MULTIPHOTON LASER SCANNING MICROSCOPY I
LSM 510 META NLO
ntroduction to Multiphoton Laser Scanning Microscopy
Carl Zeiss
03/06
B 45-0021 e
9-11
The exact dimensions of A will vary greatly depending on the properties of the objective. In general,
higher magnification, higher NA objectives will reduce A, similar to the way such objectives increase
resolution. However, other considerations such as the transmission efficiency of the objective and the
amount of dispersion produced by the objectives must also be considered as this will reduce P
avg
or
increase respectively. In addition, spherical aberration caused by the objective and sample and the
diameter of the beam at the back aperture of the objective can also affect A. Thus, empirical
determination of the best objective is often relied upon. (see Section 9.3.3 Objectives recommended for
multiphoton excitation).
The pulse length,
τ
, is a measure of the duration (length) of the pulse of photons being delivered to the
sample, measured at the full width, half maximum. Increasing the pulse length, decreases I
peak
. As
mentioned above, ultrafast lasers are able to supply short pulses with a high duty cycle. Unfortunately,
the pulse length is very difficult to measure, especially through a microscope objective. However, it is
possible to make such measurements and users should refer to Wolleschensky et al. (1998) for details.
Several factors that effect the pulse length are discussed below.
9.2.5
Optimizing the Peak Intensity without Frying the Samples
Although raising the average overall power is the easiest way to increase the peak intensity, excessive
heat can destroy both live and fixed samples, as well as produce unwanted imaging artifacts. Assuming
that one is using a laser with a fixed pulse frequency and a high magnification, high NA objective
optimized for multiphoton microscopy (see Section 9.3.3 Objectives recommended for multiphoton
excitation), the pulse length becomes the most important term to minimize.
The Spectral Bandwidth refers to the spectral frequencies of the pulse. Each short pulse produced by the
laser has a broad spectral band centered around the selected wavelength. Spectral bandwidth and pulse
length are inversely related. The wider the bandwidth, the shorter the pulse.
The Coherent Chamelon produces pulses around 140 fsec.
GVD is a temporal broadening of the pulse length as the pulse travels through normal dispersive media
such as glass (see Diels and Rudolph, 1994 for more discussion). The pulse length becomes broader in
dispersive media because the red shifted frequency components, with respect to the center wavelength,
travel faster than the blue shifted frequency components. Pulses with a broader spectral bandwidth are
more susceptible to the effects of GVD, as there is a greater difference in wavelength, and thus, velocities
within the pulse (Fig. 9-3). In addition, the amount of dispersion is related to the thickness of the
dispersive media (see Wolleschensky et al. 2001 for review). When possible, thinner glass optics and
lenses are used along the routing path.
Within the laser, a prism pair is used to adjust the spectral bandwidth of the laser pulses. However,
outside the laser, glass elements within the microscope and scan module, within the objective, and along
the routing path, can lengthen the pulse. In this case, the broader the spectral bandwidth, the more
broadening of the pulses will be caused by GVD. Thus, for direct-coupled systems without the use of a
prechirping unit, it is advantageous to adjust the laser so that the bandwidth of the pulse is at a
minimum. For instance, if the bandwidth is less than 7 nm, the pulse length at the sample will be
approximately 300 fsec. If the bandwidth is 12 nm, the pulse becomes substantially broadened by the
same dispersive elements so that the pulse length is about 700 fsec at the specimen.
