LS5003 Microscopy Basics

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1 2015 March 20, 9AM 12PM LS5003 Microscopy Basics Euiheon Chung Department of Medical System Engineering (DMSE) & School of Mechatronics Dasan building Room 311, Tel: x2753

2 Optical Imaging in BiO- scopy Lab at GIST Molecular scale (nm range) super- resolution microscopy using surface plasmon resonance Cellular scale (μm range) In vivo functional brain imaging using optogenetic tools Tissue- organ scale (mm or bigger range) whole- body molecular imaging / multi- color colonoscopy

3 Outline 1. Understanding the Nature of Light 2. Interaction of Light and Tissue 3. Geometric Optics in Imaging 4. Introduction to Optical Microscopy 5. Key Concepts in Microscopic Imaging 6. Imaging Characteristics 7. Microscopy Techniques with Various Contrast Mechanisms 8. Fluorescence Microscopy 3

4 1. Understanding the Nature of Light 2. Interaction of Light and Tissue 3. Geometric Optics in Imaging 4. Introduction to Optical Microscopy 5. Key Concepts in Microscopic Imaging 6. Imaging Characteristics 7. Microscopy Techniques with Various Contrast Mechanisms 8. Fluorescence Microscopy Note) Majority of figure materials are from A great source of information on basics of optics and microscopy 4

5 What is Light? Light is a form of electromagnetic energy ( ) Heating of illuminated objects, conversion of light into current (photoelectric effect), etc. Light energy is conveyed through particles: photon ( ) Ballistic ( ( ) ) behavior, e.g. shadow Photon : Light particle (quanta of light) Photon energy: E = hν (relates the duality nature of light) (h: Planck s constant [Js], ν: temporal frequency [1/s]) Light energy is conveyed through waves Interference ( ), diffraction ( ), polarization ( ) Quantum mechanics reconcile two point of view: wave/particle duality 5

$Characteristic parameters: Amplitude, wavelength, frequency (Hz) Index of refraction (n) in a media: n = c$

6 Light as wave phenomena FOR REF Electromagnetic wave travels or propagates in a direction that is oriented at right angles to the vibrations of both the electric (E) and magnetic (B) oscillating field vectors E & B: perpendicular & vibrate in phase (sinusoidal wave) The light wave propagates perpendicular to both E & B Characteristic parameters: Amplitude, wavelength, frequency (Hz) Index of refraction (n) in a media: n = c /v 6

7 The Spectrum of Light Visible Light Spectrum Modified from bigfootproof.com Electromagnetic radiation: electric and magnetic properties common to all forms of this wave- like energy 7 Visible light (0._ 0._ μm) represents only a small portion of the entire spectrum of electromagnetic radiation

8 Propagation of Light Waves Optical Microscopy Primer Q) Visible light from the sun? or a laser light? Chromaticity ( ) Polarization ( ) Coherence ( ) Collimation ( )

9 Light wave interactions with matter & representative characteristics Reflection ( ) Refraction ( ) Dispersion ( ) Diffraction ( ) Polarization ( ) Interference ( ) 9 NOTE: scattering : some form of radiation (i.e. light, sound) are forced to deviate from a straight trajectory by one or more localized non- uniformities in the medium through which they pass.

$Refraction of Light Q) How should you target fish when spearfishing?$ $Refractive index n = (speed of light in vacuum)/(that in medium) = c/v e.g. Air 1.0003, Water 1.$

10 Refraction of Light Q) How should you target fish when spearfishing? Q) Day talks are overheard by birds While night talks are by rats. TRUE? Refractive index n = (speed of light in vacuum)/(that in medium) = c/v e.g. Air , Water Immersion oil 1.515, Glass (Crown) Snell s Law: n 1 sinθ 1 = n 2 sinθ

11 Dispersion of Light dn/dk 0 In dispersive media Wikipedia.org n = n (λ) In microscopic lenses, dispersion causes chromatic ab ( ). 11

12 Diffraction of Light flickriver.com 12

$Diffraction of$ bright line

13 Diffraction of Light The bright line outlining the silhouette of the object is ed light. 13 physics.umd.edu intimateoutdoors.wordpress.com schoolphysics.co.uk

