MAGNIFICATION V/S RESOLUTION

EMN 521

Presented by: Loveleen Kaur L-2007-BS-27-D 2007-BS-27Ph.D. (Microbiology)

INTRODUCTION
Distance of distinct vision: 25 cm for 1X Smallest distance between two points that we can resolve by our eyes : 0.1-0.2 mm. 0.1The instrument that can show images revealing detail finer than 0.1 mm could be described as a microscope. First compound microscope: Hans and Zacharis Janssen (1590) Purpose of microscope: magnification and resolution Magnification alone not sufficient Both magnification and resolution necessary

MAGNIFICATION
DEFINITIONS
Measure of increase in diameter or size Ratio of distance between two pts in image to those in object i.e. M=v/u Ratio of resolving power of eye to resolving power of microscope i.e. M= eye/ microscope
A section of a cell of Bacillus subtilis, in TEM.

VIEWING OPTIONS OF MICROSCOPES

MAGNIFICATION CONTD...
Principle: Closer an object is brought to eye, larger it becomes and more detail we can see. Overall magnification = Objective lens x Eyepiece lens The magnification of the objective depends on its focal length fo and on the distance d (called the tube length) M=d/fo M=d/fo The magnification of the eyepiece depends upon its focal length fe

RESOLUTION


Shortest distance between two points on a specimen that can be distinguished by the observer as separate entities. entities. Ability to discriminate two closely placed structures as separate. separate. Resolving power of human eye: 0.2 eye: mm. mm. Resolving power of optical microscope: microscope: 0.2 m Resolving power of electron microscope: microscope: 0.2 nm

o o o

RESOLUTIONu
Limited by the wavelength of radiation used. used. Objects in the specimen much smaller than wavelength of the radiation used; they do not interrupt the waves, and so are not used; detected. detected. Using a microscope with higher magnification will not increase resolution any further, objects closer than 200nm will be seen 200nm as one point. point. The wavelength of light is larger than wavelength of electrons; electrons; resolution of light microscope lower. lower.

RESOLUTIONu
Light passing through the lens interferes with itself creating a ring shaped diffraction pattern, known as the Airy pattern resulting in blurring of the image. image. The limit of resolution of a microscope objective refers to its ability to distinguish between two closely spaced Airy disks in the diffraction pattern

RAYLEIGH CRITERION
accepted criterion for the minimum resolvable detail In the microscopy, two peaks are considered resolved if the image satisfies Rayleigh's criterion. criterion. Rayleigh's criterion requires that the height of the image dip at least 19% 19% between the peaks If two Airy disks are separated at least by their radius, the meet the Rayleigh criterion and are resolvable as two spots. spots. Definition of lateral resolution using Rayleigh's criterion

RESOLUTIONu
The resolution depends on the angular aperture =1 =1.22 / 2n sin or =0 =0.612 / n sin or : : collecting angle of the lens. lens. medium. n : refractive index of the medium. : wavelength of light illuminating the sample. sample. aperture. n sin : numerical aperture. Due to the limitations of the values , , and n, the resolution limit of a nm. light microscope using visible light is about 200 nm. Because: Because: for the best lens : 70 (sin = 0.94), 70 94), the shortest wavelength of visible light : ( = 450nm), 450nm), and the typical high resolution lenses are oil immersion lenses (n = 1.56). i.e: =1.22450/21.56 =1.22450/21.560.94=187 nm

HIGH RESOLUTIONu
Shorter wavelengths yield higher resolution and vice versa. versa. The greatest resolving power in optical microscopy is realized with near-ultraviolet light. nearlight. resolution depends on: on: objective numerical aperture type of specimen coherence of illumination degree of aberration correction

   

USEFUL MAGNIFICATION RANGE



Depends on numerical aperture of objective. objective. A minimum magnification necessary for the detail present in an image to be resolved. resolved. Arbitrary useful magnification = 500 to 1000 NA (Objective) Additional magnification does not result in the resolution of even smaller specimen detail. detail. Exceeding the limit of useful magnification causes the image to suffer from the phenomenon of empty magnification The image becomes more magnified with no corresponding increase in detail resolution. resolution. Excessive magnification introduces artifacts, diffraction boundaries, and halos into the image that obscure specimen features

SOME BASIC CONCEPTS

REFRACTION OF LIGHT
Refractive Index : value calculated from the ratio of the speed of light in a vacuum to that in second medium of greater density. density. n = v1/v2 /v2 The incident angle ( 1) is related to the refraction angle ( 2) by the simple relationship known as Snell's law: law: n1 sin( 1) = n2 sin( 2) HighHigh-magnification objectives employ oil between the objective lens and the specimen to improve resolution. resolution. N for: for: Vacuum: Vacuum: 1 exactly Air: Air: 1.000293 Water: Water: 1.333 Immersion oil: 1.515 oil:

   

DIFFRACTION
encounters an obstacle. obstacle.

