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1 AP 5301/8301 Instrumental Methods of Analysis and Laboratory Lecture 2 Microscopy (I) Optical Prof YU Kin Man E-mail: [email protected] Tel: 3442-7813 Office: P6422 http://www6.cityu.edu.hk/appkchu/AP5301/Notes.htm Lecture 2: Outline Introduction: ─ Materials characterization techniques ─ Microscopy Optical microscopy basics ─ Basic concepts ─ Terminologies ─ Resolution ─ Aberrations Optical microscope ─ Major parts and functions ─ Common modes of analysis Bright and dark field imaging Polarized microscopy Phase-contrast microscopy Differential interference contrast microscopy Fluorescence microscopy Scanning confocal optical microscopy Some examples of applications 2 Introduction: Materials Characterization Wikipedia: Characterization, when used in materials science, refers to the broad and general process by which a material's structure and properties are probed and measured. It is a fundamental process in the field of materials science, without which no scientific understanding of engineering materials could be ascertained. The structure of a material is determined by its chemical composition and how it was synthesized (processed) A material’s properties will determine what it can be used for (applications) and the performance of the final device. Performance is the ultimate end use function At the CORE of this tetrahedron of the material and is resulted from properly is material characterization tuning properties of materials by optimizing the structure down to the atomic level through material processing (synthesis). 3 4 Introduction: this course Introduces a broad range of advanced materials characterization techniques ─ Basic theory ─ Equipment and operation ─ Applications These techniques study a wide range of material properties (thin film and bulk) ─ ─ ─ ─ ─ ─ Chemical composition Crystal structure Surface and interfaces Defects: point and extended defects Morphology Electrical, thermal and optical properties At the end of the course, you should be able to ─ Determine the appropriate method(s) to use ─ Understand experimental papers and determine if the data presented by others are valid Materials characterization techniques We can broadly classify materials characterization techniques as imaging and spectroscopic techniques ─ Imaging (microscopy): Optical microscopy, scanning electron microscopy (SEM), Transmission Electron Microscope (TEM), Scanning Tunneling Microscope (STM), Scanning probe microscopy (SPM) ─ Spectroscopy/spectrometry: involves interaction of radiation (radiative energy) with matter resulting in a plot of the response of interest as a function of wavelength or frequency (energy)─spectrum: Energy/Wavelength-Dispersive X-ray spectroscopy (EDX/WDX), XRay Diffraction (XRD), Secondary Ion Mass Spectrometry (SIMS), Electron Energy Loss Spectroscopy (EELS), Auger electron spectroscopy (AES), X-ray photoelectron spectroscopy (XPS), Ultraviolet-visible-near infrared spectroscopy (UV-vis-NIR), spectroscopic ellipsometry, Photo/Cathodo-luminescence (PL/CL) ─ Electrical/thermal probes: Four point probe, Hall effect, CapacitanceVoltage (CV), Thermal Power (Seeback) 5 6 Course Outline Week Date Topic 1 8-30 Introduction (Chu) 2 9-06 Microscopy (I): Optical microscopy 3 9-13 Microscopy (II): SEM and Scanning probe 4 9-20 TEM & Electron probe microanalysis 5 9-27 X ray diffraction 6 10-04 Electrical measurements: four point probe, Hall, C-V, thermal probe, minority carrier lifetime 7 10-11 Optical spectroscopies: spectrophotometry, photoluminescence, spectroscopic ellipsometry, modulation spectroscopy 8 10-18 Secondary Ion Mass Spectrometry (SIMS) 9 10-25 Auger Electron Spectroscopy (AES) (Chu) 10 11-01 X-ray Photoelectron Spectroscopy (XPS) (Chu) 11 11-08 Ion Beam techniques: RBS, PIXE, channeling 12 11-15 review 13 11-22 open FINAL