Zeiss LSM 510, Руководство пользователя

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MULTIPHOTON LASER SCANNING MICROSCOPY
Carl Zeiss Using the LSM 510 NLO direct coupled system LSM 510 META NLO
9-18
B 45-0021 e 03/06
9.3.3 Objectives recommended for
Multiphoton Excitation
For nonlinear microscopy, objectives should be
optimized for the following parameters: high
transmission in the NIR and the VIS-wavelength
range, long working distance with a high
numerical aperture, limited pulse broadening due
to GVD, a uniform GVD across the pupil of the
objective for the excitation wavelength range and
a small propagation time difference (PTD). As we
have already discussed, low transmission in the NIR
can lower the average power at the sample, thus
lowering the peak intensity.
Long working distance objectives with a high numerical aperture are clearly favored for deep imaging so
that the beam can be focused deep into the tissue and the emission photons can be collected efficiently.
Objectives should have limited GVD to reduce the chance that short pulses will be lengthened en route to
the sample, which will again reduce the peak intensity.
In addition to GVD, chromatic aberration of objectives leads to pulse distortions. Specifically a radius-
dependent group delay is introduced (Kempe et al., 1993; Netz et al., 2000). Therefore, different radial
portions of the beam across the pupil of the objective arrive at different times at the focal region and
cause a temporal broadening of the pulse. This results in lower peak intensity in the focal region
(Fig. 9-7). This effect is also referred to as PTD.
Table 1 summarizes the important characteristic parameters for objectives recommended for nonlinear
microscopy and for the optics inside the LSM 510 NLO including the microscope stand. The dispersion
parameters were calculated based on the material data and the thickness of each optical element on axis.
For comparison, the dispersion parameter for the optics within the LSM 510 NLO including the
microscope stand and the AOM is 7500 fsec
2
.
The variation of the dispersion parameter for different beams across the pupil of the objective is listed in
the forth column. It can be seen from both parameters that the pulse broadening is nearly independent
of the particular objective used and the position of the beam across the pupil of the objective. The pulse
broadening due to the GVD of the different objectives and the LSM 510 NLO including the microscope
stand and the AOM was calculated for 100 fs pulses at a wavelength of 800 nm and is listed in column 5.
The pulse broadening due to PTD is listed in the last column. The PTD was calculated for the whole
optical setup, since it depends on the chromatic aberrations of the complete system including the
LSM 510 NLO and the microscope stand for a wavelength of 800 nm. The PTD effect is negligible if the
pulse length is not much shorter than 100 fsec. It can be seen that the special IR corrected objectives
show a smaller PTD in comparison to the standard UV/VIS corrected objectives.
Fig. 9-7 Schematic pulse transformation by
a singlet lens showing the influence
of chromatic aberrations on the
peak intensity due to Propagation
Time Difference (PTD).