1. Short pulses in optical microscopy Ivan Scheblykin, Chemical Physics, LU Outline: Introduction to traditional optical microscopy based on single photon absorption: Fluorescence wide-field and conforcal microscopy Introduction to single molecule imaging 2-photon absorption 2-photon confocal fluorescence microscopy 3-photon absorption, second harmonic generation Microscopy which is not limited by light diffraction

2. Why do we see objects ? Changing of the properties of light coming to the sample: Light absorption Light scattering Changing of light polarization … An object emits light itself: Luminescence Second-harmonic generation ….. Many different ways to create contrast in optical microscopy

3. object Transmission image Absorption and scattering

4. object Transmission image Absorption and scattering object 100€ Excitation light Blocking filter object

5. Transmission image Absorption and scattering object 100€ Excitation light Fluorescence Blocking filter Sample is stained by a fluorescent dye

7. White board Microscope scheme

9. Numerical Apreture  Spherical angle S Light collection efficiency S/4  NA/n = 1, 50% NA/n = 0.6, 10%

12. 10 microns Wide-field fluorescence microscopeConfocal fluorescence microscope

14. 3D imaging, z-scan

15. Single molecule spectroscopy Can we see one single chromophore ? Not in absorption, because cross section is too small  = 10 -16 cm 2, 10 -8 cm = 0.1 nm However, we can detect fluorescence light emitted by the molecule!

16. 5  Sample For SMS

17. Single molecule imaging Chemical Physics, Single Molecule Spectroscopy group, LU

18. Other ways to create contrast Non-linear processes induced by strong laser light Observation of fluorescence excitated by 2-photon absorption 3-photon absorption third harmonic signal Observation of second harmonic signal Absorption, scattering

19. Two-photon absorption Theory - Maria G ö ppert-Mayer, 1929 Experimental observation – 1961 Using in microscopy – Denk, Strickler, Webb, Science 1990 Probability of excitaion (W)  (Intensity) 2 W  ( I [ptonots/cm 2 /s] ) 2 Absorbed photon Fluorescence Virtual level i f

20. One and Two-photon absorption cross sections Transition dipole moment moment Estimation of  2 (WB)

22. Two-photon excitation versus one-photon excitation 543 nm excitation 1046 nm excitation Dye solution, safranin O

23. Resolution of 2-photon microscopy XY, Z, 1/z 4 excitation probability dependence And 1/z 2 dependence of total fluorescence(WB)

25. I2I2 I1I1 The same fluorescence signal from the sample

27. Better Light collection efficiency.. Multi-photon excitation confines fluorescence excitation to a small volume at the focus of the objective. Photon flux is insufficient in out-of-focus planes to excite fluorescence. No confocal pinhole is needed. All fluorescence (even scattered photons) constitutes useful signal. Photobleaching and photodamage are limited to the zone of 2P excitation and do not occur above or beyond the focus. Larger penetration depth. IR photons travel deeper into tissue with less scattering and absorption comparing to visible photons. Scattering 1/ 4 ! In practice - approximaterly 2 times larger penetration depth. Much smaller background from impurity fluorescence when IR laser is used in comparison with VIS or UV light. 2 photon excitation spectra are usually very broad. Therefore, one laser source can be used for many different dyes having different fluorescence wavelengths. No chromatic aberration problems. Some advantages of 2-photon excitation versus one-excitation in confocal microscopy

28. Even scattered fluorescence photons are usefull in 2-photon regime

30. All the dyes are excited by the same laser! No effect of chtomatic aberration (White board)

32. Other ways to create contrast Non-linear processes induced by strong laser light Observation of fluorescence excitated by 2-photon absorption 3-photon absorption third harmonic signal Observation of second harmonic signal Absorption, scattering SHG microscopy is generally used to observe non-centrosymmetric structures SHG is forbidden where there is an inversion symmetry, and this constraint makes it a sensitive tool for the study of interfaces and surfaces One can get a signal even without using any dyes to stain the sample Number of molecules SHG is cohherent processes: Intensity  N 2 Fluorescence is noncohherent processes: Intensity  N Cross-section of SHG on a molecules is very small, but collective response from many molecules can compensate it !

33. Third harmonic generation image, No dye staining was applied

34. Optical microscopy beyond diffraction limit ????? Diffraction limit – distribution of light intensity However, if the process is nonlinear function of intensity, then the localization is not limited by the wavelength

35. Excited state depletion Excitation pulse S0S0 S1S1 Fluorescence Excitation pulse

36. Excited state depletion Excitation pulse S0S0 S1S1 Fluorescence Stimulated emission Excitation pulse STED pulse

37. Excited state depletion Excitation pulse S0S0 S1S1 Stimulated emission Excitation pulse STED pulse Fluorescence is completely suppressed by stimulated emission process. K internal relaxation >K SM >> K fluorescence Suturation condition for STED pulse: K SM =K fluorescence ; I saturation  absorption ~ 1 ns -1 Photons in STED pulse has lower energy to avoid excitation. Pulse duration should much shorter then S 1 lifetime = 1/K fluores

38. I max >> I saturation ~ Fluorescence Excitation spot f(x) - Spatial distribution of the STED pulse Saturation parameter:  = I max / I saturation x f(x) = sin 2 (2  / )  x= /100, when  =1000