EXAM Microscopy To provide a magnified image To observe features that are beyond the resolution of the human eyes (~100mm) ─ directly by light (optical) microscope using visible light ─ other microscopy imaging techniques use some other interaction probe and response signal (usually electrons) to provide the contrast that produces an image. Able to “sense” depth ─ in the light microscope, topological contrast is provided largely by shadowing in reflection, ─ in Scanning Electron Microscopy (SEM) secondary electrons are generated from different depths, giving rise to topological contrast, ─ in Transmission Electron Microscopy (TEM), no depth information is obtained. For materials characterization, light Microscope is likely to be the first imaging instrument to use ─ the cheapest “modern” instrument and take up the least physical space 7 8 The scale of things Human hair ~ 50 mm wide Schematic, central core ATP synthase Bee ~ 15 mm Red blood cells with white cell ~ 2-5 mm DNA ~2 nm wide 10 nm Atoms of silicon spacing ~tenths of nm Magnetic domains garnet film 11 mm wide stripes Cell membrane 10-1 m 100 m 0.1 m 100 mm 1 meter (m) 1 millimeter (mm) 0.01 m 1 cm 10 mm 10-3 m 0.1 mm 100 mm Biomotor using ATP 10-2 m 10-4 m 0.01 mm 10 mm 1 micrometer (mm) 10-5 m 10-6 m 0.1 mm 100 nm 0.01 mm 10 nm 1 nanometer (nm) 0.1 nm Visible spectrum MEMS (MicroElectroMechanical Systems) Devices 10 -100 mm wide Quantum corral of 48 iron atoms on copper surface positioned one at a time with an STM tip Corral diameter 14 nm Cat ~ 0.3 m The Microworld 10-7 m 10-8 m 10-9 m 10-10 m The Nanoworld Monarch butterfly ~ 0.1 m Dust mite 300 mm Head of a pin 1-2 mm Selfassembled “mushroom” Objects fashioned from metals, ceramics, glasses, polymers ... Red blood cells Pollen grain Microelectronics 9 Optical vs. electron microscopy Optical Microscope Glass ceramic transmission microscope image made with polarized light and full wave plate SEM micrographs of SMNb0.05% Mat. Res. vol.6 no.2 São Carlos Apr./June 2003. Exfoliated molybdenum disulfide on a perforated grid Microstructure of steel D2 (Metal Ravne Steel Selector) Scanning electron microscopy (SEM) image of as-grown p-type gallium nitride (p-GaN) nanowire arrays on a silicon (111) substrate Cross-section TEM image of MOCVD grown InGaAs/GaAs quantum dot superlattice solar cell (NREL) Electron Microscope Atomic resolution TEM image of nanocrystalline palladium. H. Rösner and C. Kübel et al., Acta Mat., 2011, 59, 7380-7387. Optical (light) microscopy 10 Introduction Basic principles ─ Lens formula, Image formation and Magnification ─ Resolution and lens defects Basic components and their functions Common modes of analysis: bright field and dark field Specialized Microscopy Techniques ─ Polarized light microscopy ─ Phase contrast microscopy ─ Differential interference contrast (DIC) microscopy ─ Fluorescence microscopy ─ Scanning confocal optical microscopy (SCOM) Typical examples of applications http://micro.magnet.fsu.edu/primer http://www.doitpoms.ac.uk/tlplib/optical-microscopy/index.php Introduction 11 An optical microscope: uses visible light as the illumination source, has lateral resolution down to 0.1mm (typically a few mm), can be used for almost all solids and liquid crystals, is typically nondestructive; sample preparation may involve material removal, is mainly used for direct visual observation; preliminary observation for final characterization with applications in geology, medicine, materials research and engineering, industries, etc. Typical microstructural features observed in materials science are grains, precipitates, inclusions, pores, whiskers, defects, twin boundaries, etc. http://www.youtube.com/watch?v=bGBgABLEV4g&feature=endscreen&NR=1 using a microscope Light microscope: parts Base – Base supports the microscope which is horseshoe shaped Illuminator - This is the