MULTIPHOTON LASER SCANNING MICROSCOPY
Carl Zeiss
Introduction to Multiphoton Laser Scanning Microscopy
LSM 510 META NLO
9-8
B 45-0021 e
03/06
9.2.2
Increased signal-to-noise, enhanced vitality, and deep optical sectioning in MPLSM
One of the greatest benefits of multiphoton excitation is that excitation is practically limited to the focal
plane. This effect increases signal-to-noise and decreases phototoxicity. In single photon excitation, the
excitation of a dye is directly proportional to the average power (Ex
~
P
avg
). Thus, excitation takes place in
the whole cone of focus and optical resolution is accomplished by using a confocal aperture. For
multiphoton excitation, however, excitation of the dye is proportional to the squared intensity (Ex
~
I
2
), as
mentioned above. For a focused beam, intensity (I) can be described as average power (P
avg
) divided by
the cross-sectional area of the beam (A), so for multiphoton excitation, the excitation is proportional to
the average power divided by the area of the beam, squared (Ex
~
[P
avg
/A]
2
). Thus, as the beam diameter
becomes smaller (such as at the focal plane) excitation is increased and excitation out of the plane of
focus becomes highly improbable and falls off with the axial distance from the focal plane with the
power of 4. This explains why multiphoton excitation is mainly limited to the focal plane (Fig. 9-2).
Moreover, the cross-sectional area of the beam is dependent on the NA of the objective. Objectives with
a larger NA can focus light to a smaller beam waist, which is why high NA objectives are preferred for
multiphoton excitation microscopy.
Since out-of-focus fluorescence, which usually
contributes to background noise in the image, is
created inefficiently, MPLSM can be a better
technique for imaging fine structures masked by
background noise. Optical sectioning can be
performed without the use of the pinhole to
eliminate out-of-focus fluorescence and nearly all
of the fluorescence produced at the plane of focus
can be used to make the image. Although a
pinhole is not normally needed using MPLSM, it is
possible to use the confocal pinhole together with
multiphoton excitation to prevent highly scattered
photons from reaching the detector and to
improve optical sectioning. This can be done on an
LSM 510 NLO by carefully adjusting the collimation
lens and the z-position of pinhole 1 (Refer to the
alignment protocol in subsequent sections).
It is tempting to think of the decrease in back-
ground signal as an increase in resolution. This is a
common misconception. In fact, due the longer
excitation wavelength the optical resolution along
the optical axis is worse in comparison to the
resolution in a classical confocal LSM. Objects
obscured by background fluorescence, may appear
brighter or more defined using MPLSM, but this is
not due to an increase in resolution, but rather a
reduction in background noise, resulting in better
contrast.
One-Photon Excitation
Multiphoton Excitation
avg
P
Ex
~
2
~
⎟⎟
⎠
⎞
⎜⎜
⎝
⎛
A
P
Ex
avg
Fig. 9-2
One-photon vs. Multiphoton
excitation. Two images of an
objective are shown for each
example. In each case, the first
indicates the shape of the focused
beam after passing through the
objective. The second indicates the
fluorescence that would be
observed if the beam was focused
through a cuvette containing a
homogeneous solution of
fluorescent dye.
