1. Multiphoton and Spectral Imaging

2. Multiphoton microscopy Predicted by Maria Göppert-Mayer in 1931 Implemented by Denk in early 1990s Principle: Instead of raising a molecule to an excited state with a single energetic photon, it cam be raised to an excited state by the quasi-simulatneous absorption of two (2-photon) or 3 (3- photon) less energetic photons

3. Multiphoton-photon Jablonski diagram

4. Multiphoton In multiphoton microscopy, the intermediate state is not a defined state, and so is quantum forbidden However, in quantum mechanics, forbidden is not absolute Therefore, the requirement for quasi- simultaneity Practically, it means within ~10 -18 seconds In single photon, probablility of excitation is proportional to I; in two-photon, it is proportional to I 2

5. Excitation volume http://www.loci.wisc.edu/multiphoton/mp.html

6. Advantages of multiphoton microscopy Fluorescence excitation is confined to a femtoliter volume – less photobleaching Excitation wavelengts are not absorbef by fluorophore above plane of focus Longer excitation wavelengths penetrate more deeply into biological tissue Inherent optical sectioning

7. Increased contrast in multiphoton Centonze,V.E and J.G.White. (1998) Biophysical J. 75:2015-2024

8. Light sources Light flux necessary for multiphoton microscopy can be achieved by femtosecond pulsed IR lasers Ti-Sapphire lasers tunable from 700-900 nm http://micro.magnet.fsu.edu/primer/java/las ers/tsunami/index.htmlhttp://micro.magnet.fsu.edu/primer/java/las ers/tsunami/index.html

9. Spectra Physics Mai Tai, Coherent Chameleon Tuning Ranges 680-1080 nm Sealed box units; no adjustments necessary Computer controlled tuning Stable pointing as you scan spectrum

10. Dyes for multiphoton microscopy Multiphoton excitation spectra for dyes is an active field of exploration Generally, 2PE peaks are broad General rule: start a little more energetic than λ max for single photon For example: EGFP: λ max for single photon = 488; λ max for two photon 900 nm

11. 2PE Spectra

12. Detector configuration for multiphoton Molecular Expressions web site Note, in particular the descanned detector and the Whole Area PMT Detector = Nondescanned detector.

13. Descanned detector Uses same scan mirror to descan beam as was used to scan it. Better alignment with confocal However, only collects the amount of light represented by the projection of the mirror onto the specimen: less sensitivity Do not forget to open up the confocal pinhole, because the nature of multiphoton restricts excitation to a femtoliter volume

14. Nondescanned detector Because our excitation volume is restricted to a femtoliter volume, and is automatically an optical section, we do not need to descan Cone projected onto specimen is much wider, so much more sensitivity However, also much more sensitive to stray light

15. Confocal spectral imaging In many case, the spectra of dyes overlap either in their excitation spectrum, their emission spectrum, or both. What can we do? Excitation overlap – for instance, tetramethylrhodamine excitation spectrum overlaps that of fluorescein, so if we use the 488 and 543 lines simulatanously, we see overlap Solution: –Choose different dyey (fluorescein and Texas red) –Multitracking (sequential scanning) – excite at 488 while the fluorescein image is being collected and at 543 while the rhodamine is.

16. What about emission? Molecular Probes Choose different dyes

17. Sometimes you cant avoid overlap Autofluorescence frequently overlaps fluorescein emission NADH/Flavoprotein: on 2-P excitation at 800 nm, the 450 nm NADH emission is clean, but the 550 nm flavoprotein emission band has about 30% NADH emission Fluorescent proteins

18. Example: Lambda stack of cells expressing either CFP or GFP on chromatin

19. What do we do? Acquire a Lambda stack of our image Acquire a lambda stack of our reference dyes, or, alternatively, identify areas in the image that will be pure. Mathematicall, through linear unmixing, apply linear algebra to separate the individual dye spectra from the multispectral image

20. Linear Unmixing Different amounts of pink and blue generate different spectra

21. Pairwise comparison of dyes that can or cannot be unmixed Note that for pairs that cannot be unmixed (ie, DiO and eGFP), the shape of the spectra are very similar

22. Unmixing: fluorescein phalloidin and Sytox green

23. Problems with linear unmixing It takes a lot longer to acquire lambda stacks than single images The software – at least on the Leica – is not transparent to use Solutions Zeiss META Both use a prism to separate Nikon CSIthe spectrum to multiple channels Both have software that is easier to use