light source located below the specimen. Iris diaphragm - Regulates the amount of light into the condenser. Condenser - Focuses the ray of light through the specimen. Stage - The fixed stage is a horizontal platform that holds the specimen. Nosepiece - The portion of the body that holds the objectives over the stage. Objective - The lens that is directly above the stage. Coarse focusing knob - Used to make relatively wide focusing adjustments to the microscope. Fine focusing knob - Used to make relatively small adjustments to the microscope. Body - The microscope body. Ocular eyepiece - Lens on the top of the body tube. It has a magnification of 10× normal vision. 12 Light microscopes: then and now 13 http://www.youtube.com/watch?v=sCYX_XQgnSA&feature=related <2min http://www.youtube.com/watch?v=1k659rtLrhk <2min http://www.youtube.com/watch?annotation_id=annotation_100990&f eature=iv&src_vid=L6d3zD2LtSI&v=ntPjuUMdXbg http://www.youtube.com/watch?v=X-w98KA8UqU&feature=related 14 Basic concepts Magnification: the ratio of the size of an object seen under the microscope to the actual size observed with unaided eye. ─ The total magnification of a microscope is calculated by multiplying the magnifying power of the objective lens by that of eye piece Resolving power: the ability to differentiate two close points as separate ─ The resolving power of human eye is 0.25 mm ─ The light microscope can separate dots that are 0.25µm apart. ─ The electron microscope can separate dots that are 0.5nm apart Limit of resolution (resolving power) : is the minimum distance between two points to identify them separately. Abbe diffraction limit: 𝑤𝑎𝑣𝑒𝑙𝑒𝑛𝑔𝑡ℎ 𝑜𝑓 𝑙𝑖𝑔ℎ𝑡 resolving power, RP= 2 × 𝑛𝑢𝑚𝑒𝑟𝑖𝑐𝑎𝑙 𝑎𝑝𝑒𝑟𝑡𝑢𝑟𝑒 𝑜𝑓 𝑡ℎ𝑒 𝑜𝑏𝑗𝑒𝑐𝑡𝑖𝑣𝑒 𝑙𝑒𝑛𝑠 Focal length Numerical aperture(NA): 𝑁𝐴 = 𝑛 sin 𝜃, where n is the index of refraction of the medium, 𝜃 is the maximal half-angle of the cone of light that can enter or exit the lens Numerical Aperture (NA) 𝑁𝐴 = 𝑛(sin ) Imaging Medium Air n=1.0 Immersion oil n=1.515 http://www.youtube.com/watch?v=RSKB0J1sRnU oil immersion objective use in microscope at~0:33 15 Basic concept: Absorption When light passes through an object the intensity is reduced depending upon the color absorbed. Thus the selective absorption of white light produces colored light. The Beer–Lambert law relates the attenuation of light to the properties of the material through which the light is traveling. 𝐼𝑡 (𝜆) = 𝐼𝑜 (𝜆)𝑒 − 𝛼(𝜆)𝑥 where 𝐼𝑡 (𝜆)𝑎𝑛𝑑 𝐼𝑜 (𝜆) are the transmitted and incident light intensity and wavelength 𝜆, respectively, 𝛼(𝜆) is the absorption coefficient of the material and 𝑥 is the thickness of the material. The absorbance of an object quantifies how much of the incident light is absorbed by it. It is a measure of attenuation. Absorbance of a material, denoted A, is given by 𝐼𝑜 𝐴 = 𝑙𝑜𝑔10 𝐼𝑡 16 17 Basic concept: Refraction The bending of a wave when it enters a medium where its speed is different due to difference in the index of refraction (densities). A ray from less to more dense medium is bent toward the surface normal, with greater deviation for shorter wavelengths. The index of refraction is defined as the speed of light in vacuum 𝑐 divided by the speed of light in the medium: 𝑛 = ≥ 1, where c and v 𝑣 are the speed of light in vacuum and in the medium, respectively. Snell's Law relates the indices of refraction n of the two media to the directions of propagation in terms of the angles to the normal. 