Phenomena which occur when a wave

The apparent bending of waves around small obstacles and the spreading out of waves past small openings. openings. Occurs with all waves: sound waves, waves: water waves, and electromagnetic waves (visible light, x-rays and radio waves). light, waves) Resolution is limited by diffraction, which results in secondary, weak wavefronts which interfere with the primary wavefront when light passes through a small aperture. As a result a aperture. small spot is imaged, not as a small spot, but as a spot surrounded by a series of concentric circles (called Airy disks). disks).

Colors seen in a spider web are partially due to diffraction

Diffraction through wide and narrow slits

Airy discs

NUMERICAL APERTURE
the angle over which the objective is capable of receiving light from the specimen. specimen. is a measure of the ability of objective of microscope to gather light and resolve fine specimen detail at a fixed object distance. distance. NA = n sin(a) n = refractive index of the medium a = one-half angular aperture of the oneobjective. objective. Greater the numerical aperture, more the resolution

SPHERICAL ABERRATION
prohibits the lens from focusing all the incident light from the same location on an object to a precise point. point. The defect is most noticeable for light rays striking the outer edges of the mirror. mirror. images of objects as seen in spherical mirrors are often blurry. blurry. corrected by use parabolic mirror of a

CHROMATIC ABERRATION
Optics: caused by a lens having a : different refractive index for different wavelengths of light (the dispersion of the lens). lens). seen as "fringes" of color around the image, because each color in the optical spectrum cannot be focused at a single common point on the optical axis. axis. reduced by increasing the focal length of the lens or using achromatic lens. lens. Electron microscopy: : spreading or reduced coherence of the beam due to scattering as individual electrons strike gas molecules in the vacuum. vacuum.
Achromatic lenses using two or more pieces of glass with different refractive indexes can reduce or eliminate chromatic aberration

Chromatic Aberration focal length varies with color wavelength

RESOLUTION V/S MAGNIFICATION IN ELECTRON MICROSCOPE

ELECTRON MICROSCOPE


uses electrons to illuminate a specimen and create an enlarged image. image. have much greater resolving power and much microscopes. higher magnifications than light microscopes. can magnify specimens up to 6-10 lakh times. times. greater resolution and magnification due to electron, the wavelength of an electron, which is photon. smaller than that of a light photon. uses electrostatic and electromagnetic lenses in forming the image by controlling the electron beam to focus it at a specific plane relative to the specimen Electron microscope constructed by Ernst Ruska in 1933

AN ELECTRON
a fundamental subatomic particle that carries a negative electric charge. charge. mass is approximately 1 / 1836 of that of the proton mass :9.11 1031 kg electric charge : 1.602 1019 C common electron symbol : e stable on theoretical grounds : mean lifetime is 4.61026 years dual character: behaves both as particle and wave character: the exact momentum and position of the actual electron cannot be simultaneously determined (Heisenberg uncertainty principle) principle)

MAGNIFICATION IN SEM
controlled over a range of about 5 orders of magnitude from x25 or less to x 250,000 or more. 250, more. function of condenser and objective lenses is to focus the beam to a spot, and not to image the specimen. specimen. Magnification controlled by the current supplied to the x,y scanning coils, and not by objective lens power. power.

RESOLUTION OF THE SEM

  

Depends on the size of the electron spot wavelength of the electrons the electron-optical system which produces the scanning beam. electronbeam. The spot size and the interaction volume are both large compared to the distances between atoms, so the resolution of the SEM is not as high as in TEM. TEM. Advantages: Advantages: ability to image a comparatively large area of the specimen; specimen; ability to image bulk materials; materials; variety of analytical modes available for measuring the composition and proprties of the specimen. specimen. Resolution obtained: 1 - 20 nm. obtained: nm. The world's highest SEM resolution : Hitachi S-5500,0.4nm at 30kV and 1.6nm at 5500, 30kV 1kV. kV.

