Zeiss LSM 510, Руководство пользователя

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MULTIPHOTON LASER SCANNING MICROSCOPY
LSM 510 META NLO Using the LSM 510 NLO direct coupled system Carl Zeiss
03/06
B 45-0021 e 9-19
Objective Working
Distance
[mm]
Dispersion Parameter [fsec]
2
On axis Variation
Pulse broadening
factor of 100 fsec
pulse
Max. PTD
[fsec] (*)
IR-Achroplan 40x/0.8 W 3.61 1714 ± 20 1.14 -3
IR-Achroplan 63x/0.9 W 2.00 1494 ± 15 1.11 -9
Plan Neofluar 40x/1.3 oil 0.20 2328 ± 30 1.23 9
Plan Apochromat 20x/0.75 0.61 1531 ± 10 1.12 10
Table 1 Summary of specific parameters for objectives recommended for 2-Photon applications. All
data were measured at 800 nm. (*) Propagation time difference (PTD) is calculated for the
whole optical setup including the LSM 510 NLO and the microscope stand.
Negative values indicate that pulses at the edge of the pupil are delayed with respect to pulses traveling
on axis.
With regard to transmission, objectives for biomedical applications traditionally have been optimized for
UV and visible range imaging. Thus, many are not corrected for transmission of NIR or IR light. The
transmission of an objective depends strongly on the design of the antireflection coating. Typical
transmission curves for two different objectives are shown below. The Achroplan series is coated with
UV/VIS-antireflection coating giving optimal performance from 350 nm to approximately 800 nm. In
contrast, the IR-Achroplan series has a VIS/IR coating that enables high transmission from 420 nm to
1400 nm. The Achroplan objectives are best for multiphoton applications, where excitation is below
850 nm and the fluorescence from the specimen is in the ultraviolet or lower visible wavelength range.
These criteria describe the imaging parameters for
many ion indicators, nuclear dyes such as DAPI or
Hoechst, Blue Fluorescent Protein, and Fluorescein
or Alexa 488. The IR-Achroplan objectives are best
for multiphoton applications requiring an
excitation wavelength greater than 850 nm and
where the fluorescence emission is above 420 nm
(Fig. 9-8). These latter criteria apply to many dyes,
such as DiI, and many of the fluorescent proteins,
such as CFP, GFP, YFP, and dsRed. However, some
important fluorochromes can be imaged well with
either objectives.
Rhodamine, for example, has a multiphoton
absorption peak at 840 nm, and like Alexa 568 it
can be excited well at 800 nm. Thus, the examples
are given to indicate ideal imaging criteria;
however, non-optimized parameters can often be
used to produce high-quality images.
400 600 800 1000 1200 1400
0
20
40
60
80
100
Tr
ans
m
is
si
on [
%]
Wavelength [nm
]
IR-Achroplan
Achroplan ......
Fig. 9-8 Typical transmission curves of an IR-
Achroplan and an Achroplan.