𝑛1 sin 𝜃1 = 𝑛2 sin 𝜃2 𝑛1 𝑛2 = sin 𝜃2 sin 𝜃1 = 𝑣2 𝑣1 18 Basic concept: Refraction Material n Vacuum 1.0000 Air 1.000277 Ice 1.31 Water 1.333 Ethyl alcohol 1.362 Lucite 1.47 Glass 1.52 Polystyrene 1.59 Diamond 2.417 http://www.youtube.com/watch?v=jQDRNb-E-cY ~1.00–2:20 http://micro.magnet.fsu.edu/primer/java/refraction/refractionangles/index.html Basic concept: Diffraction Diffraction manifests itself in the apparent bending of waves around small obstacles and the spreading out of waves past small openings. Diffraction provides a powerful tool for studying the geometry of objects that are too small to be viewed directly. Will be covered in more detail when we talk about x-ray diffraction 19 Basic concept: Dispersion Chromatic dispersion is the change of index of refraction with wavelength. ─ Generally the index decreases as wavelength increases (𝒏 ↓ 𝝀 ↑) ─ Blue light (~400 nm) travels slower in the material than red light (~700nm). Dispersion is the phenomenon which gives you the separation of colors in a prism. It also gives the generally undesirable chromatic aberration in lenses. http://hyperphysics.phy-astr.gsu.edu/hbase/hph.html 20 Light microscope: a little history In 1590 F.H Janssen & Z. Janssen constructed the first simple compound light microscope. In 1665 Robert Hooke developed a first laboratory compound microscope. Later, Kepler and Galileo developed a modern class room microscope. In 1672 Leeuwenhoek developed a simple microscope with a magnification of 275x. He is mistakenly called "the inventor of the microscope“ In 1880 Abbe and Zeiss developed oil immersion systems and were able to make the a Numeric Aperture (N.A.) to the maximum of 1.4 allowing light microscopes to resolve two points distanced only 0.2 microns apart. Remember that: RP= 𝜆 2×𝑁𝐴 21 A typical light microscope 22 Basics: focusing by a curved surface In entering an optically more dense medium (𝑛𝟐 > 𝑛𝟏), rays are bent toward the normal to the interface at the point of incidence. Curved (converging) glass surface normal Air 𝑛𝟏 𝑛𝟐 F f F - focal point Focal plane f – focal length 23 Basics: principal focal length 24 For a thin double convex lens, refraction acts to focus all parallel rays to a point referred to as the principal focal point. The distance from the lens to that point is the principal focal length f of the lens For a double concave lens where the rays are diverged, the principal focal length is the distance at which the back-projected rays would come together and it is given a negative sign. The lens strength in diopters is defined as the inverse of the focal length in meters. 1 𝑃(𝑑𝑖𝑜𝑝𝑡𝑒𝑟) = 𝑓(𝑚) http://hyperphysics.phy-astr.gsu.edu/hbase/geoopt/foclen.html Basics: converging (Bi-Convex) lens Most lenses are spherical lenses: their two surfaces are parts of the surfaces of spheres. A lens is biconvex (or double convex, or just convex) if both surfaces are convex. The line joining the centers of the spheres making up the lens surfaces is called the axis of the lens. If the lens is biconvex, a collimated beam of light passing through the lens converges to a spot (a focus) behind the lens─a positive or converging lens. 𝒇 is the focal length of the lens, 𝒏 is the refractive index of the lens material, 𝑹𝟏 is the radius of curvature (with sign) of the lens surface closest to the light source, -ve +ve 𝑹𝟐 is the radius of curvature of the lens surface farthest from the light source, 𝒅 is the thickness of the lens (the distance along the lens axis between the two surface vertices) 25 26 Basics: Lensmaker’s equation 1 1 1 𝑛−1 𝑑 = (𝑛 − 1) − + 𝑓 𝑅1 𝑅2 𝑛𝑅1 𝑅2 The reciprocal of the focal length, 1/𝑓, is the optical power of the lens. If the focal length is in meters, this gives the optical power in diopters (inverse meters) For a thin lens where 𝑑 ≪ 𝑅1 and 𝑅2 : 1 𝑓 ≈ (𝑛 − 1) 1 𝑓 1 𝑅1 For a thin bi-convex lens with equal curvatures: ≈ http://www.youtube.com/watch?v=R-uMcngNsSk http://www.youtube.com/watch?v=KYrsmzM9I_8 http://www.youtube.com/watch?v=Am5wJUEiNAI − 1 𝑅2 2(𝑛−1) 𝑅 converging (convex) lens<6:10 diverging (concave) lens how it’s made: optical lenses 27 Image formation 𝑜 𝑖 Principal ray F F For a lens of negligible thickness, in air, the distances are related by the thin lens formula 1 𝑆1 + 1 𝑆2 = 1 𝑓 or 1 𝑖 1 𝑜 + = 1 𝑓 