Content of the lecture

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 Confocal Imaging Nonlinear Techniques Dyes EM Aux • New • Input pinhole • Exit pinhole • Scanning head • Detector – PMT www.olympusconfocal.com

3. Confocal microscope vs. widefield. Confocal Imaging Nonlinear Techniques Dyes EM Aux www.olympusconfocal.com

4. Confocal microscope. Images examples. Confocal Imaging Nonlinear Techniques Dyes EM Aux www.olympusconfocal.com

5. Confocal microscope. Scanning unit. Confocal Imaging Nonlinear Techniques Dyes EM Aux www.olympusconfocal.com

6. Confocal microscope. Disk-scanning. Confocal Imaging Nonlinear Techniques Dyes EM Aux Mechanical television in 1920s

7. Confocal microscope vs. widefield. 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 Confocal Imaging Nonlinear Techniques Dyes EM Aux Widefield Confocal • The whole picture is taken at once • Eyepiece image • Potentially fast imaging • High photodamage • High background noise (secondary fluorescence) • “Cheap”

8. Nonlinear techniques Nonlinear Techniques Confocal Imaging Dyes EM Aux λ IVR λ <10-17s <10-17s λ/2 λ/2 λ λ absorption scattering Two-photon microscopy Second harmonic generation Virtual state

9. Nonlinear techniques. Other schemes... Nonlinear Techniques Confocal Imaging Dyes EM Aux Multi-photon microscopy Multi-harmonic generation Virtual state λ λ IVR λ λ λ/3 λ/3 λ λ absorption scattering

10. Nonlinear techniques. Other schemes. Confocal Imaging Nonlinear Techniques Dyes EM Aux Coherent Anti-Stock Raman Scattering Microscopy λs λp λas λp ν=1 ν=0

11. Light attenuation spectra. Confocal Imaging Nonlinear Techniques Dyes EM Aux

12. Two-photon microscopy. Confocal Imaging Nonlinear Techniques Dyes EM Aux Absorption probability at the focus Total absorption probability Svoboda & Yasuda, 2006

13. Two-photon microscopy. Confocal Imaging Nonlinear Techniques Dyes EM Aux

14. Confocal vs. 2PH (non voltage-sensitive) Less photo-damaging Deeper penetration Light pathway/scheme Femtosecond laser Requires proper lenses (not a problem for 2PH now) Confocal Imaging Nonlinear Techniques Dyes EM Aux Confocal MPH • Usually better resolution

15. Second Harmonic Generation (SHG) Confocal Imaging Nonlinear Techniques Dyes EM Aux Induced Polarization: Signal intensity:

16. Second Harmonic Generation (SHG) Confocal Imaging Nonlinear Techniques Dyes EM Aux Dombeck et al, 2006

17. Second Harmonic Generation (SHG) Confocal Imaging Nonlinear Techniques Dyes EM Aux Sacconi et al, 2006

18. Voltage-sensitivity mechanisms Confocal Imaging Nonlinear Techniques Dyes EM Aux • Conformational changes in the system dye-molecule – membrane • In the case of scattering techniques (SHG, CARS,..) another mechanism – alteration of thealignment degree with voltage

19. 2PH vs. SHG High marker concentration (~N2) Mostly forward direction Narrow emission spectrum Short life-time (<ps) Excellent membrane contrast Confocal Imaging Nonlinear Techniques Dyes EM Aux 2PH SHG • Low marker concentration • Forward & backward directions • Relatively wide fluorescence spectrum • Relatively long lifetime (~ns) • Poor membrane contrast

20. Combination of 2PH and SHG Confocal Imaging Nonlinear Techniques Dyes EM Aux Nikolenko et al, 2003 Moreaux et al, 2001

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

22. Electron microscopy Confocal Imaging Nonlinear Techniques Dyes EM Aux JEM-1400 www.biologie.uni-hamburg.de

23. JEM-1400

24. Electron microscopy Confocal Imaging Nonlinear Techniques Dyes EM Aux Briggman, Denk, 2006

25. Electron microscopy. Examples. Confocal Imaging Nonlinear Techniques Dyes EM Aux 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 Confocal Imaging Nonlinear Techniques Dyes EM Aux • 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

28. END? END! Confocal Imaging Nonlinear Techniques Dyes EM Aux

29. Lateral resolution Confocal Imaging Nonlinear Techniques Dyes EM Aux • Defining the resolution using line-gratingobjects – distance between the maximum and the minimum in “Fraunhoffer slit” diffraciton. ? Is it true only for WF illumination? • 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≥ rAiry (assumes that the sources radiate coherently).

30. Axial resolution Confocal Imaging Nonlinear Techniques Dyes EM Aux 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

31. Depth of field Confocal Imaging Nonlinear Techniques Dyes EM Aux 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