20
Spectral Imaging
Excitation Fingerprinting
By means of tunable excitation lasers such as those
used in multiphoton systems, it is possible to detect
also the excitation spectra of fluorochromes. These
can be used for unmixing as an alternative to emis-
sion spectra.
Multiphoton systems are a special class of confo-
cal laser scanning microscopes, distinguished from
classical one-photon systems essentially by an
additional light source, known as a multiphoton
or NLO (non-linear optics) laser.
The infrared (IR) light emitted by such lasers can
penetrate tissues to greater depths than visible
light can. Due to its low phototoxicity, IR light is
suitable for long-time observation of live samples.
Usually, the emission wavelength of these lasers
can be varied continuously to excite the respective
fluorochrome used in the multiphoton mode.
In
Excitation Fingerprinting,
this property is used
for the acquisition of excitation lambda stacks. For
that purpose, the multiphoton laser is controlled
by the LSM software to shift its excitation wave-
length by a defined interval before every new
image. The image stacks thus recorded can be
used for the unmixing of spectral components
differing by their excitation properties, analogously
to the (emission) lambda stacks described before.
For more information on multiphoton microscopy,
refer to the literature cited on the rear cover.
The procedure for Excitation Fingerprinting
1
Define an excitation lambda stack (wavelength range and interval size)
in the
Excitation Fingerprinting
macro
2
Record the excitation lambda stack
3
Define the reference spectra via single-labeled specimen regions,
single-labeled reference samples, or by using the
ACE
function
4
Run
Linear Unmixing