ECE 480 – Introduction to Nanotechnology

1. ECE 480 – Introduction to Nanotechnology Emre Yengel Department of Electrical and Communication Engineering Fall 2013

2. Introduction and History • Magnification: objects are made to appear larger than they are • Resolution: the ability to see close together objects as distinct • Microscopes are an important tool of biologists, and are used to study cells, tissues, and microorganisms • Credit for the first microscope is usually given to Zacharias Jansen, in Middleburg, Holland, around the year 1595. • The above early microscope found in Middleburg, Holland, corresponds to our expectations of the Jansen microscopes.

3. Early Light Microscopes

4. Basic Properties of Light Light can be described as both the waveand a fluxofparticles.

5. Basic Properties of Light • Light as electromagnetic wave with mutually perpendicular E, B components characterized by wavelength,, and frequency, , in cycles/s. Wave velocity =  x . [=500nm--> =6x1014 cycles/s]

6. Basic Properties of Light • Amplitude • Wavelength • Frequency • Phase • Polarization

7. Polarization

8. Basic Properties of Light Light/matter interaction • Particles point of view: • Absorption • Emission • Scattering • Waves point of view: • Refraction • Reflection • Absorption • Diffraction (Change of Phase and Polarization)

9. Basic Properties of Light Index of refraction for different media at 546 nm • light is refracted (bent) when passing from one medium to another • refractive index:a measure of how greatly a substance slows the velocity of light • direction and magnitude of bending is determined by the refractive indexes of the two media forming the interface Air 1.0 Water 1.3333 Cytoplasm 1.38 Glycerol 1.46 Crown Glass 1.52 Immersion Oil 1.515 Protein 1.51-1.53 Flint Glass 1.67 n increases with decreasing 

10. Lenses • focus light rays at a specific place called the focal point • distance between center of lens and focal point is the focal length • strength of lens related to focal length • short focal length more magnification

11. C F F C Optics of Thin Lens Focus d F Thin Lens: C=2F

12. 2F F F 2F 2F F F 2F Optics of Thin Lens Three different scenarios: 2F F 2F F

13. The Compound Microscope • The purpose of the microscope is to create magnification so that structures can be resolved by eye and to create contrast to make objects visible.

14. eyepiece The Compound Microscope • Compound microscope combines two lens systems: the objective, which forms a real image, and the eyepiece (forms an image at infinity). The total magnification is the product of two lenses magnification. specimen objective

15. Lenses of the Compound Microscope • Oculars (eyepieces): provide some magnification, focus image at eye (can be 1 or 2 of them) • Objectives: located on rotating nosepiece, provide magnification and resolving power (may be 3 to 6 of them) • Condenser: located under stage, focuses light on the specimen. It may also contain an iris diaphragm that controls the size of the cone of light entering the condenser. (our ‘scopes don’t have a condenser)

16. The Compound Microscope • Critical illumination • The condenser focuses the light onto the specimen plane

17. Objective Specifications

18. Resolution Lateral Resolution in Fluorescence Depends on Resolving Overlapping “Airy Disks” Rayleigh Criteria: Overlap by r’, then dip in middle is 26% below Peak intensity

19. Resolution • Maximum resolution: dmin = 0.61  / N.A. • dmin is the minimum distance between objects that can be seen as distinct (in μm) •  is the wavelength (for light, 380 -760 nm = 0.38 - 0.76 μm) • N.A. is the Numerical Aperture of the objective lens • N.A. = n sin α where n = index of refraction of medium, α = < subtended by the lens • Example: green light ( = 0.52 μm): dmin= (0.61)(0.52)/1.4 = 0.23 μm (Hence, the wavelength of light limits the resolving power of light microscopes !)

20. Resolution • Resolution (dmin) improves (smaller dmin) if λ ↓ or n↑ or α↑ • Assuming that sin α = 0.95 (α = 71.8°)

21. Resolution Resolution is better at shorter wavelengths: higher objective NA and/or higher condenser NA High NA and/or shorter  Low NA and/or longer 

22. Magnification The overall magnification is given as the product of the lenses and the distance over which the image is projected: where: D = projection (tube) length (usually = 250 mm); M1, M2 = magnification of objective and ocular. 250 mm = minimum distance of distinct vision for 20/20 eyes.

