multiphoton and spectral imaging. multiphoton microscopy predicted by maria göppert-mayer in 1931...
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Multiphoton and Spectral Imaging
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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
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Multiphoton-photon Jablonski diagram
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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
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Excitation volume
http://www.loci.wisc.edu/multiphoton/mp.html
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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
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Increased contrast in multiphoton
Centonze,V.E and J.G.White. (1998) Biophysical J. 75:2015-2024
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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
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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
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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
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2PE Spectra
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Detector configuration for multiphoton
Molecular Expressions web site
Note, in particular the descanned detector and the “Whole Area PMT Detector” = Nondescanned detector.
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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
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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
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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.
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What about emission?
Molecular Probes
Choose different dyes
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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
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Example: Lambda stack of cells expressing either CFP or GFP on
chromatin
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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
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Linear Unmixing
• Different amounts of pink and blue generate different spectra
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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
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Unmixing: fluorescein phalloidin and Sytox green
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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