1. Confocal & Two-photon Microscopy

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

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

4. Nonlinear optical excitation, I 2p  P 2 1. Two-Photon Microscopy (vs One-photon) Figure 3. Quadratic dependence.

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

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

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

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

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

12. 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

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,

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 !

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)

16. 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)

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

18. 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

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

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

21. 3. Examples 1 4 352 OP Top Bottom 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. Side-view3D-reconstruction 50  m

25. 4. Application : Fluorescence Correlation Microscopy Where  F(t)=F(t)- D ~ r 0 2 / 4  D

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

27. Steady state intensity image Time resolved intensity image Autofluorescence of human skin : 2-photon image Ca image of Cortex neutron : 2-photon image