Bio 407. Applied microscopy. Introduction into light microscopy. José María Mateos. Center for Microscopy and Image Analysis

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1 Center for Microscopy and Image Analysis Bio 407 Applied Introduction into light José María Mateos

2 Fundamentals of light Compound microscope Microscope composed of an objective and an additional lens (eyepiece, occular, tube lens) Robert Hooke ca 1670 olympusmicro.com

3 Fundamentals of light Compound microscope Microscope composed of an objective and an additional lens (eyepiece, occular, tube lens) Compound microscope, zeiss-campus.magnet.fsu.edu

4 Object F objective Objective F eyepiece Primary image Fundamentals of light Eyepiece The objective forms a magnified image of the object near (or in) the eyepiece The intermediate image is examined by the eyepiece and eye forming a real image on the retina Virtual image seen by eye

Fundamentals of light Conjugate focal planes Primary image

5 illumination light path four aperture planes Iris image-forming light path four field planes Retina Bio 407 Fundamentals of light Conjugate focal planes Primary image Back focal plane Sample plane Aperture diaphragm Field diaphragm Lamp filament

6 Fundamentals of light Köhler illumination

7 Fundamentals of light Magnification: What is the maximal magnification? Is there a limit in useful magnification? => Why is there a limit in resolution? Compound microscope, Robert Hooke ca 1670

Radio Human eye 100 m Properties of light Cells Infrared 10 m Red blood cells

8 Resolution Limit Wavelength Object 1 mm MRI, CT Why is there a limit in resolution? Radio Human eye 100 m Properties of light Cells Infrared 10 m Red blood cells Visible Light microscope Ultraviolet 1 m Bacteria 100 nm Mycoplasma Viruses 10 nm Proteins x, -rays 1 nm Amino acids Electron microscope Bio nm Atoms

9 Why is there a limit in resolution? Properties of light

10 Why is there a limit in resolution? Light interactions with matter

$Diffraction and$

11 Why is there a limit in resolution? Diffraction and interference

$Aperture angle single-slit diffraction$

12 Why is there a limit in resolution? Aperture angle single-slit diffraction pattern

$Diffraction image of a point source of light The image of$ created by interference in the image plane The pattern is

central spot contains 84% of light The image is called:

13 Diffraction image of a point source of light The image of a self-luminous point in a microscope is a pattern created by interference in the image plane The pattern is a central bright spot surrounded by a series of rings The central spot contains 84% of light The image is called: Airy disk (after Sir George Airy ( )) cambridgeincolour.com

$Image formation in the light microscope Diffraction patterns in the back focal plane Generation of an image by interference requires collection of two$

14 Image formation in the light microscope Diffraction patterns in the back focal plane Generation of an image by interference requires collection of two adjacent orders of diffracted light! a,b: no image is formed c: image is formed d: image with high definition due to multiple diffracted orders collected

$Resolution and refractive index The objective aperture must capture light from a wide angle for maximum resolution (diffracted or emitted$

15 Resolution and refractive index The objective aperture must capture light from a wide angle for maximum resolution (diffracted or emitted light) NA = n sin α α: half angle of the cone of specimen light accepted by the objective n: refractive index of medium between lens and specimen

16 Why is there a limit in resolution? Resolution and aperture angle Aperture of objective determines the resolution, not the magnification!

17 Why is there a limit in resolution? Resolution and aperture angle Objective with high aperture (NA 1.25) Aperture of objective determines the resolution, not the magnification! Objective with low aperture (NA 0.3)

18 Resolution and Rayleigh criterion Resolving power of microscope: 0.61 λ Concept: an image of an extended object consists of a pattern of overlapping diffraction spots Resolution: the larger the NA of the objective, the smaller the diffraction spots (airy disks).