  

FACTORS AFFECTING RESOLUTION IN SEM (AND TEM)

ACCELERATING VOLTAGE AND RESOLUTION

difference in potential between the filament and the anode. anode. As the voltage is increased, the electrons travel with higher velocity and wavelenght decreases (as =h/mv) Increasing AV will decrease the spherical aberration of the system and therefore increase the resolution. resolution. Very high AV reduces resolution. resolution. Resolution is dependent on the spot size. size. By increasing the AV, backscattered electrons, emitted from a larger area of the sample, interact with the sample, producing secondary electrons away from the original spot size thereby reducing the resolution of the image . A higher AV increases secondary yield from all parts of the specimen due to a greater beam penetration, reducing the edge effect and diminishing the contrast. contrast. AV of 5-15 KeV are used with most biological specimens. specimens.
Decrease in resolution and contrast when a diatom is imaged using higher accelerating voltage (20kV) Fine detail of a diatom imaged at a low accelerating voltage of 5kV is visible A diatom imaged using different accelerating voltages.

CHARGE ACCUMULATION
Specimens need to be conductive when examined using a SEM. SEM. When a specimen is nonconductive a negative charge from the electron beam tends to accumulate, thus effecting the final image. image. effects abnormal contrast, appearing of abnormal lines or shifts/breaks within the image. image. Nonconductive samples need to be coated with a thin layer of metal making the surface conductive metals and semiconductors are conductive and can be examined without coating. coating.
Pollen samples showing the effect of charge accumulation. No effect of charge accumulation is visible (A). Pollen showing the type of charging that produces abnormal lines within the image (B).

WORKING DISTANCE AND DEPTH OF FIELD

Depth of field (DOF): the portion of a (DOF): scene that appears sharp in the image Effected by the working distance At a short working distance the sample will be scanned with a wide cone of electrons resulting in an image with little depth of field. field. At a longer working distance the sample will be scanned with a narrow cone of electrons resulting in an image with an increased depth of field A proper relationship between working distance and depth of field necessary
A head of a fruit fly imaged using different working distances. At a short working distance (WD = 8) only part of the head is in focus because of a limited depth of field (A). At a longer working distance (WD = 48) more is in focus due to an increase in depth of field (B).

STIGMATISM AND RESOLUTION

stigme : 'mark' or 'spot' ( Latin). Latin). refers to the image-formation property imageof an optical system which focuses a single point source in object space into a single point in image space. space. If the electron beam is not round but elliptical, extraneous information obtained by using this elliptical spot will be collected by the detector and rendered in a round spot on the final image. image. This phenomenon, called astigmatism, causes a reduction in resolution. resolution. The stigmator knobs control distortions in the roundness of the spot that is scanned over the sample. sample. When alternating between over and under focus, astigmatism can be recognized by a smeared look in two different directions. directions.

A good image of the copy paper with no stigmatism present (A). As stigmatism increases, resolution decreases. Little stigmatism present (B). More stigmatism present (C). stigmatism present and sample scanned in over or under focus, a smeared image produced (D and E).

CONDENSER LENS CURRENT AND RESOLUTION

The current in the condenser lens changes the spot size or diameter of the beam of electrons that scans the sample. sample. More detailed information will be collected when the electron beam scans the same area with a smaller spot size. size. An increased current or a higher number for the condenser lens (CL) setting, will produce a smaller spot size and result in a better resolution
A smaller spot size or smaller diameter of the beam of electrons will resolve more detailed structures of sample as compared to a beam with a big spot size.

Diatom sample imaged using different CL settings. A small spot size or a higher condenser lens setting (CL = 14) will result in an image with more detail or better resolution (A). A bigger spot size or lower CL setting will resolve less detail (B).

TRANSMISSION ELECTRON MICROSCOPY

involves a high voltage electron beam emitted by an electron gun. Electron beam is accelerated by anode gun. at +100keV (40 to 400 keV) w.r.t cathode, focused by 100k electrostatic and electromagnetic lenses, and transmitted through a specimen. When it emerges specimen. from the specimen, the electron beam carries information about the structure of the specimen that is magnified by the objective lens system of the microscope. microscope. TEM resolution is about an order of magnitude greater than the SEM resolution. resolution.

