Practical aspects of Microscopy

Transcription

1 Practical aspects of Microscopy 1 Basic Conventional Optical Microscopy Scanning Electron Microscopy Related techniques Some slides borrowed from Prof. Ashish Garg s lecture on Structure and Characterization of Materials

2 2 Microscopy Techniques Microscopy is a field of investigation which is used to study objects which are too small to be easily viewed by the human eye. Viewing and studying objects that range in size from millimeters (1 mm = 10-3 meter) to nanometers (1 nm = 10-9 meter) intrigues everyone and is currently applied to every field of science and technology in use today. Microscopes, devices which magnify, come in a wide range of forms and use a multitude of illumination sources ( light, electrons, ions, x-rays and and mechanical probes) and signals to produce an image. A microscope can be as simple as a hand held magnifying glass or as complex as a multi-million dollar research instrument. Using these tools, a microscopist explores the relationship of structure and properties of a wide variety of materials in order to better understand the reasons why a particular item behaves the way it does.

3 Microscopy Techniques 3 Microscopy can be broadly subdivided into two categories Optical microscopy Conventional light microscopy, Fluorescence microscopy, confocal/multiphoton microscopy and Stimulated emission depletion microscopy Electron probe microscopy Scanning electron microscopy (SEM), Transmission electron microscopy (TEM), Scanning transmission electron microscopy (STEM), Focus ion beam microscopy (FIB)

4 Optical Microscope 4 Simple Light Microscope Reflected Light Microscope Contrast Modes of Analysis Bright Field/ Dark Field/ Phase Field/ Polarized Light

5 Conventional Optical Microscopy 5 This is an optical instrument containing one or more lenses that produce an enlarged image of an object placed in focal plane of the lens Magnification is given by M= v/u 1. Transmission: beam of light passes through the sample 2. Reflection: beam of light reflected off the sample surface e.g. Metallurgical or reflected light microscope to study surface of opaque materials

6 6 A simple light microscope The eyepiece is placed such that the image formed by the objective falls at first focal point of the eyepiece. The light thus emerges as parallel rays. The distance between the focal plane of the objective (f1) and the focal plane of the eyepiece (f2) is called the tube length (l). The object to be viewed is placed just outside the focal point at the left side of the objective lens. The enlarged image is formed at a distance l + f1 from the objective.

7 An Optical Microscope (Reflected Light) 7 The illuminating system Source of light illuminating the sample The specimen stage holds the sample in position and controls the x, y and z coordinates of the area under observation The imaging system Transfers a magnified and undistorted image to the plane of observation and to the recording medium.

8 Applications of Optical Microscopy Optical micrographs of 1040 steel after polishing with a sequence of diamond grits: (a) Rough-grinding to achieve a planar surface; (b) after polishing with 6 mm diamond grit; (c) after polishing with 1 mm diamond grit; (d) after polishing with 1/4 mm diamond grit.

9 Applications of Optical Microscopy The same 1040 steel after etching for different lengths of time in a very dilute nitric acid: (a) under etched; (b) a good etch; (c) over etched

10 Metallography guidelines 10 If a mounted specimen is used, an adherent mount is very important, else bleeding can occur The sample must be free of scratches, disturbed metal and any kind of embedded contaminants. The specimen must be thoroughly cleaned before etching. After etching, the sample should be rinsed in HOT water, followed by an alcohol rinse and dried under HOT air. If additional etching time is required, the specimen should be re-rubbed a few seconds on a final polishing wheel

11 11 camera Beam splitter Reflected light Transmitted light Olympus BX51 Research Microscope Cutaway Diagram

12 Contrast Contrast is defined as the difference in light intensity between the specimen and the adjacent background relative to the overall background intensity. Image contrast, C is defined by Ssample Sbackground S C= = Ssample SA Ssample and Sbackgroud are intensities measured from specimen and background, e.g., A and B, in the scanned area. 12

13 13 Angle of Illumination Bright filed illumination The normal method of illumination, light comes from above (for reflected OM) Oblique illumination light is not projected along the optical axis of the objective lens; better contrast for detail features Dark field illumination The light is projected onto specimen surface through a special mirror block and attachment in the objective the most effective way to improve contrast. Light stop Imin Imax C= Imax-Imin Imax C-contrast

14 Bright Field Imaging 14 Simplest and most common White light and contrast in the sample is caused by absorbance of some of the transmitted/reflected light Advantages Simplicity of setup with only basic equipment required. Stainess Steel 330 Limitations Very low contrast of most biological samples. Low apparent optical resolution due to the blur of out of focus material. Source: Wikipedia

15 Dark field Imaging 15 An illumination technique used to enhance the contrast Illuminates the sample with light that will not be collected by the objective lens, and thus will not form part of the image. This produces the classic appearance of a dark, almost black, background with bright objects on it.

