An opening a = λ would put the first minima at θ = 90

Transcription

1 Microscopy

2 Outline Resolution & definitions Fluorescence microscopy Some other optical microscopy techniques Electron microscopes X-ray microscopy Scanning tunneling microscopes 2

3 Microscopy history First primitive microscopy H.+ Z. Janssen 1590 Yeast cells, blood cells A. van Leuwenhoek 1700 Bacteria L. Pasteur 1850 Theory of resolution E. Abbe, C. Zeiss 1872 Electron microscope E. Ruska 1933 Scanning probe microscopy G. Binnig, H. Rohrer 1980

4 Plane wave diffraction in a slit An opening a = λ would put the first minima at θ = 90 If the space between the aperture and the screen has index of refraction n an opening a = λ/n would put the first minima at θ = 90 Almost all optical components are reciprocal, i.e. if we reverse all beams that hit the screen (now using these beams as the source), light will be focussed at the aperture If the space between the aperture and the screen has index of refraction n the focus can now be a factor of n smaller

5 Plane wave focal spot D Focal ω d plane Focal = 2 0 f Stefan Andersson-Engels

6 Ideal focus: Airy spot Fig. Electrical field at focus. The Airy disk the best focused spot of light that a perfect lens with a circular aperture can make, limited by the diffraction of light

7 Plane wave focal spot D f Focal ω d plane Focal = 2 0 r distance from the center of the focal plane perpendicular to the beam propagation direction At the first minimum 1 Ι/Ι 0 r min 3.83 f λ = = D π f λ D Stefan Andersson-Engels krd/2f πdr/λf

8 Resolution - when are two peaks considered resolved?

9 Rayleigh resolution criterion On the limit of resolution. The maximum of one peak overlaps the first minimum of the other. The intensity is reduced 26% in the centre.

10 Numerical aperture (NA) The numerical aperture with respect to a point P depends on the half-angle, θ 1, of the maximum cone of light that can enter or exit the lens and the ambient index of refraction. NA = n 1 sin θ 1 = n 2 sin θ 2.

11 High magnification optical microscope Numerical aperture =NA = n*sin(u) Resolution limit Δx = 1.22 * λ/(2*na) Highest possible resolution λ/2 Fig 10.2

12 Point spread function (PSF) - how a delta peak is imaged PSF adapted from Stefan Andersson-Engels

13 Imaging resolution Image The sketch on the right shows the remaining modulation when imaging a sinusodial intensity pattern Intensity Stefan Andersson-Engels Object

14 Modulation transfer function (MTF) The modulation transfer function (MTF) describes the modulation depth as a function of spatial frequency

15 Depth of field (DOF) DOF is the range over which an object can be translated while still remaining sharp at the image plane A high NA gives a small DOF High NA Image plane Low NA

16 Outline Resolution & definitions Fluorescence microscopy Some other optical microscopy techniques Electron microscopes X-ray microscopy Scanning tunneling microscopes 16

17 Outline Fluorescence microscopy Confocal microscopy Fluorescence markers 2-photon microscopy Time domain imaging Fourier transform microscopy Sub-diffraction resolution microscopy 17

18 iphone gadget Optical microscopes

19 Scanning confocal fluorescence microscope A confocal configuration improves resolution and in particular longitudinal resolution It also decreases interference or background from scattered light A disadvantage is that the image needs to be scanned

20 Outline Fluorescence microscopy Confocal microscopy Fluorescence markers 2-photon microscopy Time domain imaging Fourier transform microscopy Sub-diffraction resolution microscopy 20

21 Bovine pulmonary artery epithelial cells, with functional markers fluorescing in different wavelengths Bovine pulmonary artery epithelial cells

22 Functional fluorescent substances Fig 10.7

23 Two-wavelength fluorescence microscopy Fig 10.8

24 Fluorescence microscopy Dyes sensitive to Ca ion concentration and to ph value Fig 10.6

25 Selectable light source fluorescence microscopy Fig 10.9

26 Ratio fluorescence microscopy using ph-sensitive dye Fig 10.10

27 Outline Fluorescence microscopy Confocal microscopy Fluorescence markers 2-photon microscopy Time domain imaging Fourier transform microscopy Sub-diffraction resolution microscopy 27

28 excitation Two-photon induced fluorescence Fluorescence Excited state population and thereby also fluorescence intensity, I F, proportional to excitation light intensity, I, squared, I F I 2 Advantages: The spatial resolution of two-photon fluorescence is better than for single-photon fluorescence The two-photon fluorescence is spectrally well separated from scattered excitation light Excitation light may be in the infrared and penetrate further into tissue without being absorbed Disadvantage: Excitation is much weaker

29 Two-photon fluorescence microscopy Intrinsic 3D resolution No photobleaching out of focus

30 Two-photon fluorescence in rodent lung tissue

31 Outline Fluorescence microscopy Confocal microscopy Fluorescence markers 2-photon microscopy Time domain imaging Fourier transform microscopy Sub-diffraction resolution microscopy 31

32 Two-photon fluorescence microscopy spectral and temporal domain Fig 10.13

33 Detection of weak signals with sub ps time resolution Svanberg, Atomic & Molecular spectroscopy, page

34 Two-photon fluorescence microscopy spectral and temporal domain Fig 10.13

35 Lifetime imaging 2 hours after tumor marker (ALA) application (Color scale) Basal cell carcinoma studies with tumor marker Advantages with lifetime imaging as compared to conventional imaging: Insensitive to fluctuations in light intensity Insensitive to geometrical factors Advantages with lifetime imaging as compared to spectral imaging: Tissue or markers that are spectrally similar might be distinguished by their life times