http://www.youtube.com/watch?v=-k1NNIOzjFo&feature=related to~3:42 28 Magnification: angular The standard close focus distance is taken as 25 cm A simple magnifier achieves angular magnification by permitting the placement of the object closer to the eye than the eye could normally focus. The angular magnification is given by: 𝑀𝛼 = 𝛼′ 𝛼 For small angles: 𝛼′ 𝛼 ≈ 25 𝑓 −𝑆2 i 𝑆1 𝛼′ 𝛼 ≈ ℎ′ 25 ℎ 25 = ℎ′ ℎ = 25 ; 𝑜 using the lens formula: + 1; angular magnification 𝑀𝛼 = 25 𝑓 25 𝑜 = 25 𝑓 + 25 25 +1 (when the image is at the close focus point of 25 cm) Our eyes are most relaxed while focus at infinity, i.e. 𝑖 = ∞ 25 𝑀𝛼 = 𝑓 A large magnification requires a lens with a small focal length http://hyperphysics.phy-astr.gsu.edu/hbase/geoopt/simmag.html 29 Magnification: Linear The linear magnification or transverse magnification is the ratio of the image size to the object size. If the image and object are in the same medium it is just the image distance divided by the object distance. 𝑜 𝑖 ℎ𝑜 ℎ𝑖 ℎ𝑖 −𝑖 −𝑆2 𝑀= = (𝑜𝑟 ) ℎ𝑜 𝑜 𝑆1 A negative sign is used on the linear magnification equation as a reminder that all real images are inverted. 30 Compound microscope A compound microscope uses a very short focal length objective lens to form a greatly enlarged image. This image is then viewed with a short focal length eyepiece (Ocular) used as a simple magnifier. 𝑓𝑒 𝑓𝑜 𝑠1 𝑠1′ Magnification by the objective 𝑚0 = 𝑠1′ /𝑠1 Since 𝑠1′ 𝐿 and 𝑠1 𝑓𝑜 , therefore magnification of objective 𝑚𝑜 = − Magnification of the eyepiece 𝑚𝑒 = 25 𝑓𝑒 Overall magnification 𝑀 = 𝑚𝑜 𝑚𝑒 = − 𝐿 𝑓𝑜 (assuming the final image forms at ) 𝐿 25 𝑓𝑜 𝑓𝑒 http://www.youtube.com/watch?v=kcyF4kLKQTQ at~1:57 http://www.youtube.com/watch?v=RKA8_mif6-E 31 Microscope resolution The resolution of an optical microscope is defined as the shortest distance between two points on a specimen that can still be distinguished by the observer as separate entities. The diffraction pattern resulting from a uniformlyilluminated circular aperture has a bright region in the center, known as the Airy disk, which together with the series of concentric bright rings around is called the Airy pattern. The lens' circular aperture is analogous to a twodimensional version of the single-slit experiment. Light passing through the lens interferes with itself creating a ring-shape Airy pattern The limit of resolution of a microscope objective refers to its ability to distinguish between two closely spaced Airy disks in the diffraction pattern. 𝐬𝐢𝐧 = /𝒅 32 Resolution– Rayleigh Criteria The angular resolution of an optical system can be estimated (from the diameter of the aperture and the wavelength of the light) by the Rayleigh criterion: Two point sources are regarded as just resolved when the principal diffraction maximum of one image coincides with the first minimum of the other. 𝜆 𝜃 = 1.22 𝑑 θ is the angular resolution (radians), λ is the wavelength of light, and d is the diameter of the lens' aperture. The factor 1.22 is derived from a calculation of the position of the first dark circular ring surrounding the central Airy disc of the diffraction pattern Resolution –Linear separation To express the resolution in terms of a linear separation r, we have to consider the Abbe’s theory Path difference between the two beams passing the two slits is 𝑑 sin 𝑖 + 𝑑 sin 𝛼 = 𝜆 Assuming that the two beams are just collected by the objective, then i = and 𝑑𝑚𝑖𝑛 = /2sin If the space between the specimen and the objective is filled with a medium of refractive 𝜆 index n, then wavelength in medium 𝜆𝑛 = 𝑛 𝜆 𝑑𝑚𝑖𝑛 = = 2𝑛 sin 2(𝑁𝐴) 𝑁𝐴 = 𝑛 sin 𝛼 𝑖𝑠 called numerical aperture For circular aperture: 𝑑𝑚𝑖𝑛 = 1.22𝜆 2(𝑁𝐴) = 0.61𝜆 (𝑁𝐴) 𝑑𝑚𝑖𝑛 ~0.3 𝜇𝑚 for a midspectrum of 0.55mm 33 Axial resolution – Depth of Field Another important aspect to resolution is the axial (or longitudinal) resolving power of an objective, which is measured parallel to the optical