Multiphoton Microscopy

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Multiphoton Microscopy

• BMEN 489/689

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Multiphoton Advantages

• The localized excitation provides high spatial
  resolution
• Inherent z-axis resolution improves sensitivity
  and three-dimentaional optical sectioning
• Reduced photodamage/ photobleaching
• Increased penetration depth in specimen
• Provides selective excitation of fluorophores by
  two and three photons
• Increased detection sensitivity of fluorophores
  by reducing autofluorescence or background
• Elimination of confocal aperture

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Multiphoton Process

• Predicted in 1930 by Maria Göppert-Mayer
• Simultaneous absorption of two photons in a
  single quantized event (about 10-15 to 10-18s)
• Need high photon flux (0.1 10 MW/cm2)

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Multiphoton Source

• Due to light source constraints, process not
  observed until 1960s with advent of the laser
• Can use a high power CW laser OR a high
  repetition rate, short pulsed laser (typically
  10s of MHz 10fs to 5ps)

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Multiphoton Targets

• Commonly use dyes that are also used in one
  photon fluorescence microscopy

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Multiphoton Excitation Volume

• Multiphoton sources excite the sample only within
  the focal volume
• This greatly reduces the amount of fluorescence
  from the sample

http//www.mi.infm.it/biolab/tpe/tutor/mpemic.htm
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Multiphoton Photobleaching

• Due to the decreased excitation volume, the
  sample will not photobleach outside of the focal
  volume
• Other techniques (such as confocal microscopy)
  bleach the sample throughout the light path

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Experimental Example

• Dr. Yeh uses multiphoton microscopy to image
  collagen in samples
• Note the second harmonic signal as well

Zoumi et al., PNAS 99, 11015 (2002)
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Experimental Example
Zoumi et al., PNAS 99, 11015 (2002)
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2-Photon vs Confocal-Big Objects

• Both confocal and multiphoton microscopy can
  image in 3-D, but they do it differently

Egner et al., J. Microsc 206, 24 (2002)
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2-Photon vs Confocal-Small Objects
Egner et al., J. Microsc 206, 24 (2002)
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