Biomedical Engineering Reference
In-Depth Information
Fig. 1.4.
An optical setup of a two-photon multi focus fluorescence microscope with
a microlens and pinhole-array scanner
of the microlenses. In principle, simultaneous rotation of the two disks scans
a specimen in 3 ms at 1800 rpm. However, in our current setup, the image
acquisition time is limited to 33.3 ms because of the imaging speed of the
CCD camera. Fluorescence imaging through the pinhole array is expected
to yeild twice higher resolution compared with that of a nonconfocal system
[3,9]. Although, even without a detection pinhole, multi-photon excitation
microscopes have a three-dimensional resolution equivalent to that of confo-
cal single-photon excitation microscopes, the lateral resolution is almost the
same as that of conventional fluorescence microscopes because of the use of
near-infrared light for excitation. The scattered fluorescence blurs the images
in observing both shallow and deep parts of the specimen. From the compa-
risons between images obtained by the confocal and the nonconfocal system,
the use of the pinhole array is useful irrespective of the observation depth
and works more effectively for observing stronger scattering specimens. The
elimination of the scattered fluorescence enables us to observe deeper parts
of the specimen than without it. Except for the high spatial resolution, these
advantages brought about by confocal detection do not appear in a typical
multi-photon excitation microscope with single-focus scanning.
1.2.4
Calcium Ion Dynamics Revealed
by Multi-Photon Microscopy
Modulation of the intracellular Ca
2+
concentration ([Ca
2+
]
i
) constitutes a
fundamental mechanism of signal transduction in excitable cells. A spon-
taneous increase in Ca
2+
concentration can occur at a single focus or at
multiple foci within a cell, and can lead to propagation of an elevation of
