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Confocal & Two-photon Microscopy

Confocal & Two-photon Microscopy. Contents. 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,

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Confocal & Two-photon Microscopy

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  1. Confocal & Two-photon Microscopy

  2. Contents • 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

  3. 1. Two-Photon Microscopy (vs One-photon) Figure 2, Relevant time scale. Figure 1. Jablonski diagram

  4. 1. Two-Photon Microscopy (vs One-photon) • Nonlinear optical excitation , I2p P2 Figure 3. Quadratic dependence.

  5. 1. Two-Photon Microscopy (vs One-photon) • 3D localized uncaging and photobleaching in subfemto-liter volume Z-axis Z-axis Figure 4. Excitation region in one & two-photon microscopy

  6. 1. Two-Photon Microscopy (vs One-photon) Figure 3. Photobleaching in one & two-photon microscopy

  7. 1. Two-Photon Microscopy (vs One-photon) One-photon Two-photon Figure 5. Z-direction scanning spectra in One & Two-photon microscopy

  8. 2     r Er • r m><m i> <f Er •  P ~ r - mi m 1. Two-Photon Microscopy (vs One-photon) • Quantum Theory of Two-photon excitation * Multi-Photon transition probability (P)

  9. 1. Two-Photon Microscopy (vs One-photon) • Two-Photon transition Probability * Time-averaged two-photon fluorescence intensity per molecule 

  10. for 0 < t <  1. Two-Photon Microscopy (vs One-photon) a. Continuous Wave Laser where b. Pulsed Laser where 1 for < t < fp

  11. 1. Two-Photon Microscopy (vs One-photon) • Architecture of Two-Photon microscopy Beamrouting Supplement Mira Safety Box Mira-900 Verdi DMIR

  12. 1. Two-Photon Microscopy (vs One-photon) Leica confocal systems : TCS SP2 1. The spectral detector for brilliant confocal 2D, 3D images by emitted fluorescence 2. Two-photon confocal microscopy combined with femtosecond laser  Thickness, depth and more precise images measurement by 3D sectioning

  13. 1. Two-Photon Microscopy (vs One-photon) • Light source : 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 !!)  Mira 900 : 76MHz, 180fs, 400~500mW,

  14. 1. Two-Photon Microscopy (vs One-photon) • Advantages of Two-photon • Deep-specimen imaging • Lower absorption & scattering coefficient due to IR • : Deeper penetration effect ! • b. Excitation only in a subfemtoliter-sized focal volume • : It reduce photodamage !

  15. 2. Resolution and Contrast (confocal vs two-photon) 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 !

  16. 2. Resolution and Contrast (confocal vs two-photon) • Three-dimensional distribution of light near the focus of lens •  Point Spread Function Where, The intensity PSF ( related to its FWHM) ,

  17. 2. Resolution and Contrast (confocal vs two-photon) * Confocal system Pointwise-illumination Pointwise-detection Cf) Uniform detector Cf)

  18. 2. Resolution and Contrast (confocal vs two-photon) Table1, FWHM FWHM extent (m) PSF Lateral Axial Detection (det) 0.20 0.84 Illumination (ill) 0.19 0.78 0.14 0.57 Confocal=illdet 0.93 Two-photon=(ill)2 0.23 2p-confocal=(ill)2xdet 0.16 0.63 Figure 3. Calculated Point Spread Function

  19. 2. Resolution and Contrast (confocal vs two-photon) * Lateral Resolution * Axial Resolution

  20. 2. Resolution and Contrast (confocal vs two-photon) Depth discrimination !

  21. 3. Examples Top Bottom 1 2 3 5 4 OP TP Z-scanning range : ~28m (5 sections, 7.0417 m step)

  22. 3. Examples : Neuron cell imaging Lysosome (DND-189) Nucleus (Propidium iodide)

  23. 3. Examples : Neuron cell imaging

  24. 3. Examples : Neuron cell imaging 50m 50m Side-view 3D-reconstruction

  25. 4. Application : Fluorescence Correlation Microscopy Where F(t)=F(t)-<F(t)> D ~ r02 / 4D

  26. 4. Application : Life-time two-photon imaging

  27. 4. Application : Life-time two-photon imaging Steady state intensity image Time resolved intensity image Autofluorescence of human skin : 2-photon image Ca image of Cortex neutron : 2-photon image

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