19 Amplitude and phase objects: only amplitude objects can be seen by eye! Bright field Phase contrast Phase contrast

20 Amplitude and phase objects: only amplitude objects can be seen by eye! Phase contrast

21 Phase contrast

22 Bright Field Microscopy Phase Contrast Microscopy Objective Objective with Phase Ring Condenser Condenser Phase Ring Alignment: Köhler illumination Condenser aperture: close max 20% Field aperture: illuminaton of field of view Alignment: Köhler illumination Condenser aperture fully open Field aperture: illuminaton of field of view Adjust correct phase rings

23 Differential interference (DIC)

24 Bright Field Microscopy Differential Interference Microscopy Phase Contrast Microscopy Polarizer Wollaston Prism Objective Objective Objective with Phase Ring Condenser Condenser Condenser Wollaston Prism Polarizer Phase Ring Alignment: Köhler illumination Condenser aperture: close max 20% Field aperture: illuminaton of field of view Alignment: Köhler illumination Condenser aperture: close max 20% Field aperture: illuminaton of field of view Adjust polarizers and wollaston prisms Alignment: Köhler illumination Condenser aperture fully open Field aperture: illuminaton of field of view Adjust correct phase rings

25 Bio 407 Fluorescence

26 Fluorescence

27 Bio 407 Fluorescence

28 Bio 407 Fluorescence

29 Fluorescence

30 Bio 407 Fluorescence

Source Polarizer Wollaston Prism Condenser Wollaston Prism Phase Ring

31 Setup Light microscopes Bright Field Microscopy (including DIC / Phase Contrast) Fluorescence Microscopy Ocular Fluorescence Light Source Polarizer Wollaston Prism Condenser Wollaston Prism Phase Ring Polarizer Light Source Objectives Sample Plane Z Focus Fluorescence Filter Cube

Bright Field Microscopy Differential Interference Microscopy Phase Contrast Microscopy Fluorescence Microscopy Polarizer Wollaston Prism Fluorescence Cube Objective Objective Objective with Phase

32 Bright Field Microscopy Differential Interference Microscopy Phase Contrast Microscopy Fluorescence Microscopy Polarizer Wollaston Prism Fluorescence Cube Objective Objective Objective with Phase Ring Objective Condenser Condenser Condenser Wollaston Prism Polarizer Phase Ring Alignment: Köhler illumination Condenser aperture: close max 20% Field aperture: illuminaton of field of view Alignment: Köhler illumination Condenser aperture: close max 20% Field aperture: illuminaton of field of view Adjust polarizers and wollaston prisms Alignment: Köhler illumination Condenser aperture fully open Field aperture: illuminaton of field of view Adjust correct phase rings Alignment: Correct alignment of fluorescence lamp

33 Widefield Bio 407 Confocal laser scanning

34 Confocal laser scanning Increase signal to noise ratio Widefield Higher Z-resolution Less out-of-focus blur Confocal

35 Theory Reality 0.1 µm bead focal plane Bio 407 Confocal laser scanning Spatial resolution in x,y and z Objects are 3 dimensional The resulting image will also be a 3D image in the image space an image of an extended object consists of a pattern of overlapping diffraction spots Crossection 1 µm

$extended object consists of a pattern of overlapping diffraction spots In widefield$

36 Confocal laser scanning Spatial resolution in x,y and z Objects are 3 dimensional The resulting image will also be a 3D image in the image space an image of an extended object consists of a pattern of overlapping diffraction spots In widefield the out of focus information is increasing the background and results in low contrast images

37 focal plane Theory 0.2 µm or smaller structure Bio 407 Confocal laser scanning Spatial resolution in X,Y and Z 1 µm Reality Crossection d = 0.61 λ / NA Imaging EGFP in living cells has a resolution of approximately 200 (XY) and 500 nanometers (Z)

38 Confocal laser scanning Increase signal to noise ratio Higher Z-resolution Less out-of-focus blur Only small volume at once Large volumes need long acquisition times

39 Confocal laser scanning Sample is excited by point focus laser Emitted fluorescent from focus is focus at pinhole and reaches detector Emitted fluorescent from out-of-focus is also out-offocus at pinhole and largely excuded from detector