14 Interference of Light 14

15 Light wave interactions with matter & representative characteristics Reflection ( ) Return of wavefront into the medium from which it originated at an interface of different media Refraction ( ) Change in direction of a wave due to a change in its transmission medium (i.e. speed) Dispersion ( ) Wave velocity dependent on frequency (or wavelength) Diffraction ( ) Bending of waves when encounter an obstacle Polarization ( ) The orientation of electromagnetic waves Interference ( ) Superposition of two or more waves resulting in a new wave pattern Richard Feynman said that no- one has ever been able to define the difference between interference and diffraction satisfactorily. It is just a question of usage, and there is no specific, important physical difference between them when there are only a few sources, say two, called interference, if there is a large number of them, diffraction is more often used. (Lectures on Physics, Vol 1, 1963, pg. 30-1). 15 NOTE: scattering : some form of radiation (i.e. light, sound) are forced to deviate from a straight trajectory by one or more localized non- uniformities in the medium through which they pass. Questions?

16 1. Understanding the Nature of Light 2. Interaction of Light and Tissue 3. Geometric Optics in Imaging 4. Introduction to Optical Microscopy 5. Key Concepts in Microscopic Imaging 6. Imaging Characteristics 7. Microscopy Techniques with Various Contrast Mechanisms 8. Fluorescence Microscopy 16

Light propagation in tissue Q) Why biological tissue appear turbid (, )? http://live- billiards.com- download.

17 Light propagation in tissue Q) Why biological tissue appear turbid (, )? billiards.com- download.net/ How does light propagate in tissue? What limitations do the physics of light scattering or absorption impose on optical imaging techniques? 17

18 Overview of the interactions between Light and Tissue Backscattering Diffuse scattering Absorption Tissue Elastic Scattering ( ) Scattered and un- scattered waves with the same wavelength and no energy loss Scattering affects angle/ polarization/ phase Absorption ( ) Conversion of light energy into another type (e.g. heat) Note: Scattering and absorption are wavelength- dependent Non- elastic interactions Fluorescence, phosphorescence, Raman scattering, etc. 18

Why is the Sky Blue while the Cloud is White?

- 4 Anisotropy constant, g 0 Mie scattering Larger particles, ϕ

of λ Forward scattering, g > 0 http://math.ucr.

19 Why is the Sky Blue while the Cloud is White? FOR REF Rayleigh scattering Small particles, ϕ << λ μ s (λ) ~ λ - 4 Anisotropy constant, g 0 Mie scattering Larger particles, ϕ ~ λ μ s (λ) ~ roughly indep. of λ Forward scattering, g > astr.gsu.edu/hbase/atmos/blusky.html (N/A any more) 19

$Biological Origins of Light Scattering FOR REF Photon scattering with cell organelles Tissue components n, refractive index$ 38 1.41 Melanin n = 1.6 1.

20 Biological Origins of Light Scattering FOR REF Photon scattering with cell organelles Tissue components n, refractive index Extracellular matrix n = Cell cytoplasm n = Cell nucleus n = Mitochondria/organelles n = Melanin n = Biological structures of various sizes for photon scattering Optical scattering from light interaction with biological structures More scattering with i) increased size & ii) increased Δn Q) primary scatterer inside cells? 20

Light propagates ~ 100 μm before scattered Scattering decreases as wavelength increases

21 Features of Light in Tissue: Optical Window Oximetry.org internationalcancertherapy.com Q) How does Pulse Oximetry work? Light propagates ~ 100 μm before scattered Scattering decreases as wavelength increases Absoption by Hemoglobin (Hb), melanin in visible spectrum Optical windows (or therapeutic window): um, um, 2.2 um Questions? 21

22 1. Understanding the Nature of Light 2. Interaction of Light and Tissue 3. Geometric Optics in Imaging 4. Introduction to Optical Microscopy 5. Key Concepts in Microscopic Imaging 6. Imaging Characteristics 7. Microscopy Techniques with Various Contrast Mechanisms 8. Fluorescence Microscopy 22

23 Optical Imaging: General concept 23

24 Reflection and Refraction at an interface 24

$(assume the refractive index of the water as n) Draw the rays from the actual fish to the observer s eye, and also the virtual rays where apparent fish is perceived by the observer$

