Multiphoton and Spectral Imaging Multiphoton microscopy

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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 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

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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.
752015-2024
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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/tsu
  nami/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
  ?max for single photon
• For example EGFP ?max for single photon 488
  ?max 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?
Choose different dyes
Molecular Probes
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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

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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