40 Confocal laser scanning

41 Confocal laser scanning

42 Confocal laser scanning Select fluorochromes according to laser lines

43 Confocal laser scanning X-Y Galvoscanner

44 Confocal laser scanning Photomultiplier (PMT)

45 Bio 407 Confocal laser scanning 3D + Time

46 Confocal laser scanning Widefield vs laser scanning confocal microscope

47 Confocal laser scanning Widefield vs laser scanning confocal microscope

48 Confocal laser scanning Widefield vs laser scanning confocal microscope

49 Confocal laser scanning Widefield vs laser scanning confocal microscope

50 Confocal laser scanning Widefield vs laser scanning confocal microscope

51 Confocal laser scanning Fluorescence recovery after photobleaching FRAP

52 Glutamate Receptor movement in NMJ of drosophila Bio 407 Confocal laser scanning Fluorescence recovery after photobleaching FRAP Füger et al., Nature Protocols 2, (2007)

53 Confocal laser scanning Fluorescence recovery after photobleaching FRAP

54 Confocal laser scanning Fluorescence resonance energy transfer FRET Emission spectrum donor overlapping Excitation spectrum acceptor Förster distance range 10 nm Gines and Davidson Florida State University

55 Spinning disk Increase acquisition speed

56 Spinning disk Increase acquisition speed Gines, Rainey and Davidson Florida State University

57 Selective Plane Illumination Microscopy SPIM 4D imaging Light-sheet-imaging technique Better signal-to-noise ratio Low photodamage Huisken et al., Science 305, (2004)

58 Selective Plane Illumination Microscopy SPIM Widefield compared with SPIM Widefield SPIM Huisken et al., Science 305, (2004)

59 Total internal refletion fluorescence TIRF Signal to noise ratio Fellers and Davidson, Florida State University Ross and Schwartz Nikon Instruments

$Refractive index between glass and sample Critical angle to produce evanescent field Axelrod, Long and Davidson Univ Michigan and Florida State$

60 TIRF Signal to noise ratio Nanometer range Evanescent wave excites only fluorophores near the glass farther away from the interface are not excited Refractive index between glass and sample Critical angle to produce evanescent field Axelrod, Long and Davidson Univ Michigan and Florida State University

61 TIRF Prism method Objective lens method Fellers and Davidson, Florida State University Ross and Schwartz Nikon Instruments

62 Bio 407 Multiphoton Imaging deep into tissue

63 Multiphoton Imaging deep into tissue

64 Multiphoton Two photons that arrive simultaneously at a molecule combine their energies to promote the molecule to an excited state, and leads to fluorescence emission.

molecule to an excited state, and leads to

65 Multiphoton Two photons that arrive simultaneously at a molecule combine their energies to promote the molecule to an excited state, and leads to fluorescence emission. Helmchen and Denk, Nature Methods 2005

66 Multiphoton Imaging in scatter tissue All fluorescent photons provide useful signal. Helmchen and Denk, Nature Methods 2005

67 Multiphoton Deep tissue two-photon Helmchen and Denk, Nature Methods 2005

68 Bio 407 Multiphoton

69 Bio 407 Multiphoton laser scanning microscope

70 Multiphoton

$Super resolution Beyond the diffraction limit d = 0.$

71 Super resolution Beyond the diffraction limit d = 0.61 λ / NA Imaging EGFP in living cells has a resolution of approximately 200 (XY) and 500 nanometers (Z)

$diffraction limit d = 0.$ 61 λ / NA Imaging EGFP in living cells

72 Super resolution Beyond the diffraction limit d = 0.61 λ / NA Imaging EGFP in living cells has a resolution of approximately 200 (XY) and 500 nanometers (Z)

$objetives Diffraction limit (Abbe, 1873) Lateral (XY) 200nm Axial (Z) 500nm Abbe, E. Arch. Mikrosk. Anat.$

