Multiphoton and Spectral Imaging
Mar 30, 2015
Multiphoton and Spectral Imaging
Multiphoton microscopy
• Predicted by Maria Göppert-Mayer in 1931
• Implemented by Denk in early 1990’s
• 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
Multiphoton-photon Jablonski diagram
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 I2
Excitation volume
http://www.loci.wisc.edu/multiphoton/mp.html
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
Increased contrast in multiphoton
Centonze,V.E and J.G.White. (1998) Biophysical J. 75:2015-2024
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/lasers/tsunami/index.html
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
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 λmax for single photon
• For example: EGFP: λmax for single photon = 488; λmax for two photon ≈ 900 nm
2PE Spectra
Detector configuration for multiphoton
Molecular Expressions web site
Note, in particular the descanned detector and the “Whole Area PMT Detector” = Nondescanned detector.
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
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
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.
What about emission?
Molecular Probes
Choose different dyes
Sometimes you can’t 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
Example: Lambda stack of cells expressing either CFP or GFP on
chromatin
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
Linear Unmixing
• Different amounts of pink and blue generate different spectra
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
Unmixing: fluorescein phalloidin and Sytox green
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 CSI the spectrum to multiple
channels
Both have software that is easier to use