MULTIPHOTON LASER SCANNING MICROSCOPY
Carl Zeiss
Using the LSM 510 NLO direct coupled system
LSM 510 META NLO
9-22
B 45-0021 e
03/06
As mentioned above, photostability is an important factor when choosing a dye. Photobleaching is
minimal in out-of-focus regions using MPLSM, but bleaching still occurs at the point of focus. In fact,
some dyes that are relatively stable using one-photon excitation, have more rapid bleaching rates using
MPLSM (Patterson and Piston, 2000). This phenomenon is not fully understood, so the bleaching rate of
dyes of interest should be tested for each application. In cases where rapid bleaching occurs, we have
found that ProLong (Molecular Probes, Eugene, OR) has helped stabilize the signal in fixed samples.
Table 2 is a rough guide of fluorochromes recommended by the Biological Imaging Center at Caltech.
Currently, there is no comprehensive reference for all dyes excited by two- or three-photon excitation for
the user to refer to for information on the peak absorbance wavelength or relative brightness of a given
dye.
The information given below is based on empirical observation and does not reflect a serious, scientific
investigation of all dyes, nor does it indicate the exact optimal excitation wavelength for each dye in
question. Instead, this list is offered as a rough guideline of what is known to work for various imaging
applications and the user is encouraged to empirically determine which dyes are most useful for their
own applications. Clearly the success of any fluorescence imaging experiment relies on how well the
target is labeled. The same is true for MPLSM.
9.3.5
Samples your mother should have warned you about
While multiphoton excitation has many advantages, there are also some disadvantages to using high
intensity NIR or IR light for fluorochrome excitation. Probably one of the biggest disadvantages is the heat
that is generated via the absorption of NIR photons by water. The local heating that is caused can affect
vital cellular processes, such as cell replication (König et al., 1996) and other growth abnormalities in
sensitive specimens. Thus, great care should be taken to maximize PMT settings so that minimal power
levels can be used while imaging. Particular care should be taken when imaging cells or specimens that
contain molecules that absorb light such as melanin.
In some cases, the absorption of NIR photons can cause dramatic local heating effects and cause cells to
explode. This effect has been seen frequently in zebrafish embryos, but can be avoided by blocking
melanin production with chemical agents, such as PTU or by using albino embryos.