16 16 Contrasting examples of dark field (left) versus bright field (right) illumination of SS330. Dark field shows grain boundaries while bright field shows grain structure See more at:

17 17 Polarized light microscopy is utilized to distinguish between singly refracting (optically isotropic) and doubly refracting (optically anisotropic) media

18 18 Polarized Light Microscope Configuration

19 Phase Contrast Microscopy Conversion of phase shifts (typically invisible) in light that passes through a transparent specimen to brightness changes in the image which are now visible. Source: Wikipedia 19

20 Phase Contrast Microscopy 20 Phase contrast is based on the destructive interference of light scattered from small features associated with a difference in height h. Based on the idea that the scattered amplitude is approximately π/2 out-of-phase with the specularly reflected beams. A phase shift ring ensures that the background light is also put out-of-phase by π/2 Bumps and hollows in the surface topology will appear either brighter or darker than the background in the image because of interference with background light Colorless and transparent specimen, such as living cells and micro-organisms

21 21 Example (a cell) Useful in biology bright field image phase contrast image Source: Wikipedia

22 Limitations: Resolution 22 DIFFRACTION AND RESOLUTION LIMIT (

23

24 Numerical Aperture (NA) 24 Angular aperture ( 72 degrees) Even an aperture in front of the lens can change the value of NA α One half of A-A NA of an lens is a measure of its ability to gather light and resolve fine specimen detail at a fixed object distance. NA = n(sin α) n: refractive index of the imaging medium between the front lens of objective and specimen cover glass Dr. Shashank Shekhar Materials Characterization

25 Limitations: Depth of Field 25

26 26 n : index of refraction of the medium between the objective and the object. So smaller NA is better for depth-offield, but what are we losing?

27 27 If Disc of confusion is smaller than resolution, then does it matter that the focus is not proper?

28 28 Depth of Field Since the resolution is finite, the object need not be in the exact object plane in order to remain in focus: there is an allowed depth of field d. Similarly, the image may be observed without loss of resolution if the image plane is slightly displaced: there is an allowed depth of focus D. d= nλ n + e 2 NA M.NA First term is Diffraction limited d-o-f λ: Wavelength of illumination Second term is geometry related d-o-f n: Refractive index of the imaging medium NA: Objective numerical aperture M: Objective lateral magnification, e: Smallest distance that can be resolved by a detector that is placed in the image plane of the objective.

29 (d-o-f) Optical Microscopy vs Scanning Electron Microscopy 29 25µm radiolarian OM Small depth of field Low resolution SEM Large depth of field High resolution J.I. Goldstein et al., eds., Scanning Electron Microscopy and X-Ray Microanalysis, (Plenum Press,NY,1980).

30 30 Scanning Electron Microscope 1938 ( Von Ardenne) cope/sem/ Principles of Electron Microscopy

31 Electron Microscopy 31 Electrons from a source interact with electrons in specimen yielding a variety of photons and electrons via elastic and inelastic scattering processes. These are the signals that are used to make images and measure or characterize the structure composition of our specimens

32 SEM: Why electrons? 32 (1) Wavelength: (Wave nature of Electron) h λ= = mv h 1 + ev 2m0 ev ( ) 2 2m0 c (1.5 / V )1/ 2 nm At 10 kv, typical of many applications of SEM, the wavelength is only nm, appreciably less than the interatomic distances in solids.

33 SEM: Why electrons? 33 (2) Charge: (Particle nature of Electron) Being charged particles, electrons can be deflected in an electromagnetic-field and thus can be brought to focus to very small region over the sample. Compared to it, the photons in a beam of light (electromagnetic waves) being neutral can not be focused in this fashion.

34 34 SEM contains broadly Gun Column of lenses Specimen chamber SE detector Control Console

35 Beam Specimen Interaction 35

36 Beam-Specimen Interaction 36 Elastic (a): incident electron s direction altered by Coulombic field of nucleus. Direction may be changed by (ave 2-5 ) but velocity remains virtually constant. <1 % of beam energy transferred. Inelastic (b): incident electron E0 = accelerating voltage (of electrons emitted from gun); usually KeV for SEM transfers some energy (up to all, E0) to tightly bound inner-shell electrons and loosely bound outer-shell electrons. (Goldstein et al, 1992, p.72)

37 Elastic and inelastic scattering 37 Slide from University of Tennesse

38 Ranges and interaction volumes 38

39 Secondary Electrons 39 Produced by inelastic interactions of high energy electrons with electrons of atoms in the specimen which cause the ejection of the electrons from the atoms After undergoing additional scattering events while travelling through the specimen, some of these ejected electrons emerge from the surface of the specimen.

40 Backscattered Electrons 40 High energy beam electrons may suffer single or multiple elastic scattering events in the solid, escaping from the material. The fraction of beam electrons that scatter back was found experimentally to vary directly as a function of composition (atomic number Z). This provides a valuable imaging tool: a rapid means to discriminate phases that have different mean Z values. Intensity (grey level) varies from black (voids/epoxy), to plagioclase, olivine, basaltic glass, with Ti-magnetite the brightest phase.

41 Backscattered Electrons 41

42 Topographic Contrast 42 Image from Characteriation Facility Manual, University of Minnosota

43 Other Signals in SEM 43

44 Other Signals in SEM: X-ray (for EDS and WDS) and Auger Electrons 44 Time 1 K shell L shell (=photoelectron ) HV electron knocks inner shell (K here) electron out of its orbit An electron from a higher energy orbital (L here) falls in to fill the void (time=2). 2 Blue Lines indicate subsequent times: 1 to 2, then 3 where there are 2 alternate outcomes Excess of energy released as a photon 3 EDS/ WDS The photon can exit, either by ejecting another outer shell electron as an Auger electron, or as X-ray AES (Goldstein et al, 1992, p 120)

45 45 Elemental distribution over a surface can be obtained EDS

46 Electrons and X-rays don t get them confused! 46 X-rays have no mass, no charge; electrons have charge (key!) and a small mass X-rays can be produced both by accelerating HV electrons -- and with other X-rays -- in a vacuum and colliding them with a target. The resulting X-ray spectrum contains (1) continuum or continuous background (Bremsstrahlung), (2) occurrence of sharp lines (characteristic X-rays), and (3) a cutoff of continuum at a short wavelength/high kev.