1. Multi-photon Fluorescence Microscopy

2. Topics Basic Principles of multi-photon imaging Laser systems Multi-photon instrumentation Fluorescence probes Applications Future developments

3. Multi-photon Excitation A non-linear process Excitation caused by 2 or more photons interacting simultaneously Fluorescence intensity proportional to (laser intensity) n, n = number of photons fluorescence localised to focus region

4. History - Multi-photon Originally proposed by Maria Goeppert-Mayer in 1931 First applications in molecular spectroscopy (1970s) Multi-photon microscopy first demonstrated by Denk, Strickler and Webb in 1989 (Cornell University, USA) With Cornell, Bio-Rad is the first to commercial develop the technology in 1996

5. Multi-photon microscopy The only contrast mode is fluorescence ( IR transmission/DIC is possible) Lateral and axial resolution are determined by the excitation process Red or far red laser illumination is used to excite UV and visible wavelength probes (e.g.. 700nm for DAPI)

6. Multi-Photon Excitation Physical Principles

7. Consequence of multi photon excitation 1-Photon2-Photon * Excitation occurs everywhere* Excitation localised that the laser beam interacts with samples* Excitation efficiency proportional the square of laser intensity * Excitation efficiency proportional to the intensity * Emission highest in focal region where intensity is highest

8. Classical and confocal fluorescence Multi-photon fluorescence

9. Key points for multi photon excitation Wavelength of light used is approximately 2 x that used in a conventional system. (i.e. red light can excite UV probes) Excitation process depends on 2-Photons arriving in a very short space of time (i.e. 10 seconds) Special kind of laser required -16

10. Lasers for MP Mode-locked femto-second lasers

11. CW and Pulsed Lasers CW Pulsed Short Pulse Advantage Fluorescence proportional to 1/pulse width x repetition rate

12. Laser Options Coherent, Verdi-Mira (MiraX-BIO) X-Wave Optics, good beam pointing, beam reducer needed Spectra Physics, Millennia/Tsunami Established system, extended tuning optics, good beam diameter Coherent Vitesse & Nd:Ylf Turn-key, fixed wavelength lasers, small footprint Coherent Vitesse XT and Spectra physics Mai Tai - small footprint, limited tuning TiS ( 100 nm range) computer controlled

13. General Laser Specifications for MP Microscopy Pulse Width<250 fsecs Repetition Rate>75 MHz Average Power>250 mW

14. Comparison of Lasers Available For Multi-Photon Microscopy

15. Why Femto-second? High output powers needed in deep imaging - higher average power generated by pico-second pulses may generate heating and tweezing effects 3P excitation of dyes (DAPI, Indo-1) with pico-second pulses practically impossible Femto-second pulses may cause 3P excitation of endogenous cellular compounds - however no evidence that this causes cell toxicity

16. Relationship between Average Power and Pulse Width

17. Ratio of 3P excitation to 2P excitation as a Function of Pulse Width

18. What about Fibre-delivery of Pulsed Lasers Advantage - alignment and system footprint Problem - average power output combined with short pulses for a tuneable laser suffer considerable power loss, and realignemnt of laser with each wavelength change ( repointing) problem less with fixed wavelength. ie NdYlf uses p-sec pulses which are then compressed by fibre

19. Instrument Design

20. C C C C Objective Lens Laser Confocal Aperture Detector Laser Emission Excitation MP Optics Instrument design

21. Scan head convertible from upright to inverted ( MP ONLY option also available) Beam Control and Monitoring Unit ( Optics Box) 2 or 4 External detector unit Fentosecond TiS laser Choice of Microscope, upright or inverted or both Radiance2000MP

22. Key specifications Adaptable to a wide range of microscopes - Nikon, Olympus and Zeiss Compatible with six femtosecond pulsed lasers Beam conditioning units range from basic functionality to flexible fully featured units Beam delivery systems for single scopes and to switch between scopes Non-descanned and descanned detector options Reduced system footprints Multi-Photon ONLY scan head version available

23. Why all this trouble? Conventional confocal has many limitations –limited depth penetration –short life times for cell observation –problems with light scatter especially in dense cells –limitations with live cell work

24. Is not UV confocal the solution? No - its the problem for many of these applications

25. Why has UV confocal seen such little popularity worldwide Despite being available for nearly 10 years, only a small number of systems have been installed Chromatic errors High Toxicity to cells and tissues Poor penetration Enhances autofluorescence Almost unusable in plant sciences High scattering User safety Limited options with lenses In two years the installed base of MP systems have doubled over all UV systems world wide.

26. Strengths of Multi-Photon Microscopy Deeper sectioning - thick, scattering sections can be imaged to depths not possible in standard confocal Live cell work - ion measurement (i.e. Ca 2+ ), GFP, developmental biology - reduced toxicity from reduced full volume bleaching allows longer observation Autofluorescence - NADH, seratonin, connective tissue, skin and deep UV excitation

28. Deep Imaging improved by..

29. Scattered Light Collection

30. Reduction of Emitted Fluorescence due to Scattering Events

31. Relationship between the Number of Scattering Events and Depth into Aortic Tissue 350nm 500nm 700nm

32. Scatter light detection improved by External light Detector From Vickie Centonze Frohlich IMR, Madison, WI

33. Reduced Photo bleaching...

35. MP Fluorochromes and Applications

36. Key issues Most commonly used probes can be imaged MP is effectively exciting at UV/blue wavelengths Excitation spectra are broader than for 1-photon Emission spectra are the same as in 1-photon excitation All probes are excited simultaneously at the same wavelength Probe combinations must be chosen so that they are separated by emission spectra Co-localization is exact even between UV and visible probes Can use objective lenses which are not full achromats (e.g. z focus shift)

37. Fluorescent Probes for MP Imaging

39. Efficient Simultaneous Detection of Multiple Labels

40. Following Dynamic Ca 2+ Changes using MP Excitation

41. Sources of Tissue Autofluorescence

42. Serotonin Distribution in Living Cells

43. Imaging of Serotonin Containing Granules Undergoing Secretion

44. MP Imaging of Drug Localisation and Metabolism

45. Non Imaging Possibilities FRAP (Fluorescence recovery after photobleaching) Photoactivation Knock out experiments FCS (Fluorescence correlation spectroscopy)

46. MP in a nutshell Multi-Photon microscopy allows optical section imaging deeper into samples than other methods, even in the presence of strong light scattering Multi-Photon microscopy allows the study of live samples for longer periods of time than other methods, reducing cytotoxic damage