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 9-21
Blue/Cyan dyes
Alexa 350 (780 nm-800 nm)
Hoechst (780 nm-800 nm or 900-1100 nm)
DAPI (780-800 nm or 900 nm-1100 nm)
CFP (800 nm-900 nm)
Green dyes
Oregon Green (800 nm-860 nm)
Alexa 488 (800 nm-830 nm)
GFP (840 nm-900 nm)
BODIPY (900-950 nm)
FITC (750 nm-800 nm)
DiO (780 nm-830 nm)
Yellow Dyes
YFP (890 nm-950 nm)
Orange dyes
DiA (800 nm-860 nm)
Red dyes
DiI (830-920 nm)
Rhodamine B (800 nm-860 nm)
Alexa 568 (780 nm-840 nm)
The web-site shows a collection of excitation cross
sections for various dyes. Currently available dyes
have multiphoton cross-section values ranging
from 40-200 GM (see Xu, 2000).
These values are only an indication of how well a
dye will perform in biological imaging using
MPLSM. A one to one correlation between the
cross-section and dye performance cannot always
be drawn. Measurements of multiphoton cross-
section are usually performed with the dye
dissolved in organic solvents or at non-
physiological conditions and the local environment
of a dye may influence absorption and emission
characteristics. Also, these values are determined
at an optimal pulse-width that cannot always be
achieved at the level of the specimen. Although
the cross-section is a valuable first approximation
of how a dye will perform, trial and error provides
the truest test for a particular application.
The multiphoton cross-section not only provides
information about how well a particular molecule
is excited by pulsed NIR light but also indicates the
multiphoton absorption peak, a value that has
been surprisingly difficult to predict. While it may
seem logical that the two-photon absorption peak
should be twice the one-photon peak, this has not
proven to be the case. An emerging rule of thumb
is that the peak is usually shorter (more blue-
shifted) than twice the one-photon absorbance
peak (Xu, 2000). However, one should also keep
in mind that the brightest signal may not be
obtained at the predicted excitation peak but at
the point where the excitation peak and the
power peak of the laser overlap.
Table 2 Recommended multiphoton
excitation wavelengths for
common dyes.
For tunable Ti: Sapphire lasers, there is a peak output at or near 800 nm, with less average power
produced at lower and higher wavelengths; as a result, it is possible to excite a given dye at a non-
optimal wavelength by using more power. However, more power may also mean more heat or bleaching,
so one should be careful to optimize the signal-to-power ratio.
In addition to how well a molecule absorbs light, the best molecules to choose are the ones that also
efficiently release light as the molecule relaxes back to the ground state. The fluorescence quantum yield
is a measure of the proportion of emission photons that are shed per excitation event.
In general, this value is the same for one- or multiphoton excitation and values for common
fluorochromes can be found in Haugland (1996).