolution Ability of a lens to separate or distinguish small objects that are close together Wavelength of light used is major factor in resolution shorter wavelength greater resolution 20
Resolution The ability to resolve fine details is called resolution. It is the minimum distance between two points such that the two points are perceived as separated image. R. P. 0.61 N. A. N. A. sin R.P.=Resolving Power, N.A=Numerical Aperture λ=wavelength of the light, β=the half acceptance angle of the lens, µ = refractive index of the lens 21
22 Resolving Power Line
Resolution The resolution limit is d 1 /2. Microscope apertures are normally referred to in terms of the semi-angle, α, which they subtend at the specimen. Where λ is the wavelength of the light μ is the refractive index of the medium between the object and the objective lens. The product, μ sin α is usually called the numerical aperture (NA). 23
Resolution In order to obtain the best resolution (i.e. the smallest r 1 ) it is obviously possible to decrease λ or increase μ or α. sin α can be increased towards 1 by using as large an aperture as possible and μ can be increased by using an oil immersion objective lens. 24
SEM over OM Mag Depth of Field Resolution OM: 4x 1400x 0.5mm ~ 0.2mm SEM: 10x 500Kx 30mm 1.5nm The SEM has a large depth of field, which allows a large amount of the sample to be in focus at one time and produces an image that is a good representation of the three-dimensional sample. The combination of higher magnification, larger depth of field, greater resolution, compositional and crystallographic information makes the SEM one of the most heavily used instruments in academic/national lab research 25 areas and industry.
Depth of Field The range of positions for the object for which our eye can detect no change in the sharpness of the image is known as the depth of field. In most microscopes this distance is rather small and therefore in order to produce sharp images the object must be very flat. If a non-flat object (or a transparent object of appreciable thickness) is viewed at high magnification using a light microscope then some out-of-focus regions will be seen. 26
Depth of Field Optical micrograph SEM micrograph 27
28 Depth of Field
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Depth of Field The only effective way to increase the depth of field is to decrease the convergence angle, which is controlled in most cases by the objective aperture Figure 1.10 shows. Notice that conditions which maximize the depth of field simultaneously make the resolution worse (equation 1.4). It will become apparent later that the use of electrons for microscopy brings a number of advantages, among which are an improvement in both resolution and depth of field. The reason for this is that high energy electrons have a much smaller wavelength than light and the microscopes are usually operated with very small values of α. 30
31 Depth of Field
Depth of Focus A term which is often confused with depth of field is the depth of focus. This refers to the range of positions at which the image can be viewed without appearing out of focus, for a fixed position of the object.. it will not make any difference to the sharpness of the image if the object is anywhere within the range h shown in Figure 1.11. 32
Depth of Field: OM vs SEM Optical Microscope 0.5mm 33 SEM 30mm
Issue: Depth of Focus OPTICAL SEM 34
Electrons versus Light Light as electromagnetic radiation with a wavelength λ and of electrons as atomic particles. Both types of description (wave and particle) of course apply to both light and electrons: 1.The first obvious difference between electrons and light is that their wavelengths differ by a factor of many thousands. 35 Thus light may be described in terms of photons or as radiation of wavelength 400-700 nm, While electrons can also be considered as radiation with wavelengths (useful in microscopy) between about 0 001 and 0 01 nm.
