Biomedical Engineering Reference
In-Depth Information
Fig. 4.5.
Exemplary optical sectioning obtained with two-photon excitation at
720 nm. The sample is Colpoda cists, and the signal is produced by autofluores-
cence of the membranes
background and an increase in the image contrast. This compensates the de-
crease in resolution due to the longer wavelength compared to the one used
in conventional excitation (4.7). However, the use of infrared wavelength in-
stead of UV-visible allows deeper penetration as the long wavelength used
in TPE (and in general in MPE) will be scattered less than the UV-visible
light [55, 76]. Because of the high intensity delivered in the focal point (com-
pared to conventional excitation), it must be noted that the laser power must
be finely controlled in order to prevent photo-damage effects [73]. On the
other side, the confinement of the excitation processes allows to photochem-
ically modify the sample properties with a high 3D spatial control, opening
new frontiers in the context on micro- and nano-surgery [77, 78]. Finally, the
localization of the photobleaching effect allows to acquire 3D maps of the
sample for longer times than in conventional and in confocal architecture, as
when observing a specific slice of the sample, the out-of-focus contributes will
not be affected by photobleaching [70].
4.6 Two-Photon Optical Setup
The basic setup for multi-photon imaging includes the following elements:
a high-intensity ultra-fast infrared laser source (femto- or picosecond pulse
width with a 80-100 MHz repetition rate); a laser beam scanning system
