Biomedical Engineering Reference
In-Depth Information
(a) Two-photon (confocal)
(b) Two-photon (nonconfocal)
Fig. 1.3.
x
-
y
cross-section images obtained by
a
the confocal system and
b
the
nonconfocal system
because of backward scattering of the fluorescence coming from the deeper
parts of the specimen. This means that the use of confocal pinholes is also
effective for observing not only deep parts of a specimen but also the shallow
parts of it. Although the resolution of the CCD camera is not high enough to
observe the enhancement of the lateral resolution in our current setup, the
reduction of the scattered fluorescence helps one to recognize the details of
the observed specimen.
The use of a pinhole array in a multi-point multi-photon excitation micros-
cope increases the lateral and axial resolution and eliminates fluorescence
scattered or emitted by reabsorption within a specimen. We demonstrated
that the pinhole array eliminates the scattered fluorescence effectively and
brings about higher contrast of images and a sharp depth-discrimination
property.
1.2.3
Instrumentation
Figure 1.4 shows a schematic of a multi-point multi-photon excitation
microscope. A collimated laser beam from a mode-locked Ti : Sapphire laser
(Spectra-Physics, Tsunami, wavelength = 800 nm, pulse width = 80 fs, repe-
tition rate = 82 MHz) is incident to a microlens-array disk and focused onto
a pinhole-array disk. Each focused beam passes through each pinhole and is
focused in a specimen by an objective lens so that the pinholes are imaged on
a focal plane of the objective lens as multi-photon excitation points of fluore-
scence. Fluorescence from a specimen is introduced onto an intensified CCD
camera (Hamamatsu, C-2400-35) by a dichroic mirror that is placed between
the two disks and has around 95% reflectivity at a 400-580 nm wavelength
of light.
Simultaneous rotation of the two disks scans a specimen to realize confo-
cal fluorescence imaging of the specimen. The diameters of the microlenses
and the pinholes are 250
m, respectively. The microlenses and
the pinholes are arranged in a helical order to achieve uniform illumination
on a specimen and produce 12 images with one rotation of the disks. Both
disks are precisely aligned so that each pinhole is placed on each focal point
μ
mand50
μ
