Zeiss LSM 510, Руководство пользователя

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MULTIPHOTON LASER SCANNING MICROSCOPY
Carl Zeiss Introduction to Multiphoton Laser Scanning Microscopy LSM 510 META NLO
9-6
B 45-0021 e 03/06
9.2 Introduction to Multiphoton Laser Scanning Microscopy
Multiphoton laser scanning microscopy (MPLSM) has become an important technique in vital and deep
tissue fluorescence imaging. In MPLSM, fluorescent molecules are excited by the simultaneous absorption
of two or more near infrared (NIR) photons. Multiphoton excitation has a quadratic dependence,
producing excitation only at the focal plane; thus, out-of-focus fluorescence does not contribute to image
background, and photodamage outside the plane of focus is greatly reduced. In practical terms, MPLSM
makes it possible to acquire images with a high signal-to-noise ratio by using a wavelength that is less
harmful to live cells. The use of NIR light makes it possible to image deeper in the specimen, due to less
scatter and absorption of the incident light. However, multiphoton excitation depends on some special
criteria that differ from those needed for single photon excitation events. Here we will provide a
simplified explanation of the physics of multiphoton excitation.
9.2.1 Multiphoton excitation – How does it work?
In single photon excitation, a fluorescent molecule or fluorochrome (also called a chromophore) absorbs a
high energy photon of light within a certain wavelength range and then, within nanoseconds, releases a
photon of longer wavelength (lower energy). The absorption of a photon results in the excitation of the
molecule, by displacing an electron within the molecule from the ground state to an excited state. Thus,
for a single photon excitation event, excitation is directly proportional to the incident photon flux of the
source, since each photon has an equal probability of exciting a molecule in the ground state. As the
molecule relaxes back to the ground state, some energy is lost through non-radiative exchange (heat or
vibration within the molecule), but the rest is shed as a photon of light.
UVA
NIR
NIR
Emission
Emission
Single-Photon Absorption
Two-Photon
b
NIR
NIR
Emission
Three-Photon Absorption
NIR
Fig. 9-1 The principle of multiphoton excitation
The energy loss accounts for the Stokes shift seen between the excitation and the emission wavelength
and explains why the emission maxima is always of a lower energy, more red-shifted, from the excitation
maxima. Multiphoton excitation of the fluorochrome is induced by the combined effect of two or more,
lower energy, NIR photons. As a rule of thumb the energy of the two photons is roughly half the energy
of the photons needed for single photon absorption, although there are clear exceptions to this rule.
Multiphoton excitation can be achieved by two photons of the same or different wavelengths, but with a
single laser source, two photons of the same wavelength are used.
The probability of multi-photon excitation is proportional to the incident photon flux density which is the
intensity squared (I
2
), because a quasi-simultaneous absorption of two photons is necessary. It follows,
that for three-photon excitation, the probability of three-photon absorption is the intensity cubed (I
3
). The
emission characteristics of the excited fluorochrome is unaffected by the different absorption processes
(Fig. 9-1).