Biomedical Engineering Reference
In-Depth Information
detection pathway) becomes important. Ideally, one would like
to employ an objective with high NA, large field of view (low
magnification) and large back aperture. Such objectives recently
became available from different manufacturers (Olympus, Nikon,
Zeiss, etc.).
Salzberg et al.
(68)
determined the effective depth of focus for a
0.4 NA objective lens by recording an optical signal from a neu-
ron when it was in focus and then moving the neuron out of
focus by various distances. They found that the neuron had to be
moved 300
4.3.3. Depth of Focus
in Wide-Field Imaging
m out of focus to cause a 50% reduction in signal
size. Using 0.5 NA optics, Kleinfeld and Delaney
(74)
found that
100
μ
μ
m out of focus led to a reduction in signal size of 50%.
Light scattering can limit the spatial resolution of an optical mea-
surement.
Figure 3.12
illustrates the results of experiments car-
ried out on tissue from the salamander olfactory bulb. The top
section indicates that when no tissue is present, essentially all of
the light (750 nm) from a small spot falls on one detector. The
bottom section illustrates the result when a 500
4.3.4. Light Scattering
and Out-of-Focus Light
in Wide-Field Imaging
m-thick slice of
olfactory bulb is present. The light from the small spot is spread
to about 200
μ
m. Mammalian cortex appears to scatter more
than the salamander olfactory bulb. Thus, light scattering will
cause considerable blurring of signals in vertebrate preparations.
Presumably, this effect will be more severe at lower wavelengths
because scattering by nervous tissue is inversely related to wave-
length squared
(75)
.
A second source of blurring is signal from regions that are
out of focus. For example, if the active region is a cylinder (a
column) perpendicular to the plane of focus, and the objective
is focused at the middle of the cylinder, then the light from the
focal plane will have the correct diameter at the image plane.
However, the light from the regions above and below is out
of focus and will have a diameter that is too large. The middle
section of
Fig. 3.12
illustrates the effect of moving the small
spot of light 500
μ
m out of focus. The light from the small
spot is spread to about 200
μ
m. Thus, in preparations with
considerable scattering or with out-of-focus signals, the actual
spatial resolution is likely to be limited by the preparation and
not by the number of pixels in the imaging device.
μ
The confocal microscope
(76)
substantially reduces both the scat-
tered and out-of-focus light by using a pinhole in the detection
pathway. Two-photon microscopes also reduce out-of-focus light
as well as out-of-focus phototoxicity and photobleaching
(77)
.In
this case, however, the excitation of the fluorophore is restricted
to the focal plane. With both types of microscope, one can obtain
4.3.5. Confocal and
Two-Photon Microscopes
