1. Multi-Photon Principles - topics- l Confocal complements Multi-Photon. l What is an emission event ? l Single photon event. l Stokes’ shift and what you see. l Multi-photon event.

2. Confocal and the Multi-Photon l The visible light confocal and multi-photon systems complement each other. l The multi-photon cannot completely replace the visible light confocal but it does replace UV systems and even extends their applications.

3. Why ? l The dyes used in the visible light confocals are more numerous and better understood than those available for multi-photon at present. l A major strength of the multi-photon system is its ability to reduce damage to live cells when exciting UV dyes.

4. ….and the future l The number of dyes available for multi- photon is increasing but there are problems in getting them to work as well as visible light confocal dyes. l For example, the red excited dyes simply don’t work with multi-photon excitation.

5. ….So why bother ?

6. Advantages of Multi Photon l Much deeper imaging than confocal l Much lower cytotoxicity l Much slower bleaching of the fluorophore l Much higher contrast than UV l Much wider selection of dyes than UV

7. Emission Event l An emission event is where an incident photon causes a material to fluoresce. l There are single photon and multi-photon events.

8. Emission Event l Remember from high school that all atoms and molecules have ENERGY LEVELS. l Between these are forbidden bands. l ….and that an electron cannot exist between these levels.

9. Emission Event l Well that’s not true ! l An electron CAN exist between the energy levels for a very short time (about 10 -14 sec).

10. Pictorial Comparison l.l.

11. Single Photon Event l The incident photon has an energy which is larger than the separation of two energy levels in an atom. l This allows it to excite an electron directly from a lower energy level to a higher one. l Any extra energy is lost, usually as heat or vibration.

12. Single Photon Event l The excited electron can fall back to its rest state, or some intermediate state, giving up the energy it gained from the incident photon. l This energy is emitted as light of longer wavelength (lower energy) than the incident photon had.

13. Single Photon Event l This all happens quickly, about 10 -7 seconds. l This process is called fluorescence.

14. Single Photon Event Summary l An incident photon gives excitation of an electron directly from one energy level to a high energy level. l This electron then falls from the higher energy level back to a lower state and emits one new photon. l No diversions or complications.

15. ___Stokes’ Shift l The difference between the wavelength (energy) of the incident photon and the new one given off by the electron is called the Stokes Shift. l The emitted photon ALWAYS has a longer wavelength than the incident photon.

16. Stokes’ Shift l Stokes’ shift is associated with atoms. l A similar wavelength shifting is associated with molecules. This is called Raman Shifting. l The wavelength of a Raman shifted photon can be longer OR shorter than the incident photon.

17. Stokes’ Shift means…. l If the exciting photon is of blue light, the emitted light will be something from green to red. l Similarly if the exciting wavelength is UV, the emitted light will be visible. l To see the emission with the naked eye, the excitation MUST have a wavelength which is visible or UV.

18. Multi-Photon Event l If there are enough incident photons in the same place at the same time (or very nearly), a multi-photon event can occur. l A multi-photon event does NOT require the electron to be excited directly from one energy level to another. l The electron can be excited part of the way by the first photon.

19. Multi-Photon Event l Then the electron can be excited the rest of the way by the arrival of later photons. l The subsequent photons MUST arrive about 10 -14 seconds after the first. l The energy of each arriving photon is summed to bridge the gap between energy levels in the atom.

20. Multi-Photon Event l This means that a visible image can be generated from a fluorescent dye when it is excited by IR (infra-red) light. l It is impossible to produce a single photon image with IR. l ALL images produced with IR are the result of multi-photon events.