Electrons versus Light 2. Another major difference is that electrons are very much more strongly scattered by gases than is light. This is so severe an effect that in order to use electrons in a microscope all the optical paths must be evacuated to a pressure of better than 10- l opa (about 10-7 of atmospheric pressure) The electrons would scarcely penetrate a few millimetres of air at atmospheric pressure. 36
Electrons versus Light 3. A further major difference between electrons and light is that Electrons carry a charge. Not only does this mean that electromagnetic fields can be used as lenses for electrons but it opens up the possibility of easily scanning a beam of electrons back and forth. Both types of electron microscope, transmission and scanning, the use of electromagnetic lens But in optical microscope use of glass lens 37
Conclusion Electron microscopy therefore offers 1. Higher resolution, 2. Higher magnification, 3. Greater depth of field and 4. Greater versatility than the light microscope, Although at a rather higher price. 38
Aberrations in Optical & EM systems Resolution and depth of field it has been assumed that all the components of the microscope are perfect and will focus the light from any point on the object to a similar unique point in the image. This is in fact rather difficult to achieve because of lens aberrations. Two types of aberration Chromatic aberrations which depend on the spectrum of wavelengths in the light and monochromatic or achromatic aberrations which affect even light of a single wavelength. The effect of each aberration is to distort the image of every point in the object in a particular way, leading to an overall loss of quality and resolution in the image. 39
Chromatic Aberrations Chromatic aberrations occur when a range of wavelengths is present in the light (e.g. in 'white' light) Ray diagram illustrating the introduction of chromatic aberration by a single lens. light of shorter wavelength (blue) is brought to a focus nearer the lens than the longer wavelength (red) light. The smallest 'focused' spot is the disc of least confusion at C. 40
Aberration Corrections All aberration corrections are designed to reduce in size this disc of confusion. In the light microscope there are two ways in which chromatic aberrations can be improved, 1. Either by combining lenses of different shapes and refractive indices or 41 2. By eliminating the variation in wavelength from the light source by the use of filters or special lamps. Both methods are often used if the very best resolution is required,
Monochromatic Aberrations Monochromatic aberrations arise because of the different path lengths of different rays from an object point to the image point. The simplest of these effects is spherical aberration All the monochromatic aberrations are reduced if only the central portion of the lens is used, i.e. if the lens aperture is 'stopped down'. 42
The objective aperture The objective aperture controls the convergence angle A small aperture will reduce the effect of spherical aberration (through a small) and increase the depth of focus, but will limit the beam current. 43 43
Spherical Aberration Lens imperfections lead to different focal lengths in centre and at edges of lens 44 44
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Distortion This will occurs if the magnification of the lens changes for rays off the optical axis. 1. The two possible cases are when magnification increases with distance from the optical axis, leading to pincushion distortion, 2. When magnification decreases with distance from the optical axis, leading to barrel distortion The appearance of a square grid in the presence of (a) barrel and (b) pincushion distortion. 46
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Diffraction Diffraction occurs when a wavefront encounters an edge of an object. This results in the establishment of new wavefronts 48
Diffraction When this occurs at the edges of an aperture the diffracted waves tend to spread out the focus rather than concentrate them. This results in a decrease in resolution, the effect becoming more pronounced with ever smaller apertures. 49
Apertures Advantages 1. Increase contrast by blocking scattered electrons 2. Decrease effects of chromatic and spherical aberration by cutting off edges of a lens Disadvantages 1. Decrease resolution due to effects of diffraction 2. Decrease resolution by reducing half angle of illumination 3. Decrease illumination by blocking scattered electrons 50
Astigmatism If a lens is not completely symmetrical objects will be focused to different focal planes resulting in an astigmatic image 51
Astigmatism The result is a distorted image. This can best be prevented by having as near to perfect a lens as possible but other defects such as dirt on an aperture etc. can cause an astigmatism 52
Astigmatism Lens defect caused by magnetic field asymmetry Astigmatism The inability of the lens to bring to focus both vertical and horizontal lines on the same plane. 53 can be corrected using stigmators! 53
54 Aberrations
Stigmatism and Resolution The shape of electron beam affects SEM image resolution: when the beam is round, or without stigmatism, the image shows small features (high resolution) as seen in Fig. a; when the beam is not round, or with stigmatism, the image details become unclear (lower resolution) as seen in Fig. b. without stigmatism with stigmatism When the image has stigmatism, changing beam focus may result in elongated feature: Figs. c and d were recorded when the beam were under and over focus, respectively. 55 Under focus Over focus 55
Astigmatism -x&y focus at different planes -fix by adjusting stigmators 56 56
Assignment 1. If a small object is placed 2 mm away from a convex lens of focal length 1 mm, how far from the lens will the image be formed? 2. Where is the image produced by a thin convex lens when the object is at the focal point? 3. How many convex lenses, with focal length 1 mm and object distance (u) 1 1 mm, are needed to give a final image with magnification 1 million times? 4. In a light microscope an object is placed 2 mm away from a lens of diameter 2 mm. The object is in air (refractive index = 1) and the wavelength of the (green) light is 520 nm. What is the best possible resolving power of this microscope? 5. Calculate the position of the image and its magnification when an object is held 10 cm from a convex lens of focal length 8 cm. 6. Calculate the depth of field for a resolving power of 1 μm in a microscope with a final aperture of diameter 1 mm and a working distance of 20 mm. What is the depth of focus at a magnification of100lx? 7. If lenses with maximum useful magnifications of 40x are available, how many lenses are needed to achieve magnifications of: 100 x, 10000 x, 1 million x? 8. What are the dimensions of C s? Deduce the approximate value of C s for (a) an electron microscope capable of 0 1 nm resolution, and (b) a light microscope capable of 0 5 mm resolution. 9. Chromatic aberrations can be virtually eliminated by using electrons of a very small range of wavelengths.' Why, in a TEM, can chromatic aberrations never be 57 completely eliminated?