25 Spearfishing and Apparent depth When you are spearfishing, what would be the apparent depth h of a fish if it actually is h below the surface? (assume the refractive index of the water as n) Draw the rays from the actual fish to the observer s eye, and also the virtual rays where apparent fish is perceived by the observer Use Snell s law 25 Photo: onebigphoto.com

26 Optical imaging : Using a lens Each point source from the object plane focuses onto a point image at the image plane (Note: image inversion) 26

27 Optical system example: human eye as a camera Analogy with DSLR (digital single- lens reflex) camera: Iris ( ): iris diaphragm ( ) Retina ( ): CCD (charge- coupled detector) sensor 27 wiki

$system do not focus perfectly due to Diffraction- limited resolution Aberration: an imperfection in image formation thus result in image blurring$

28 Ideal optical imaging system Ideal imaging system: Each point in the object is mapped onto a single point in the image In real world, the imaging system do not focus perfectly due to Diffraction- limited resolution Aberration: an imperfection in image formation thus result in image blurring 28

29 Geometrical optics of a simple lens: ray tracing exercise f: focal length F,F: focal points d o : object lens distance d i : lens- image distance Object- image math: Lens equation 1/f = 1/d o + 1/d i Magnification = h i / h o = d i / d o 29

Location of a real and virtual images in a light microscope Real image: can be seen on a screen placed in the image plane Virtual image: cannot be observed on a

30 Location of a real and virtual images in a light microscope Real image: can be seen on a screen placed in the image plane Virtual image: cannot be observed on a viewing screen or recorded on a film Viewing an image in a microscope A real image is formed on the retina, but is perceived as a virtual image located in front of the eye 30

31 1. Understanding the Nature of Light 2. Interaction of Light and Tissue 3. Geometric Optics in Imaging 4. Introduction to Optical Microscopy 5. Key Concepts in Microscopic Imaging 6. Imaging Characteristics 7. Microscopy Techniques with Various Contrast Mechanisms 8. Fluorescence Microscopy 31

Developments of Light Microscopes homunculus olympusmicro.

microscope With Johan Ham, first observed human sperm (1677)

illuminating lamp device Zeiss microscope (1930s): Knobs,

32 Developments of Light Microscopes homunculus olympusmicro.com Von Leeuwenhoek microscope (1600s): Simple magnifier microscope With Johan Ham, first observed human sperm (1677) Hooke microscope (1670s): Simple compound microscope Sample illuminating lamp device Zeiss microscope (1930s): Knobs, nosepieces, eyepieces, and mechanical stage Very functional and still in use today 32

33 Image formation in microscopy #1 : past century 1/a + 1/b = 1/ f Standard finite tube length: US: b = 160 mm EU (Royal Micros. Soc.: RMS): b = 210 mm : good for fluorescence or scanning microscope 4f imaging system 33

34 Image formation in microscopy #2 Finite- tube length magnification: M FT = - b/a Note: a, working distance Infinity- corrected magnification: M INF = - L tb / L ob 34

35 Optical paths in bright field microscopy #1 Bright field microscopy Simplest white light trans- illumination Contrast from absorbance difference from sample e.g. histopathology 35

36 Optical paths in bright field microscopy #2 36

37 Light sources: which one to choose for shining? FOR REF Olympusmicro.com zeiss- campus.magnet.fsu.edu/ 37

38 Light sources: emergence of new shining tools FOR REF Consider wavelength characteristics, brightness, stability, coherence & uniformity! LED is an emerging technology. zeiss- campus.magnet.fsu.edu/ 38

39 1. Understanding the Nature of Light 2. Interaction of Light and Tissue 3. Geometric Optics in Imaging 4. Introduction to Optical Microscopy 5. Key Concepts in Microscopic Imaging 6. Imaging Characteristics 7. Microscopy Techniques with Various Contrast Mechanisms 8. Fluorescence Microscopy 39

Numerical Aperture (NA): light collecting power - - Defined by E. Abbe (1840-1906) Dimensionless number Aperture stop: A physical elements that limits NA NA vs.