axis and is most often referred to as depth of field. Depth of field (F in mm) is the range of distance at the specimen parallel to the illuminating beam in which the object appears to be in focus Depth of focus (f in mm) is the range of distance at the image plane in which an object appears to be in focus Depth of focus varies with numerical aperture (NA) and magnification (M) of the objective ─ high NA systems have deeper focus depths but lower depth of field M M NA NA f f F F http://www.youtube.com/watch?v=FvC2WLUqEug at~3:40 http://micro.magnet.fsu.edu/primer/java/nuaperture/index.html 34 35 Optical aberrations The influences which cause different rays to converge to different points are called aberrations Aberrations reduce resolution of a microscope http://hyperphysics.phy-astr.gsu.edu/hbase/geoopt/aberrcon.html Aberration Character Correction Spherical Monochromatic, on-and -off axis, image blur Bending, high index, aspherics, gradient index, doublet Coma Monochromatic, off-axis only, blur Bending, spaced doublet with central stop Oblique astigmatism Monochromatic, off-axis, blur Spaced doublet with stop Curvature of field Monochromatic, off-axis Spaced doublet distortion Monochromatic, off-axis Spaced doublet with stop chromatic Heterochromatic, on- and off-axis, blur Contact doublet, spaced doublet Spherical aberration For lenses made with spherical surfaces, rays which are parallel to the optic axis but at different distances from the optic axis fail to converge to the same point The image appears hazy or blur and slightly out of focus. For a single lens, it can be minimized by bending the lens into its best form. For multiple lenses, spherical aberrations can be canceled by overcorrecting some elements. 36 Chromatic Aberration The focal length depends on refraction and the index of refraction 𝑛 for blue light (short wavelengths) is larger than that of red light (long wavelengths) Axial - Blue light is refracted to the greatest extent followed by green and red light, a phenomenon commonly referred to as dispersion Lateral - chromatic difference of magnification: the blue image of a detail was slightly larger than the green image or the red image in white light, thus causing color ringing of specimen details at the outer regions of the field of view. Achromatic doublet─a strong positive lens made from a low dispersion glass like crown glass coupled with a weaker negative high dispersion glass like flint glass calculated to match the focal lengths An achromatic doublet does not completely eliminate chromatic aberration, but can eliminate it for two colors 37 Aberrations due to lens imperfection Astigmatism occurs when rays travelling along two perpendicular planes have different image distances for a sharp focus ─ The off-axis image of a specimen point appears as a disc or blurred lines instead of a point. Comatic aberration occurs due to imperfection in the lens or other components resulting in off-axis point sources such as stars appearing distorted, appearing to have a tail (coma) like a comet. Curvature of Field - When visible light is focused through a curved lens, the image plane produced by the lens will be curved The image appears sharp and crisp either in the center or on the edges of the viewfield but not both 38 Light microscope: basic components and functions 1. Eyepiece (ocular lens) 2. Revolving nose piece (to hold multiple objective lenses) 3. Objective lenses 4. Focus knobs coarse 5. Focus knobs Fine 6. Stage (to hold the specimen) 7. Light source (lamp) 8. Condenser lens and diaphragm 9. Mechanical stage (move the specimen on two horizontal axes for positioning the specimen) 39 Functions of the Major Parts Lamp and Condenser: project a parallel beam of light onto the sample for illumination Sample stage with X-Y movement: sample is placed on the stage and different part of the