MPM Imaging with Laser Scanning Microscopy
In confocal microscopy,
the out of focus fluorescence that is generated everywhere by one-photon
excitation with a continuous visible laser is excluded from the image
by collection of the fluorescence back through the same scanning
pathway as the illumination followed by a confocal aperture through
which only light originating in the focal plane can pass.
In multiphoton microscopy the same pathway is used for the illumination by
a mode locked laser generating a train of 100 femtosecond pulses at 80 MHz
with wavelengths tunable from 700-1050 nm. Since the fluorescence is generated
only in the focal volume there is no out of focus excitation and the three-dimensional
resolution is intrinsic. Therefore, efficient direct external detection of
the fluorescence can be accomplished by selecting the fluorescence by a dichroic
mirror close behind the objective lens.
Schematic Representation of the optics of Laser Scanning Microscopy showing both confocal “descanned” detection and direct external detection for multiphoton microscopy.
Next Functional
imaging in live preparations using "conventional" fluorescent
probes
