Advanced Optical Microscopy

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1 Nanosystems I - Seminar TU München 8th December 2008

2 1 Introduction to Classical Optical Microscopy Denitions in Optical Microscopy Contrast and Contrast Enhancement

3 1 Introduction to Classical Optical Microscopy Denitions in Optical Microscopy Contrast and Contrast Enhancement 2 Total Internal Reection Confocal Microscopy Stimulated Emission Depletion Microscopy

4 1 Introduction to Classical Optical Microscopy Denitions in Optical Microscopy Contrast and Contrast Enhancement 2 Total Internal Reection Confocal Microscopy Stimulated Emission Depletion Microscopy 3

5 Denitions and Resolution Contrast and Contrast Enhancement 1 Introduction to Classical Optical Microscopy Denitions in Optical Microscopy Contrast and Contrast Enhancement 2 3

6 Denitions and Resolution Contrast and Contrast Enhancement Denitions and formulae by Classical I mean microscopy with an external, non-monocromatical and non-coherent light source Nikon MicroscopyU webpage

7 Denitions and Resolution Contrast and Contrast Enhancement Denitions and formulae by Classical I mean microscopy with an external, non-monocromatical and non-coherent light source Resolution is the shortest distance between two points, that still can be separated Nikon MicroscopyU webpage

Denitions and Resolution Contrast and Contrast Enhancement Wikipedia - Nikon MicroscopyU webpage Denitions and formulae by Classical I mean microscopy with an external,

8 Denitions and Resolution Contrast and Contrast Enhancement Wikipedia - Nikon MicroscopyU webpage Denitions and formulae by Classical I mean microscopy with an external, non-monocromatical and non-coherent light source Resolution is the shortest distance between two points, that still can be separated Numerical Aperture is dened as NA = n sinα, Typically < 1

shortest distance between two points, that still can be separated Numerical Aperture is dened as NA

9 Denitions and Resolution Contrast and Contrast Enhancement Denitions and formulae by Classical I mean microscopy with an external, non-monocromatical and non-coherent light source Resolution is the shortest distance between two points, that still can be separated Numerical Aperture is dened as NA = n sinα, Typically < 1 Magnication is the enlargement of the incoming angular MA = tan(ɛ) tan(ɛ 0 ) ɛ ɛ 0

10 Denitions and Resolution Contrast and Contrast Enhancement Easiest way of illumination Nikon MicroscopyU webpage

11 Denitions and Resolution Contrast and Contrast Enhancement Nikon MicroscopyU webpage Easiest way of illumination Possible Resolution: Abbe condition d min = 200 nm λ 2 NA

12 Denitions and Resolution Contrast and Contrast Enhancement buttlery wing scales Olympus Microscopy Webpage Easiest way of illumination Possible Resolution: Abbe condition d min = 200 nm λ 2 NA Advantages: easy realisation, good contrast of dark colors

13 Denitions and Resolution Contrast and Contrast Enhancement Easiest way of illumination Possible Resolution: Abbe condition d min = 200 nm λ 2 NA Advantages: easy realisation, good contrast of dark colors Disadvantages: bad contrast of light colors (biological samples), overillumination Nikon MicroscopyU webpage

14 Denitions and Resolution Contrast and Contrast Enhancement Creating picture only by scattering events, thus there is no base illumination Wikipedia

15 Denitions and Resolution Contrast and Contrast Enhancement Creating picture only by scattering events, thus there is no base illumination Resolution: Abbe Condition d min = λ 2 NA Wikipedia

16 Denitions and Resolution Contrast and Contrast Enhancement Creating picture only by scattering events, thus there is no base illumination Resolution: Abbe Condition d min = λ 2 NA Advantages: Eective on highly transmitting samples (biological samples), less artifacts (no halos) Wikipedia

Denitions and Resolution Contrast and Contrast Enhancement Nikon MicroscopyU webpage Creating picture only by scattering events, thus there is no base illumination Resolution: Abbe Condition

17 Denitions and Resolution Contrast and Contrast Enhancement Nikon MicroscopyU webpage Creating picture only by scattering events, thus there is no base illumination Resolution: Abbe Condition d min = λ 2 NA Advantages: Eective on highly transmitting samples (biological samples), less artifacts (no halos) Disadvantages: Intense illumination needed, blind for low scattering samples

18 Denitions and Resolution Contrast and Contrast Enhancement Contrast Enhancement Contrast is created by dierence in reection, absorption, refraction, polarisation, and more