73 Center for Microscopy and Image Analysis Superresolution Resolution depends on: wavelength and opening angle of objetives Diffraction limit (Abbe, 1873) Lateral (XY) 200nm Axial (Z) 500nm Abbe, E. Arch. Mikrosk. Anat. Entwicklungsmech. 1873

74 Superresolution

75 Superresolution

76 222nm Superresolution

77 222nm Superresolution

78 Superresolution Resolution limit, defined as the minimum distance that two point-source objects have to be in order to distinguish the two sources from each other 222nm

79 Center for Microscopy and Image Analysis Superresolution Structured illumination José María Mateos

80 Superresolution Structured illumination

81 Superresolution Structured illumination Sample structure Illumination pattern Image (Moiré) Algorithm (calculation of sample structure) Gustafsson 2005

82 Superresolution Structured illumination

83 Superresolution Structured-illumination Superresolution white paper SR-SIM Zeiss

84 Superresolution Structured illumination Advantages Uses standard epifluorescence microscope No special fluorophores required Live cell imaging possible Large field of view 3D-possible (but decreased speed, etc.) Disadvantages Resolution ~100 nm

85 In STED, an initial excitation pulse is focused on a spot. The spot is narrowed by a second, donutshaped pulse that prompts all excited fluorophores in the body of the donut to emit (this is the emission depletion part of STED). This leaves only the hole of the donut in an excited state, and only this narrow hole is detected as an emitted fluorescence. Bio 407 Super resolution Stimulated emission depletion STED Abbott Nature 459, (2009)

86 Super resolution Stimulated emission depletion STED

87 Super resolution Stimulated emission depletion STED Based on confocal Hell et al.,

88 Super resolution Confocal Stimulated emission depletion STED Based on confocal STED Sample courtesy Martin Engelke, Urs Greber, Institute of Zoology, University of Zurich

89 Advantages Standard fluorescent proteins and fluorophore tagged antibodies can be used High frame rate for live cell imaging Resolution: x, y ~20 nm, z ~50 nm 3D possible Bio 407 Super resolution Stimulated emission depletion STED Disadvantages Special lasers required/desired (supercontinuum laser) Small field of view (confocal imaging pixel size)

90 Imaging single molecules Bio 407 Super resolution Statistical STORM Stochastic optical reconstruction Position of a single molecule can be localized to 1 nm accuracy or better if enough photons are collected and there are no other similarly emitting molecules within ~200 nm (Heisenberg 1930, Bobroff 1980). PALM Photoactivated localization GSD Ground state depletion

91 Super resolution Photoactivated localization PALM single-molecule techniques stochastic photoswitching where most of the molecules remain dark llippincott-schwartz Nature 459, (2009)

92 Super resolution Photoactivated localization PALM single-molecule techniques stochastic photoswitching where most of the molecules remain dark Price and Davidson Florida State University

93 Super resolution Photoactivated localization PALM single-molecule techniques stochastic photoswitching where most of the molecules remain dark Price and Davidson Florida State University

94 Super resolution Photoactivated localization PALM single-molecule techniques stochastic photoswitching where most of the molecules remain dark Price and Davidson Florida State University

95 Advantages Large field of view (imaging with CCD camera) Resolution: x, y ~25 nm, z ~ 65 nm 3D possible Disadvantages Photoactivatable proteins/fluorophores required (EosGFP etc.) Slow for high accuracy/resolution Special lasers may be required Live-cell difficult (illumination, bleaching, time) Bio 407 Super resolution Photoactivated localization PALM

96 Center for Microscopy and Image Analysis Superresolution Structured illumination José María Mateos

Introduction to light microscopy

Center for Microscopy and Image Anaylsis Introduction to light microscopy Basic concepts of imaging with light Urs Ziegler ziegler@zmb.uzh.ch Light interacting with matter Absorbtion Refraction Diffraction