sample can be viewed due to the X-Y movement capability Focusing knobs: since the distance between objective and eyepiece is fixed, focusing is achieved by moving the sample relative to the objective lens 40 41 Major Parts: objective lens Objective: does the main part of magnification and resolves the fine details on the samples (𝑚𝑜 ~10 – 100) 𝑑𝑚𝑖𝑛 = 0.61𝜆/𝑁𝐴 Objectives are the most important components of a light microscope: they are responsible for image formation, magnification, the quality of images and the resolution of the microscope Major parts: eyepiece Lens Eyepiece: is a cylinder containing two or more lenses; its function is to bring the image into focus for the eye with typical magnification up to 20x 𝑴 = (𝑳/𝒇𝒐 )(𝟐𝟓/𝒇𝒆 ) Eyepieces (Oculars) work in combination with microscope objectives to further magnify the intermediate image 42 Common light microscopes Depending on the nature of samples, either a transmitted or reflected optical microscope can be used Transmitted OM transparent specimens ─ thin section of rocks, minerals and single crystals Reflected OM - opaque specimens ─ most metals, ceramics, semiconductors 43 Common modes of analysis Instead of using the full illumination of the light source (bright field), it is sometimes useful to illuminate the sample with peripheral light by blocking the axial rays─dark field microscopy. This produces the classic appearance of a dark, almost black, background with bright objects on it Advantages: ─ A simple procedure which can be used on live transparent specimens ─ The images appear spectacular and are visually impressive. ─ Even allows for the visualization of objects that are below (!) the resolution of the microscope. Disadvantages: ─ very sensitive to dirt and dust located in the light path ─ not suitable for all specimens ─ The intensity of the illumination system must be high 44 Polarized Light Microscopy Polarized light microscopy involves illumination of the sample with polarized light. Designed for specimens that are visible primarily due to their optically anisotropic character. Image contrast arises from the interaction of plane-polarized light with a birefringent (or doubly-refracting) specimen to produce two individual wave components that are each polarized in mutually perpendicular planes. The light components are recombined with constructive and destructive interference when they pass through the analyzer. Reveals detailed information concerning the structure and composition of materials 45 Phase contrast microscopy Phase contrast microscopy uses a special condenser and objective lenses to convert phase differences (not visible) into amplitude differences (visible) The image contrast is improved in two steps: ─ The background light is phase-shifted by −90° by passing it through a phase-shift ring, leading to an increased intensity between foreground and background ─ To further increase contrast, the background is dimmed by a gray filter ring 46 Differential interference contrast microscopy 47 Differential interference contrast (DIC) microscopy enhances contrast by creating artificial shadows (pseudo three-dimensional) using polarized light as if the object is illuminated from the side Differential interference contrast converts gradients in specimen optical path length into amplitude differences that can be visualized as improved contrast in the resulting image. The specimen optical path difference is determined by the product of the refractive index difference (between the specimen and its surrounding medium) and the geometrical distance (thickness) traversed by a light beam between two points on the optical path. Blue-green Algae Phase contrast DIC Fluorescence microscopy 48 A fluorescence microscope uses fluorescence to generate an image The specimen is illuminated with light of a specific wavelength (or wavelengths) which is