40 Numerical Aperture (NA): light collecting power - - Defined by E. Abbe ( ) Dimensionless number Aperture stop: A physical elements that limits NA NA vs. optical resolution Physical meaning: NA limits optical information entering the imaging system NA indicates the resolving power of a lens With larger NA Collect more light Provide brighter image Provide shallower depth of field Provide better lateral resolution 40

41 Image of a Point Source of Light: PSF Point Objective Tube lens CCD PSF source Radius of Airy disk (or Rayleigh criterion) d = 1.22λ (f/d) = 0.61 λ / NA where f: focal length D: lens diameter Airy disk Intensity PSF: point- spread function, the response of an imaging system to a point source The image of a self- luminous point object is a diffraction pattern created by the action of interference in the image plane. Q) What would be the images of 10 nm, 50 nm, 100 nm objects? 41

42 NA and spatial resolution PSF: point- spread function The narrower the PSF, the higher the resolution Key parameter for defining optical resolution (further discussion later) How to define resolution with given PSF? 42

43 1. Understanding the Nature of Light 2. Interaction of Light and Tissue 3. Geometric Optics in Imaging 4. Introduction to Optical Microscopy 5. Key Concepts in Microscopic Imaging 6. Imaging Characteristics 7. Microscopy Techniques with Various Contrast Mechanisms 8. Fluorescence Microscopy 43

Contrast The visibility of a structure in an image depends on, among other factors, the contrast C: C = _ / _ While <I> is the average background image intensity, ΔI is the intensity

44 Contrast The visibility of a structure in an image depends on, among other factors, the contrast C: C = _ / _ While <I> is the average background image intensity, ΔI is the intensity variation in the region of interest. Exa) Interferometric visibility visibility = amplitude /average Practice: Show that visibility = (I max I min ) / (I max + I min ) En.wikipedia.org 44

45 Signal- to- Noise Ratio (SNR) Contrast does not represent a fundamental limitation on visualization since it can be artificially enhanced by, for example, subtracting part of the background (thresholding) or raising the intensity to some power. Statistical noise does, however, represent a fundamental limitation. The signal- to- noise ratio (SNR) is defined as SNR = _ / _ Where σ BG denotes the standard deviation of the background intensity (i.e. the noise representing the root- mean- squared (rms) value of the intensity fluctuations. 45

Field of View The field of view (FOV) in an image refers to

A tradeoff often exists between FOV and spatial resolution.

resolution. Q) How to determine the FOV in an imaging system?

46 Field of View The field of view (FOV) in an image refers to the extent of the image field that can be seen all at once. A tradeoff often exists between FOV and spatial resolution. For example, zooming in a camera compromises the FOV for resolution. Q) How to determine the FOV in an imaging system? Q) Scale bar in an image? diglloyd.com community.pictureline.com 46

47 Frame Rate The frame rate is defined as the number of frames of an animation that are displayed per second, measured in frames per second (fps); it measures how rapidly an imaging system produces consecutive 2D images. At or above the video rate (30 fps), the human eye cannot resolve the transition of images; hence, the animation appears smooth. Q) What is the right fps for real- time imaging? 47

48 1. Understanding the Nature of Light 2. Interaction of Light and Tissue 3. Geometric Optics in Imaging 4. Introduction to Optical Microscopy 5. Key Concepts in Microscopic Imaging 6. Imaging Characteristics 7. Microscopy Techniques with Various Contrast Mechanisms 8. Fluorescence Microscopy 48

49 Contrast generation in Imaging Imaging: The process of mapping a certain physical property of an object and displaying it in a visual form. Physical property (or Contrast mechanism): Absorption (e.g. light imaging, X- rays) Scattering (e.g. phase contrast imaging) Emission (e.g. fluorescence imaging) Reflectivity (e.g. ultrasound, common photography) Proton density (e.g. MRI) Concentration of radionuclides (e.g. nuclear imaging) In microscopy, intrinsic specimen absorption, scattering, and emission are quantities of interest. Resolution vs. Contrast Resolution: A property of the instrument itself Contrast: Depends on both the instrument and sample 49

50 Sample Contrast USAF resolution target 50

51 Dark- field microscopy 51

Phase Contrast Microscopy converting phase into intensity Frits Zernike: The Nobel Prize in Physics (1953) Allows label- free, noninvasive investigation of live cells (major breakthrough) How quick

52 Phase Contrast Microscopy converting phase into intensity Frits Zernike: The Nobel Prize in Physics (1953) Allows label- free, noninvasive investigation of live cells (major breakthrough) How quick we are to learn- that is, to imitate what others have done or thought before- and how slow to understand that is, to see the deeper connections. Slowest of all, however, are we in inventing new connections or even in applying old ideas in a new field. [Zernike] Science 1955 How I discovered phase contrast 52

