Zeiss LSM 510, Руководство пользователя

Страница: 569 / 606

MULTIPHOTON LASER SCANNING MICROSCOPY I
LSM 510 META NLO ntroduction to Multiphoton Laser Scanning Microscopy Carl Zeiss
03/06
B 45-0021 e 9-7
While this appears conceptually simple, two difficulties, at the level of the fluorochrome, confound our
understanding of this process. First, it is difficult to predict whether a molecule will efficiently absorb the
two lower energy photons simultaneously. Drastic differences in multiphoton absorption between
different molecules have been identified and it is difficult to predict by the structure of a molecule how
well it will efficiently absorb simultaneous low energy photons, although some theories are emerging (see
Albota et al., 1998; Rumi et al., 2000 for review). Second, the wavelengths for maximum multiphoton
excitation are very difficult to predict. Deriving the multiphoton excitation wavelength maximum is clearly
not as simple as doubling the single photon excitation wavelength maximum. Both of these criteria must
be measured and are reflected in the multiphoton cross-section (usually referred to as
δ
) for a given
fluorochrome (see Xu, 2000 for review).
The cross-section data indicate how well the molecule absorbs multiphoton energy at different
wavelengths of NIR light. What is both interesting and perplexing about this data is that several
molecules that all emit green light, i.e. excited at roughly the same wavelength via single photon
absorption, can have multiphoton excitation maxima that are very different. For instance, although
Fluorescein and GFP both emit green light, the multiphoton cross-section peak for Fluorescein is 770-790
nm, but is centered around 900 nm for GFP (S65T) (Xu, 2000) whereas, the single photon excitation
maxima for these two molecules are both around 470-490 nm. These questions represent an intense area
of investigation for physicists and chemists who specialize in multiphoton absorption (see Section 9.3.4
Choosing fluorescent probes for MPLSM).