Non-Linear Microscopy - Biophotonics - page 61

(i.e. a pair of galvanometric mirrors); high numerical aperture objectives (NA
1.0-1.4) in order to deliver high peak intensity in the focal point; and a highly
e cient detection system.
Laser source plays an essential role in the production of MPE signal. Be-
cause of relatively low cross-section of the non-linear processes, high photon
flux is required, > 10 24 photons cm 2 s − 1 at the focal plane [79]: considering the
spectral range of 600-1,100 nm this means a peak intensity in the MW cm − 2 -
GW cm − 2 . By using an ultra-fast pulsed laser in combination with a high
numerical aperture objective, this results in a mean laser power of 50 mW or
less. This allows for a su cient peak intensity to induce TPE, with a mean
power levels that are biologically tolerable [72, 80].
The most common ultra-fast pulsed lasers are Ti:sapphire femtosecond
laser sources [40], which can be tuned in wavelength between 700 and 1,050 nm
allowing most of the common fluorescent molecules to be excited in TPE
regime. Typical parameters for Ti:sapphire lasers include an average power of
700 mW-1 W, pulse width of 100-150 fs and repetition rate of 80-100 MHz.
Other laser sources for MPE include Cr-LiSAF, pulse-compressed Nd-YLF in
the femtosecond regime, and mode-locked Nd-YAG. Picosecond pulse width
can also be reached by using a pulse stretcher. It must be noted that due to
time-energy uncertainty principle, ∆ E ∆ T ≥ h/ 2, a shorter pulse width will

return in a broader emission in terms of wavelengths: by considering ∆ T = τ p ,
∆ λ/λ =∆ E/E and E = hc/λ , we obtain

λ 2
2 πτ p c ,

∆ λ = (4.18)

which for a central laser emission wavelength of 1,000 nm and pulse width τ p =
100 fs gives an uncertainty on the wavelength of about 5 nm. Furthermore, it
must be considered that, when the laser light crosses the sample, a temporal
broadening of the pulse occurs. Direct measurement of the pulse width at
the focal plane is not an easy task [81-83]. For practical purposes, it can be
assumed that at the focal volume 1.5-2 times broadening is obtained.
Some notes also must be given to the scanning system. Generally, a x - y
raster scan is performed by means of galvanometric mirrors [84]. Therefore,
the image acquisition rate is generally limited by the mechanical properties
of the scanner. Fast scanning methods based on resonant scanners can be
performed as well; however, it must be considered that fast scanning and sub-
sequent high temporal resolution may require some compromises about spatial
resolution and sensitivity, since by scanning faster the laser will illuminate for
shorter times each point of the sample, resulting in a lower collected signal.
Axial scanning can be performed by a variety of solutions, the most common
being a single objective piezo nanopositioner or a galvanometric object table.
Two-photon architecture usually allows to switch between confocal and TPE
imaging retaining the x - y - z focal position [85]. Furthermore, the use of electro-
optical (like electro-optic modulators, EOMs) or acusto-optical devices allow