53 Phase contrast microscopy: Principle FOR REF Enhance contrast of phase objects using phase delay Placing a small metal film covering the dc component in the Fourier plane of the objective to both attenuate and shift the phase of the unscattered field. Note the presence of halo 53

54 1. Understanding the Nature of Light 2. Interaction of Light and Tissue 3. Geometric Optics in Imaging 4. Introduction to Optical Microscopy 5. Key Concepts in Microscopic Imaging 6. Imaging Characteristics 7. Microscopy Techniques with Various Contrast Mechanisms 8. Fluorescence Microscopy 54

55 Green Fluorescent Protein (GFP) & GFP labeling The structure of GFP cdna of a target molecule fused to that of GFP GFP: a protein that exhibits bright green fluorescence when exposed to blue light. Isolated from the jellyfish Aequorea victoria (Nobel prize in Chemistry 2008) campus.magnet.fsu.edu/ 55 DOI: /annurev.bioeng ,

56 Green Fish Fluorescence Imaging Various fluorescent proteins science.kukuchew.com Brainbow Nature.com Microscopyu.com 56

57 Fluorescence microscopy: Principle Note: Isotropic emission micro.magnet.fsu.edu 57

Principles of Fluorescence Fluorescence: the

(fluorophore) as a result of incident radiation

Jablonski diagram: illustrates the energy states of

58 Principles of Fluorescence Fluorescence: the radiation of light (emission) by certain substance (fluorophore) as a result of incident radiation (excitation) of a shorter wavelength. Jablonski diagram: illustrates the energy states of a molecule and transitions among them (absorption & emission mechanism) 58

59 Stoke s Shift: Red shift? Stoke s Shift: the difference between excitation & emission wavelengths between maxima of the absorption and emission spectra Q) Which one has lower energy, emitted photon or exciting photon? (exception: two- photon fluorescence) 59 Art.ca

60 Fluorescence Microscopy Jic.ac.uk Modified from the book cover of the Biology of Cancer (Robert A.Weinberg) Fluorescence adds s to the measurement! micro.magnet.fsu.edu 60

61 Fluorescence Microscope: Filter blocks FOR REF Olympusmicro.com 61

62 Anatomy of the fluorescence microscope micro.magnet.fsu.edu 62

63 Summary of Light Imaging & Microscopy 1. Understanding the Nature of Light Light as particle Light as wave Characteristics of Light Reflection Refraction Dispersion Diffraction Polarization Interference 2. Interaction of Light and Tissue Scattering Absorption NIR window 3. Geometric Optics in Imaging Reflection / Refraction Simple lens Light microscope: real and virtual image Aberrations 4. Introduction to Optical Microscopy Image formation in microscopy finite vs. infinite tube length Conjugate planes and Kohler illumination Objective lens and Light sources 5. Key Concepts in Microscopic Imaging Diffraction & interference in image formation Abbe's diffraction theory of image formation Numerical aperture and resolution Point- spread function 6. Imaging Characteristics Contrast Signal- to- Noise Ratio (SNR) Contrast- to- Noise Ratio (CNR) Field- of- view (FOV) Frame rate 7. Microscopy Techniques with Various Contrast Mechanisms Dark- field microscopy Phase contrast microscopy Differential interference contrast microscopy 8. Fluorescence Microscopy Excitation & Emission Jablonski Diagram Stokes Shift Photobleaching/ Phototoxicity/ Photodamage 63

64 Useful Information Book: Murphy & Davidson, Fundamentals of Light Microscopy and Electronic Imaging (2013) Second Edition Reading list: Abramowitz, Microscope basics and beyond (2003) - Presents many clear visual diagrams for understanding basics and concepts The following sites provides wonderful explanations with many useful figures. Molecular Expressions Olympus Microscopy Resource Center Nikon MicroscopyU for microscopy education Zeiss Education in Microscopy and Digital Imaging campus.magnet.fsu.edu/ 64

Introduction to Light Microscopy. (Image: T. Wittman, Scripps)

Introduction to Light Microscopy (Image: T. Wittman, Scripps) The Light Microscope Four centuries of history Vibrant current development One of the most widely used research tools A. Khodjakov et al. Major