MULTIPHOTON LASER SCANNING MICROSCOPY
Carl Zeiss
Using the LSM 510 NLO direct coupled system
LSM 510 META NLO
9-20
B 45-0021 e
03/06
While the Achroplan objectives clearly provide the best transmission curves with the longest working
distance, other objectives have proven to be useful for multiphoton imaging. For instance, the Plan-
Apochromat objectives have favorable properties for multiphoton imaging as Table 1 indicates. In
addition, the C-Apochromat 40x W/NA 1.2 and its 63x counterpart have proven to be very effective
objectives, perhaps due to their high NA and collection efficiency. Moreover, these objectives have a 0.23
and 0.25 mm working distance, respectively, and can be very useful for collecting large 3-D data sets.
Unfortunately, these objectives begin to decline in transmission at around 840 nm, so they are not
optimal for dyes with more red-shifted excitation wavelength absorption maxima, but can often be used
without concern for the lost photons
9.3.4
Choosing Fluorescent Probes for MPLSM
Presently, there are thousands of different fluorochrome derivatives and conjugates that can be used to
study a variety of biological events. Many of these fluorochromes have been optimized for single-photon
excitation and scientists are just beginning to concentrate on developing optimized fluorochromes for
multiphoton imaging applications (see Albota et al, 1999). Furthermore, it is difficult to predict whether
or not a particular dye will be useful for multiphoton imaging and many of us have relied on trial and
error while a greater understanding of the physical principles of multiphoton excitation is evolving. That
being said, there are some important parameters that one can use to help predict which dyes may be
best for a particular application.
Similar to the way one would choose a fluorescent probe for conventional fluorescence microscopy, the
best fluorochromes for multiphoton imaging are the ones that efficiently absorb light, reliably emit
fluorescence, and are photostable. Values such as the multiphoton cross-section and the fluorescence
quantum yield are important to consider when choosing a fluorochrome.
The multiphoton cross-section is a measure of how strongly a fluorochrome absorbs photons at a given
wavelength and is expressed in units of 10
-50
cm
4
sec/photon or 1 GM (Goeppert-Mayer unit, after the
scientist who first predicted multiphoton absorption, Maria Goeppert-Mayer). A list of cross-sections can
be found in Xu (2000) or at
www.uga.edu/caur/dyechart.pdf