36 Autofluorescence Lifetime Imaging with 250 ps resolution Signal at 50 µm tissue depth 760 nm, 170 fs, 80 MHz time-resolved single photon counting K. König et al.; Karsten Jena Univ. König

37 Lifetime imaging, color coding according to life time Fig 10.15

38 Outline Fluorescence microscopy Confocal microscopy Fluorescence markers 2-photon microscopy Time domain imaging Fourier transform microscopy Sub-diffraction resolution microscopy 38

39 Analyzers: Fourier transform spectrometer From the spectroscopic equipment lecture

40 Fourier transform fluorescence microscopy Fig 10.11

41 Outline Fluorescence microscopy Confocal microscopy Fluorescence markers 2-photon microscopy Time domain imaging Fourier transform microscopy Sub-diffraction resolution microscopy 41

42 Velcro Super resolution microscopy

43 Creating beams with a spatial donought mode Optical vortex phase plate Lens

44 Stimulated Emission Depletion Fluorescence Microscopy (STED) Hell, Betzig, Moerner, Chemistry Nobel Prize 2014

45 Dichroic mirrors

46 Stimulated Emission Depletion Fluorescence Microscopy (STED) Fig Hell, Betzig, Moerner, Chemistry Nobel Prize 2014

47 Stimulated emission depletion fluorescence microscopy (STED) Fig 10.17

48 Stochastic Optical Reconstruction Microscopy (STORM) Fig 10.18

49 Outline Resolution & definitions Fluorescence microscopy Some other optical microscopy techniques Electron microscopes X-ray microscopy Scanning tunneling microscopes 49

50 Optical tweezer Figure below: Using an optical tweezer and an organelle has been moved in a living cell Fig 10.5

51 Second harmonic generation ω 1 ω 1 Photon picture ω 2 =2*ω 1 Photons at frequency ω 1 are sent into the material and photons at frequency ω 2 =2*ω 1 are generated in a nonlinear interaction with the material. The non-linear interaction ω 2 =2*ω 1 is only allowed if the material is non-centrosymmetric. This can e.g. be the case at surfaces or for narrow structures As for two-photon fluorescence the signal is spectrally well separated from the excitation light 51

52 High resolution optical sectioning of human skin autofluorescence and second harmonic generation (SHG) Cells in autofluorescence Karsten König Collagen imaging using second harmonic generation K. König et al., Jena Univ.

53 The coherence time, τ c, for a light source of bandwidth ν is roughly τ c ~ 1/ ν and the coherence length is l c ~ cτ c Interference fringes in the interferometer above is thus just seen across a distance l c. This is also the spatial resolution of the measurememt set-up above. Fig 10.19

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55 In Vivo Ultrahigh Resolution OCT versus Histology temporal Parafovea Fovea centralis Foveola Parafovea nasal ILM / NFL GCL IPL INL HF / OPL ONL ELM IS/OS PR RPE 250 µm ILM / NFL GCL IPL INL HF / OPL ONL IS/OS PR PL RPE Choriocapillaris and Choroidea Choriocapillaris and Choroidea Gass J.D.M., 1997 Fig W. Drexler et al. Nature Medicine, Vol 7, No. 4, , 2001

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57 High-resolution OCT Living tadpole Resolution: 1 micron Femtosecond broadband source J. Fujimoto et al.

58 Outline Resolution & definitions Fluorescence microscopes Some other optical microscopy techniques Electron microscopes X-ray microscopy Scanning tunneling microscopes 58

59 Electron microscopy in brief Electron source instead of photon source Electron waves instead of photon waves Electric and magnetic lenses instead of optical lenses Why? Short wavelength means high resolution Physics of biomed imaging 2012 Courtesy: Hans Hertz

60 Principles p = h λ λ = ho 2 m E kin Ekin λ 10 ev 3.2 Å 100 ev 1.2 Å 10 kev 0.12 Å 1 MeV Å Physics of biomed imaging 2012 Courtesy: Hans Hertz

61 Measuring the energy spectrum of Svanberg, Atomic & molecular spectroscopy, Fig 5.15 electrons

62 Elemental selectivity electron microscopy

63 Energy electron loss spectroscopy: SiO 2 -covered Pb-Cr-Oxide L-edge 100 ev K-edge 532 ev L-edge 580 ev Kyoto University Fig 10.23

64 STEM Images: Nanowire: InAs and InP stripes Fig Wallenberg, Samuelsson et al.

65 Optical probes

66 Electron microscopes operate in vacuum Sample preparation: Fixation: stops biological activity preserves structure vacuum compatible Methods: Chemical or cryogenic Another potential problem for bioinvestigations: Radiation damage: May be severe Courtesy: Hans Hertz

67 Outline Resolution & definitions Fluorescence microscopes Some other optical microscopy techniques Electron microscopes X-ray microscopy Scanning tunneling microscopes 67

68 X-ray scanning microscopy Page 272

69 Water-window X-ray microscopy Fig 10.25

70 Outline Resolution & definitions Fluorescence microscopes Some other optical microscopy techniques Electron microscopes X-ray microscopy Scanning tunneling microscopes 70

71 Scanning tunneling microscope Invented in 1982 by Binnig and Rohrer, for which they shared the 1986 Nobel Prize in Physics (STM)

72 STM principle Electrons can tunnel across the sample-tip gap. Tunnel current depends exponentially on the gap distance.

73 For surface science and for moving single atoms on a surface

74 Outline Resolution & definitions Fluorescence microscopy Some other optical microscopy techniques Electron microscopes X-ray microscopy Scanning tunneling microscopes 74

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