Resolution of TEM can be about 2 The magnification of the TEM can be as high as 1,500,000X.
Wavelength/pm 5.36 3.70 2.51 1.42 0.69 8.59 Accelerating Voltage /KV 20 50 100 200 500 1000

THE RESOLUTION OF TEM

Because of using a highly focused electron beam as a probe, TEM has high spatial resolution. The wavelength, , of 100 keV electrons is only 0.0037 nm. The higher the operating voltage of a TEM instrument, the greater its lateral spatial resolution. Some commercially available 300 kV and 400 kV instruments, classified as high-voltage TEM instruments, have point to-point resolutions better than 0.2 nm.

RESOLUTION LIMITS IN TEM

Resolution of the HRTEM is limited by spherical and chromatic aberration. aberration. Software correction of spherical aberration has allowed the production of images with resolution which shows carbon atoms in diamond separated by only 0.89 ngstrms at magnifications of 50 million times. times. HRTEM an indispensable tool for nanotechnology research and development in many fields. fields.

TRANSMISSION ELECTRON ABERRATIONABERRATION-CORRECTED MICROSCOPE

Transmission Electron Aberration-corrected Microscope or AberrationTEAM is a $100 million U.S. Department of Energy research project being conducted at five US laboratories. laboratories. To develop a transmission electron microscope capable of 0.05 nanometers (0.5 x 1010m) resolution, about half the size of a 1010m) hydrogen atom. As electron microscope lenses normally produce a atom. aberration, significant amount of aberration, a complex system of lenses to correct the aberrated images is required. required. The first TEAM microscope will be based at the Lawrence Berkeley National Laboratory in Berkeley, California

ATOMIC FORCE MICROSCOPY

Invented by Binnig, Quate and Gerber in 1986. Binnig, 1986. provides a true three-dimensional surface profile. threeprofile. true atomic resolution in ultra-high vacuum (UHV) ultracan provide higher resolution than SEM can image a maximum height on the order of micrometres and a maximum scanning area of around 150 by 150 micrometres. micrometres. Piezoelectric elements that facilitate tiny but accurate and precise movements on (electronic) command enable the very precise scanning Piezoelectricity: Piezoelectricity: ability of some materials (notably crystals and certain ceramics) to generate an electric ceramics) potential in response to applied mechanical stress. stress.

AFMu
uses a laser beam deflection system laser is reflected from the back of the reflective AFM lever and onto a position-sensitive detector. positiondetector. AFM tips and cantilevers are microfabricated from Si or Si3N4. Require high SNR (S/N)

(a) A new AFM tip; inset: The end of the new tip. (b) A used AFM tip.

IMAGING MODES
  

Contact Mode : probe comes in contact with the sample highhigh-speed atomic resolution may damage the sample NonNon-contact mode: mode: low resolution NonNon-destructive Dynamic Force / Intermittant-contact / tapping mode IntermittantAFM : improved lateral resolution on soft samples NonNon-destructive

 

 

RESOLUTION IN AN ATOMIC FORCE MICROSCOPE

Traditional microscopes have only in the plane of an image. image. resolution AFM has two measures of resolution In Plane Resolution: depends on the Resolution: geometry of the probe that is used for scanning. The sharper the probe, higher scanning. the resolution of the AFM image. image. Vertical Resolution: is established by Resolution: relative vibrations of the probe above the surface. Getting the maximum vertical surface. resolution requires minimizing the vibrations of the instrument. instrument. Resolution depends on radius of curvature of probe tip. tip. Lateral Resolution: 1.1 and 1.5 nm Resolution: Vertical Resolution: 0.1 nm Resolution:

The image on the right will have a higher resolution because the probe used for the measurement is much sharper.

PROBE TIPS
The lateral resolution is determined by the minimum radius of the tip. A typical probe tip height : 10-15 m 10radius of curvature : less than 10 nm at probe tip end Cone angle of tip : 10 20O Radius of sharpest tips available commercially: 50. Can commercially: 50 provide a lateral resolution of 10 to 20. 20 Microfabricated tips available in three geometries: geometries: pyramidal, tetrahedral, and conical. conical. Conical tips can be made sharp, with high aspect ratios (the ratio of tip length to tip width, diameter to length ratio). ratio). Pyramidal tips have lower aspect ratios and tip radii of a few hundred angstroms, but are more durable. durable. tips must have an aspect ratio better than 1:10. 10.
Ultrafine tip Aspect ratio 1:40

  

Scanning electron micrographs of typical silicon probes and probe tips. (1) Standard pyramidal probe tip. (2) High-aspect-ratio probe tip. (3) Probe at end of cantilever. (4) View of a standard probe from the end of the cantilever.

Thanx