Biology Reference
In-Depth Information
A
Single-photon
2P
Excitation
Emission
Single-photon
2P
Single-photon
2P
Focal point
Photons
Excitation and emission intensities
B
Continuous single-photon laser
Time
Femtosecond pulsed 2P laser
1
00 fs
10 ns
Time
Fig. 6
Two-photon (2P) excitation microscopy, including a comparison to single-photon confocal
microscopy. (A) Schematic of illumination lightbeams, in which excitation occurs along the whole Z-
axis with single-photon confocal microscopy, though with highest intensity at the focal point, whereas
excitation is confined to a narrow area around the focal point with 2P microscopy. Correspondingly,
emission occurs along the entire z-axis with confocal microscopy; though out-of-focus light is blocked
by the confocal aperture, whereas emission is confined to a narrow area around the focal point with 2P
excitation microscopy. (B) Schematic of the continuous laser used for single-photon excitation and a
pulsed titanium:sapphire (Ti:Sapphire) laser used for 2P excitation.
Therefore, the optical sectioning and spatial and temporal resolution one may
achieve will be close to that achieved by confocal microscopy or a little poorer,
but not better (
Cox and Sheppard, 2004
). A clear benefit of this is that there need
not be a confocal aperture. Although crucial for confocal single-photon micros-
copy, confocal apertures themselves reduce light transmission, but also require
that the emission light path is returned through the scanhead including the
mirrors, which further causes a loss of light. Instead, the emitted fluorescence
after 2P excitation may be focused directly onto a PMT without having to be
descanned, as in confocal microscopy, such that 2P microscope systems are
generally more light sensitive. However, the introduction of a confocal aperture
with a pinhole into the 2P emission pathway may improve the spatial resolution
to a confocal standard.