23. Depth of Focus • We also need to consider the depth of focus (vertical resolution). This is the ability to produce a sharp image from a non-flat surface. • Depth of Focus is increased by inserting the objective aperture (just an iris that cuts down on light entering the objective lens). However, this decreases resolution.

24. Types of Microscopy • Bright Field (absorption) • Dark Field (scattering) • Phase-contrast (phase change) • Polarization (scattering by birefringent specimen) • Differential interference contrast (DIC) (gradients of optical thickness) • Fluorescent (frequency change as a result of absorption/emission by fluorophores)

25. Bright Field Microscopy • Light from an incandescent source is aimed toward a lens beneath the stage called the condenser, through the specimen, through an objective lens, and to the eye through a second magnifying lens, the ocular or eyepiece. • The condenser is used to focus light on the specimen through an opening in the stage. • After passing through the specimen, the light is displayed to the eye with an apparent field that is much larger than the area illuminated. • Typically used on thinly sectioned materials

26. Bright Field Microscopy

27. Dark Field Microscopy • To view a specimen in dark field, an opaque disc is placed underneath the condenser lens, so that only light that is scattered by objects on the slide can reach the eye. • Instead of coming up through the specimen, the light is reflected by particles on the slide. • Everything is visible regardless of color, usually bright white against a dark background.

28. Dark Field Microscopy • uses the difference in scattering abilities • block out the central light rays (leave oblique only) • Result: only highly diffractive and scattering structures are seen

29. Phase Contrast Microscopy • If the sample is colorless, transparent, and isotropic, and is embedded in a matrix with similar properties, it will be difficult to image. • This is due to the fact that our eyes are sensitive to amplitude and wavelength differences, but not to phase differences. • Phase Contrast Microscopy uses the λ/4 phase change when light passes through thin structures • Similar oblique illumination to the Dark Field method • The specimen diffracts some of the light that passes through it and introduces phase lagging λ/4 • A phase difference (λ/2) is introduced between background and diffracted light (using phase plate) → destructive interference

30. Phase Contrast Microscopy Suitable for unstained specimens

31. Polarization Microscopy • Uses polarization property of light and birefringence • Polarizer polarizes light • Analyzer passes only the light with polarization perpendicular to the source light • Birefringent material introduces 2 perpendicularly polarized components, propagating at different speed in the specimen → Δφ • Constructive interference following analyzer is possible only for phase shifted light

32. Polarization Microscopy Polarized microscopy example

33. Differential Interference Contrast (DIC) (Nomarski optics) • Addon to the polarization microscopy • Wollaston prism generates 2 || beams, π/4 polarized to polarizer and laterally displaced (this is the difference to polarization microsc., endowing optical density gradient sensitivity) • The rest is similar to pol. Micr. (except for 2nd Wollaston prism) • Result: good for edge detection

34. Differential Interference Contrast (DIC) (Nomarski optics) DIC example

35. Fluorescent Microscopy • Fluorescence • Emission light has longer wavelengths than the excitation light: Stokes shift.

36. Fluorescent Microscopy • The basic task of the fluorescence microscope: • Illuminate the specimen with excitation light • Separate the much weaker emission light from the brighter excitation light. • Only allow the emission light to reach the eye or other detector. • The background is dark, the fluorescent objects are bright • Types of Fluorescence • Auto-Fluorescence (Plants, Fungi, Semiconductors, etc) • Fluorescent dyes • Fluorochromes (Flurescein, Acredine Orange, Eosin, Chlorophyll A, … ) • Genetically coded (GFP, YFP,…)

37. Fluorescent Microscopy Human cortical neurons Human brain glioma cells

38. Fluorescent Microscopy Fluorescence/DIC combination, cat brain tissue infected with Cryptococcus

39. Fluorescent Microscopy Brainbow

40. Useful in Nanoscale? Optical Microscopy vs Scanning Electron Microscopy