19 Denitions and Resolution Contrast and Contrast Enhancement Contrast Enhancement Contrast is created by dierence in reection, absorption, refraction, polarisation, and more Phase Contrast: light passing through a transparent specimen cause small phase shifts

20 Denitions and Resolution Contrast and Contrast Enhancement Contrast Enhancement Contrast is created by dierence in reection, absorption, refraction, polarisation, and more Phase Contrast: light passing through a transparent specimen cause small phase shifts

$reection, absorption, refraction, polarisation, and more Phase Contrast: light passing through a transparent specimen cause small$

21 Denitions and Resolution Contrast and Contrast Enhancement Wikipedia Contrast Enhancement Contrast is created by dierence in reection, absorption, refraction, polarisation, and more Phase Contrast: light passing through a transparent specimen cause small phase shifts

22 Denitions and Resolution Contrast and Contrast Enhancement Contrast Enhancement Contrast is created by dierence in reection, absorption, refraction, polarisation, and more Phase Contrast: light passing through a transparent specimen cause small phase shifts Dierential Interference Contrast: interferometry due to slopes and valleys to see dierences in optical paths

$Denitions and Resolution Contrast and Contrast Enhancement Wikipedia Contrast Enhancement Contrast is created by dierence in reection, absorption, refraction, polarisation, and more Phase$

23 Denitions and Resolution Contrast and Contrast Enhancement Wikipedia Contrast Enhancement Contrast is created by dierence in reection, absorption, refraction, polarisation, and more Phase Contrast: light passing through a transparent specimen cause small phase shifts Dierential Interference Contrast: interferometry due to slopes and valleys to see dierences in optical paths

$Denitions and Resolution Contrast and Contrast Enhancement Nikon MicroscopyU webpage Contrast Enhancement Contrast is created by dierence in reection, absorption, refraction, polarisation, and$

24 Denitions and Resolution Contrast and Contrast Enhancement Nikon MicroscopyU webpage Contrast Enhancement Contrast is created by dierence in reection, absorption, refraction, polarisation, and more Phase Contrast: light passing through a transparent specimen cause small phase shifts Dierential Interference Contrast: interferometry due to slopes and valleys to see dierences in optical paths

25 TIRFM Confocal Microscopy STED 1 Introduction to Classical Optical Microscopy 2 Total Internal Reection Confocal Microscopy Stimulated Emission Depletion Microscopy 3

26 TIRFM Confocal Microscopy STED Technique uses uorescence and phosphorescence (recapitulation) Nikon MicroscopyU webpage For more information see Talk about Fluorescence

27 TIRFM Confocal Microscopy STED Technique uses uorescence and phosphorescence (recapitulation) Nikon MicroscopyU webpage

28 TIRFM Confocal Microscopy STED Technique uses uorescence and phosphorescence (recapitulation) Nikon MicroscopyU webpage

29 TIRFM Confocal Microscopy STED Technique uses uorescence and phosphorescence (recapitulation) Problem: the uorophores are not only bound to the surface but also owing in the medium surrounding it

30 TIRFM Confocal Microscopy STED Technique uses uorescence and phosphorescence (recapitulation) Problem: the uorophores are not only bound to the surface but also owing in the medium surrounding it uorescence of the surrounding, larger populated non-attached uorophores, overwhelm those attached to the surface

31 TIRFM Confocal Microscopy STED Total Internal Reection evanescent wave makes controlled uorophor activation possible

32 TIRFM Confocal Microscopy STED Total Internal Reection evanescent wave makes controlled uorophor activation possible Resolution: < 200 nm, even single molecules are visible Nikon MicroscopyU webpage

33 TIRFM Confocal Microscopy STED Total Internal Reection evanescent wave makes controlled uorophor activation possible Resolution: < 200 nm, even single molecules are visible Resolution limit: optic's ability to gather light, uorophore dimension and concentration

TIRFM Confocal Microscopy STED Nikon MicroscopyU webpage Total Internal Reection evanescent wave makes controlled uorophor activation possible Resolution: < 200 nm, even single

34 TIRFM Confocal Microscopy STED Nikon MicroscopyU webpage Total Internal Reection evanescent wave makes controlled uorophor activation possible Resolution: < 200 nm, even single molecules are visible Resolution limit: optic's ability to gather light, uorophore dimension and concentration Advantage: less disturbance of the specimen, even dynamics are visible

35 TIRFM Confocal Microscopy STED Nikon MicroscopyU webpage Total Internal Reection evanescent wave makes controlled uorophor activation possible Resolution: < 200 nm, even single molecules are visible Resolution limit: optic's ability to gather light, uorophore dimension and concentration Advantage: less disturbance of the specimen, even dynamics are visible

36 TIRFM Confocal Microscopy STED Nikon MicroscopyU webpage Confocal Microscopy scanning a uorophore-bathed probe point wise with lasers.

