Confocal & Two-photon Microscopy. Contents 1.Two-Photon Microscopy : Basic principles and...

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Transcript of Confocal & Two-photon Microscopy. Contents 1.Two-Photon Microscopy : Basic principles and...

  • Slide 1
  • Confocal & Two-photon Microscopy
  • Slide 2
  • Contents 1.Two-Photon Microscopy : Basic principles and Architectures 2. Resolution and Contrast in Confocal and Two-Photon microscopy 3. Example of two-photon images 4. Extensive application : Fluorescence Correlation Microscopy, Life-time imaging
  • Slide 3
  • Figure 2, Relevant time scale. 1. Two-Photon Microscopy (vs One-photon) Figure 1. Jablonski diagram
  • Slide 4
  • Nonlinear optical excitation, I 2p P 2 1. Two-Photon Microscopy (vs One-photon) Figure 3. Quadratic dependence.
  • Slide 5
  • 3D localized uncaging and photobleaching in subfemto-liter volume 1. Two-Photon Microscopy (vs One-photon) Figure 4. Excitation region in one & two-photon microscopy Z-axis
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  • 1. Two-Photon Microscopy (vs One-photon) Figure 3. Photobleaching in one & two-photon microscopy
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  • 1. Two-Photon Microscopy (vs One-photon) One-photon Two-photon Figure 5. Z-direction scanning spectra in One & Two-photon microscopy
  • Slide 8
  • Quantum Theory of Two-photon excitation P ~ i> r - mi 2 m * Multi-Photon transition probability (P) 1. Two-Photon Microscopy (vs One-photon)
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  • Two-Photon transition Probability * Time-averaged two-photon fluorescence intensity per molecule 1. Two-Photon Microscopy (vs One-photon)
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  • a. Continuous Wave Laser where b. Pulsed Laser where 0 < t < for 1 < t < fpfp for 1. Two-Photon Microscopy (vs One-photon)
  • Slide 11
  • Architecture of Two-Photon microscopy 1. Two-Photon Microscopy (vs One-photon) Safety Box Beamrouting Supplement Mira Mira-900 DMIR Verdi
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  • 1. Two-Photon Microscopy (vs One-photon) Leica confocal systems : TCS SP2 2. Two-photon confocal microscopy combined with femtosecond laser Thickness, depth and more precise images measurement by 3D sectioning 1. The spectral detector for brilliant confocal 2D, 3D images by emitted fluorescence
  • Slide 13
  • : Femtosecond Ti-Sapphire system with 80MHz, 100fs delivering peak powers of over 100kW !! wide tuning range, 700~1000nm !! At the entrance of scanning head, ~20mW Before the objective lens, 9~13mW At the sample, 3~5mW Picosecond, CW (required higher average power !!) Light source 1. Two-Photon Microscopy (vs One-photon) Mira 900 : 76MHz, 180fs, 400~500mW,
  • Slide 14
  • 1. Two-Photon Microscopy (vs One-photon) Advantages of Two-photon Deep-specimen imaging a.Lower absorption & scattering coefficient due to IR : Deeper penetration effect ! b. Excitation only in a subfemtoliter-sized focal volume : It reduce photodamage !
  • Slide 15
  • The resolution, defined as the minimum separation of two Point objects that provides a certain contrast between them, depends on The wavelength of the light ! Numerical Aperture of the optical arrangement ! Specimen ! 2. Resolution and Contrast (confocal vs two-photon)
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  • Three-dimensional distribution of light near the focus of lens Point Spread Function Where, The intensity PSF ( related to its FWHM), 2. Resolution and Contrast (confocal vs two-photon)
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  • * Confocal system Pointwise-illumination Pointwise-detection Cf) Cf) Uniform detector 2. Resolution and Contrast (confocal vs two-photon)
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  • Table1, FWHM 2. Resolution and Contrast (confocal vs two-photon) Figure 3. Calculated Point Spread Function FWHM extent ( m) Illumination (ill) Detection (det) LateralAxialPSF Confocal=ill det Two-photon=(ill) 2 2p-confocal=(ill) 2 xdet 0.20 0.84 0.190.78 0.140.57 0.23 0.16 0.93 0.63
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  • * Lateral Resolution 2. Resolution and Contrast (confocal vs two-photon) * Axial Resolution
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  • Depth discrimination ! 2. Resolution and Contrast (confocal vs two-photon)
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  • 3. Examples 1 4 352 OP Top Bottom TP Z-scanning range : ~28 m (5 sections, 7.0417 m step)
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  • 3. Examples : Neuron cell imaging Lysosome (DND-189) Nucleus (Propidium iodide)
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  • 3. Examples : Neuron cell imaging
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  • Side-view3D-reconstruction 50 m
  • Slide 25
  • 4. Application : Fluorescence Correlation Microscopy Where F(t)=F(t)- D ~ r 0 2 / 4 D
  • Slide 26
  • 4. Application : Life-time two-photon imaging
  • Slide 27
  • Steady state intensity image Time resolved intensity image Autofluorescence of human skin : 2-photon image Ca image of Cortex neutron : 2-photon image