MULTIPHOTON LASER SCANNING MICROSCOPY
Carl Zeiss
Introduction to Multiphoton Laser Scanning Microscopy
LSM 510 META NLO
9-10
B 45-0021 e
03/06
9.2.4
Achieving Efficient Multiphoton Excitation using Ultrafast lasers
In order to achieve efficient two- or three-photon excitation, the photons must collide with the molecule
simultaneously. For single-photon excitation, a continuous wave laser with a continuous photon flux can
be used because the probability of excitation is directly proportional to the photon flux or average power
of the source. Increasing the laser intensity (turning up the power) increases the photons delivered to the
sample, increasing excitation until all of the molecules are saturated. To deliver enough photons to
achieve simultaneous absorption of two NIR photons using a continuous wave laser would require
enormous power. Ultrafast lasers improve the efficiency of multiphoton excitation by delivering photons
in pulsed “wave packets”. The high peak intensity needed for multiphoton excitation is created by
concentrating photons into very brief pulses which are delivered to the sample over and over again at a
rapid rate, about every 13 ns, to ensure efficient dye excitation. Instead of a steady flux of photons
bombarding the fluorochrome one after another, multiple photons collide with the molecule
simultaneously. This process has the advantage of delivering a high peak intensity, to satisfy the I
2
or I
3
requirement for two- or three-photon excitation, without using enormous amounts of average power.
Many lasers are now available that can produce ultrashort pulses at high repetition rates. Titanium-
sapphire lasers, for instance, are capable of producing ~100 fsec pulses over a broad tunable wavelength
range (690 nm-1064 nm) with a high repetition rate, ~80 MHz. Similarly, solid state, doubled
neodymium doped yttrium lithium fluoride (Nd: YLF) lasers, emitting 1047 nm, 175 femtosecond pulses
at 120 MHz have also be used for multiphoton excitation.
Ti: Sapphire lasers are probably the most popular lasers because of the wavelength range that is
available. These lasers can operate in both a continuous wave (CW) mode or in a mode that emits pulsed
light. Lasers operating in this latter mode are said to be mode-locked (ML), which refers to the fact that
the laser is locking in different frequencies together to form a pulse of a particular bandwidth.
The use of ultrafast mode-locked lasers for multiphoton excitation requires to consider several additional
factors which are not necessary for continuous-wave lasers used for single-photon excitation. The length
of the laser pulse (referred to as the pulse length or pulse width) (
τ
), the peak intensity produced at the
focal plane (I
peak
), the average power of the laser at the specimen (P
avg
), the cross-sectional area of the
beam (A), and the pulse frequency (F
p
) or repetition rate are all important factors for achieving and
maintaining efficient multiphoton excitation in the sample.
Raising the average power of the laser, P
avg
(controlled by the Acousto-Optic-Modulator, AOM), will raise
the peak intensity. However, raising the average power will also increase the amount of heat generated
in the sample, which may damage vital processes or disrupt cellular structures.
The pulse frequency, F
p
, is determined by the design of the laser and is not easily manipulated. For the
Coherent Chamelon Ti: Sapphire laser, Fp is equal to ~90 MHz.
The cross-sectional area of the beam, A, is an important term to consider when dealing with light
focused through an objective. Simply put, reducing the cross-sectional area of the beam, for instance by
focusing the beam through an objective, increases the intensity at the point of focus or in other words
increases the amount of photons per area as we saw above.
