1. Content of the lecture Principles of confocal imaging. Different implementations/modes. Primer on multi-photon (MPH) and multi-harmonic (MHG) generation. Dyes  Ca2+ sensitive  Voltage-Sensitive  Photostimulation: Channel Rhodopsin, Caged glutamate  FRET  Methods of staining Electron microscopy (EM)

2. Confocal microscope. Design www.olympusconfocal.com New Input pinhole Exit pinhole Scanning head Detector – PMT Confocal ImagingNonlinear TechniquesDyesEMAux

3. Confocal microscope vs. widefield. www.olympusconfocal.com Confocal ImagingNonlinear TechniquesDyesEMAux

4. Confocal microscope. Images examples. www.olympusconfocal.com Confocal ImagingNonlinear TechniquesDyesEMAux

5. Confocal microscope. Scanning unit. www.olympusconfocal.com Confocal ImagingNonlinear TechniquesDyesEMAux

6. Confocal microscope. Disk-scanning. Confocal ImagingNonlinear TechniquesDyesEMAux Mechanical television in 1920s

7. Confocal microscope vs. widefield. The whole picture is taken at once  Eyepiece image  Potentially fast imaging  High photodamage  High background noise (secondary fluorescence)  “Cheap”  Thin sections (.5-1.5μm)  Max thickness ~50μm  High contrast and definition  Reduced photo-damaged Scanning   “slow”   “-” eyepieces   digital zooming  Limited number of laser colors  expensive WidefieldConfocal Confocal ImagingNonlinear TechniquesDyesEMAux

8. Nonlinear techniques Two-photon microscopy Second harmonic generation Virtu al state IV R <10 -17 s λ λ λ/2λ/2 absorption <10 -17 s λ λ λ/2λ/2 scattering Confocal ImagingNonlinear TechniquesDyesEMAux

9. Nonlinear techniques. Other schemes... Multi-photon microscopy Multi-harmonic generation Virtu al state IV R λ λ λ/3 absorptio n λ/3λ/3 scattering λ λ λ λ Confocal ImagingNonlinear TechniquesDyesEMAux

10. Nonlinear techniques. Other schemes. Coherent Anti-Stock Raman Scattering Microscopy λpλp λsλs λpλp λ as ν=1 ν=0 Confocal ImagingNonlinear TechniquesDyesEMAux

11. Light attenuation spectra. Confocal ImagingNonlinear TechniquesDyesEMAux

12. Two-photon microscopy. Svoboda & Yasuda, 2006 Absorption probability at the focus Total absorption probability Confocal ImagingNonlinear TechniquesDyesEMAux

13. Two-photon microscopy. Confocal ImagingNonlinear TechniquesDyesEMAux

14. Confocal vs. 2PH (non voltage-sensitive) Usually better resolution Less photo-damaging Deeper penetration Light pathway/scheme Femtosecond laser Requires proper lenses (not a problem for 2PH now) ConfocalMPH Confocal ImagingNonlinear TechniquesDyesEMAux

15. Second Harmonic Generation (SHG) Induced Polarization: Signal intensity: Confocal ImagingNonlinear TechniquesDyesEMAux

16. Second Harmonic Generation (SHG) Dombeck et al, 2006 Confocal ImagingNonlinear TechniquesDyesEMAux

17. Second Harmonic Generation (SHG) Sacconi et al, 2006 Confocal ImagingNonlinear TechniquesDyesEMAux

18. Voltage-sensitivity mechanisms 1. Conformational changes in the system dye-molecule – membrane 2. In the case of scattering techniques (SHG, CARS,..) another mechanism – alteration of the alignment degree with voltage Confocal ImagingNonlinear TechniquesDyesEMAux

19. 2PH vs. SHG  Low marker concentration  Forward & backward directions Relatively wide fluorescence spectrum Relatively long lifetime (~ns) Poor membrane contrast High marker concentration (~N 2 ) Mostly forward direction  Narrow emission spectrum  Short life-time (<ps)  Excellent membrane contrast 2PHSHG Confocal ImagingNonlinear TechniquesDyesEMAux

20. Combination of 2PH and SHG Moreaux et al, 2001 Nikolenko et al, 2003 Confocal ImagingNonlinear TechniquesDyesEMAux

21. Electron microscopes Louis d’Broyle, 1927: electrons, like photons, can behave like waves. But with very small wavelength.

22. Electron microscopy www.biologie.uni-hamburg.de Confocal ImagingNonlinear TechniquesDyesEMAux JEM-1400

24. Electron microscopy Briggman, Denk, 2006 Confocal ImagingNonlinear TechniquesDyesEMAux

25. Electron microscopy. Examples. Confocal ImagingNonlinear TechniquesDyesEMAux DEMO! : rat’s brain EM sections from Kristen Harris

26. Image obtained with SEM Geological Survey of Canada, Electron Beam Laboratory

27. EM advantages and drawbacks  Best resolution, available now  Large depth of field  Distinctive staining techniques (electron-dense (heave metal) + selective for the tissue of interest (neurons, syn)  Only slices (  postmortem)  Expensive  Very slow  Creation of the stack of images  Reconstruction of the volume Confocal ImagingNonlinear TechniquesDyesEMAux

28. END? END! Confocal ImagingNonlinear TechniquesDyesEMAux

29. Lateral resolution Defining the resolution using line-grating objects – distance between the maximum and the minimum in “Fraunhoffer slit” diffraciton. Defining the resolution using point objects – diameter of the first dark disk on the Airy diffraction image. Rayleigh criterion: the images of two equally bright spots are resolved if d ≥ r Airy (assumes that the sources radiate coherently). ? Is it true only for WF illumination? Confocal ImagingNonlinear TechniquesDyesEMAux

30. Axial resolution Has a diffraction nature as well as the lateral resolution Thus z resolution is usually substantially larger than the xy resolution. Nevertheless don’t forget – inability to resolve objects doesn’t mean that it’s impossible to know there location Confocal ImagingNonlinear TechniquesDyesEMAux

31. Depth of field The depth of field of a microscope is the depth of the image (measured along the microscope axis translated into distances in the specimen space) that appears to be sharply in focus at one setting of the fine-focus adjustment. In bright field microscopy the depth of field should be approximately equal to the axial resolution, at least in theory. In the dark field or conventional fluorescence microscopy because of the out of focus excitation of the specimen, the depth of field is much greater than the axial resolution. In confocal imaging the depth of field correspond to the axial resolution. Moreover, it was shown using information theory (Ingelstam, 1955), that the lateral resolution becomes better by a factor of sqrt(2), when the depth of field becomes vanishingly small Confocal ImagingNonlinear TechniquesDyesEMAux