Photobleaching
An unexpected result in these experiments is the reduced
photostability of the dye molecules during TP-NSOM recording. When
excited by one and two-photon excitation the molecules emitted an average
of 6 x 105 and 1 x
105 photons respectively
before irreversible photobleaching occurred. This is surprising since
photobleaching due to triplet state excitation is not expected to depend
on the mechanism of excitation.
In the case of one-photon excitation, we believe the primary
bleaching mechanism is photochemical modification of the dye while it is
in the first excited triplet state. The much lower photostability
of two-photon excited molecules suggests a different bleaching mechanism,
namely photoionization of the long lived triplet state due to absorbance
of a 800 nm photon.
We note that a recent demonstration of two-photon excitation
in the confocal geometry7
reported a similar average number of emitted photons before bleaching for
single Rhodamine B molecules.
Furthermore, we suggest that the use of lower repetition
rate lasers should improve the photostability of two-photon excited molecules.
Summary
Understanding many basic molecular systems requires an understanding
of conformational dynamics at the single molecule level. Near-field
spectroscopy provides the ability to study single biomolecules without
the ensemble averaging effects of traditional far-field optical spectroscopy.
With a resolution of 200 - 20 nm and single molecule detection sensitivity
combined with its noninvasive nature, near-field spectroscopy is well suited
to in situ and in vivo dynamic studies of protein structure
and conformation. Finally, the addition of two-photon excitation
extends the capabilities of near-field spectroscopy to studies of extended
samples.
References
return to Current Research page
return to main NSOM page