37 TIRFM Confocal Microscopy STED Confocal Microscopy scanning a uorophore-bathed probe point wise with lasers. using patterns also 3-dimensional proles are producible Wikipedia

38 TIRFM Confocal Microscopy STED Nikon MicroscopyU webpage Confocal Microscopy scanning a uorophore-bathed probe point wise with lasers. using patterns also 3-dimensional proles are producible Resolution: Limited by point size typically λ excitation due to diraction

39 TIRFM Confocal Microscopy STED Stimulated Emission Depletion Microscopy Reduction of point-size by exploiting non-linear eects

40 TIRFM Confocal Microscopy STED Stimulated Emission Depletion Microscopy Reduction of point-size by exploiting non-linear eects focus two-pulsed lasers with an excitation and a few ps afterward an emission-wavelength-tuned depletion pulse

41 TIRFM Confocal Microscopy STED Stimulated Emission Depletion Microscopy Reduction of point-size by exploiting non-linear eects focus two-pulsed lasers with an excitation and a few ps afterward an emission-wavelength-tuned depletion pulse only excited states remain and spontaneous emission is measured

TIRFM Confocal Microscopy STED Max Planck Institute for Biophysical Chemistry Stimulated Emission Depletion Microscopy Reduction of point-size by exploiting non-linear eects focus two-pulsed lasers

42 TIRFM Confocal Microscopy STED Max Planck Institute for Biophysical Chemistry Stimulated Emission Depletion Microscopy Reduction of point-size by exploiting non-linear eects focus two-pulsed lasers with an excitation and a few ps afterward an emission-wavelength-tuned depletion pulse only excited states remain and spontaneous emission is measured close to point-like resolution is possible (uorophor)

43 TIRFM Confocal Microscopy STED Max Planck Institute for Biophysical Chemistry Stimulated Emission Depletion Microscopy Reduction of point-size by exploiting non-linear eects focus two-pulsed lasers with an excitation and a few ps afterward an emission-wavelength-tuned depletion pulse only excited states remain and spontaneous emission is measured close to point-like resolution is possible (uorophor)

44 1 Introduction to Classical Optical Microscopy 2 3

45 Classical Optical Microscopy works by either full illumination of the sample (bright eld) or angular illumination causing only scattered light to be seen (dark eld)

46 Classical Optical Microscopy works by either full illumination of the sample (bright eld) or angular illumination causing only scattered light to be seen (dark eld) is done mainly by applying uorophor exciting laser light and measuring emitted uorescence light

47 Classical Optical Microscopy works by either full illumination of the sample (bright eld) or angular illumination causing only scattered light to be seen (dark eld) is done mainly by applying uorophor exciting laser light and measuring emitted uorescence light TIRFM is a more advanced method of Fluorescence Microscopy by using exponentially decaying evanescent waves

48 Classical Optical Microscopy works by either full illumination of the sample (bright eld) or angular illumination causing only scattered light to be seen (dark eld) is done mainly by applying uorophor exciting laser light and measuring emitted uorescence light TIRFM is a more advanced method of Fluorescence Microscopy by using exponentially decaying evanescent waves STED exploits non-linear optical eects to shrink the diractivly widened laser points of Confocal Microscopy

49 Classical Optical Microscopy works by either full illumination of the sample (bright eld) or angular illumination causing only scattered light to be seen (dark eld) is done mainly by applying uorophor exciting laser light and measuring emitted uorescence light TIRFM is a more advanced method of Fluorescence Microscopy by using exponentially decaying evanescent waves STED exploits non-linear optical eects to shrink the diractivly widened laser points of Confocal Microscopy We saw that the classical maximal resolution of 200 nm for Optical Microscopes can be violated by either point wise (STED) or controlled excitation (TIRFM) of a uorophor.

50 Thank You for Your Attention!

51 Nikon Webpage Olympus Microscopy Webpage Max Planck Institute for Biophysical Chemistry - Department of NanoBiophotonics

Lecture 23 MNS 102: Techniques for Materials and Nano Sciences

Lecture 23 MNS 102: Techniques for Materials and Nano Sciences Reference: #1 C. R. Brundle, C. A. Evans, S. Wilson, "Encyclopedia of Materials Characterization", Butterworth-Heinemann, Toronto (1992),