absorbed by the fluorophores, causing them to emit light of longer wavelengths (i.e., of a different color than the absorbed light). The illumination light is separated from the much weaker emitted fluorescence through the use of a spectral emission filter. Only allows observation of the specific structures which have been labeled for fluorescence Fluorescent micrograph of an amphibian cell during anaphase when the chromatids comprising each chromosome disjoin and move towards their respective poles Scanning Confocal Optical Microscopy Confocal microscopy is an optical imaging technique used to increase optical resolution and contrast of a micrograph by adding a spatial pinhole placed at the confocal plane of the lens to eliminate out-of-focus light. Scanning confocal optical microscopy (SCOM) is a technique for obtaining high-resolution optical images with depth selectivity. (a laser beam is used) The key feature is its ability to acquire in-focus images from selected depths, a process known as optical sectioning. Images are acquired point-by-point and reconstructed with a computer, allowing three-dimensional reconstructions of topologically complex objects. 49 50 Some examples of applications in materials science 51 Grain size examination Thermal Etching 1200C/30min a 1200C/2h b 20mm A grain boundary intersecting a polished surface is not in equilibrium (a). At elevated temperatures (b), surface diffusion forms a grain-boundary groove in order to balance the surface tension forces. 52 Grain size examination Objective Lens x100 Reflected OM 53 Grain growth - reflected OM 5mm Polycrystalline CaF2 illustrating normal grain growth. Better grain size distribution. 30mm Large grains in polycrystalline spinel (MgAl2O4) growing by secondary recrystallization from a fine-grained matrix Liquid phase sintering – reflective OM Amorphous phase 40mm Microstructure of MgO-2% kaolin body resulting from reactive-liquid phase sintering. 54 55 Image of magnetic domains Magnetic domains and walls on a (110)-oriented garnet crystal (Transmitted LM with oblique illumination). The domains structure is illustrated in (b). 56 Phase identification by reflective polarized OM YBa2Cu307-x superconductor material: (a) tetragonal phase and (b) orthorhombic phase with multiple twinning (arrowed) (100 x). 57 Internet resources Spherical aberration http://www.youtube.com/watch?v=MKNFW0YwDYw -Canon lens production http://www.youtube.com/watch?v=E85FZ7WLvao http://micro.magnet.fsu.edu/primer/java/aberrations/spherical/index.html Chromatic aberration http://www.youtube.com/watch?v=yH7rbRu7Av8&list=PL02D1D436A44B521A http://www.youtube.com/watch?v=H8PQ9RMUoA8 at~3:30-4:30 chromatic aberration Astigmatism http://www.youtube.com/watch?v=yQ4rDNOX7So at~3:27-4:15 http://www.youtube.com/watch?v=4RijnutOU4o http://micro.magnet.fsu.edu/primer/java/aberrations/astigmatism/index.html Comatic aberration http://www.youtube.com/watch?v=EXmaY2txEBo&list=PL02D1D436A44B521A&index=4 http://micro.magnet.fsu.edu/primer/java/aberrations/coma/index.html Curvature of field http://micro.magnet.fsu.edu/primer/java/aberrations/curvatureoffield/index.html Basic components and their functions 58 http://www.youtube.com/watch?v=RKA8_mif6-E Microscope Review (simple, clear) http://www.youtube.com/watch?v=b2PCJ5s-iyk Microscope working in animation (How to use a microscope) http://www.youtube.com/watch?annotation_id=annotation_100990&feature=iv&src_vid=L6d3zD2 LtSI&v=ntPjuUMdXbg (I) http://www.youtube.com/watch?v=VQtMHj3vaLg (II) Parts and Function of a Microscope (details) http://www.youtube.com/watch?v=X-w98KA8UqU&feature=related How to use a microscope (specimen preparation at~1:55-2:30) http://www.youtube.com/watch?v=bGBgABLEV4g How to care for and operate a microscope 59 Do review problems (1-9) on OM http://www.doitpoms.ac.uk/tlplib/optical-microscopy/questions.php