Biology Reference
In-Depth Information
mask) and nonconjugate image to provide further potential enhancements in
dynamic image quality (
Heintzmann et al., 2003
).
The real power of the programmable matrix approach lies in the ability to
redefine the array composition, without any additional hardware changes. This
enables complete flexibility for optimization of the illumination pattern for the
sample and experiments in hand—from low speed high resolution to high speed
lower resolution studies.
X. Advantages and Disadvantages of Confocal Microscopy
From an experimental point of view, confocal Ca
2
þ
imaging o
ers many benefits
over conventional fluorescence microscopy, especially with respect to spatial and
temporal resolution and the ability to optically section the specimen along the
Z-axis down to a resolution of 0.5-1
m
m, enabled by the presence of a confocal
aperture with a pinhole in the light path that e
V
ectively gives this imaging modality
its uniqueness, as well as its name. Importantly, confocal microscopy may be
performed in live cells that are electrically, mechanically, and by all other mea-
sures, viable. Moreover, confocal microscopy does not impair the ability to
manipulate the surrounding environment in terms of solutions in which the cells
or specimen are bathed, solution pens, temperature devices, application of phar-
macological drugs and inhibitors, patch pipettes, electrical and mechanical manip-
ulators, etc., as long as these do not interfere with the actual light pathway.
However, the significant drawbacks with confocal microscopy relate firstly to the
unavoidable scattering as well as chromatic aberration with subsequent focal plane
o
V
setting that reduces confocal signal collection (
Bliton et al., 1993
) of the illumi-
nation light as it penetrates the specimen, especially since wavelengths of
V
300-600 nm are commonly used for excitation, as those are very prone to
scattering; e
40-50
m
m (or less,
depending on the laser output and the spectral properties of the specimen), and
secondly to the fact that although only the emitted fluorescence produced at or
very near to the focal point will be recorded, excitation will still occur along the
whole Z-axis of the specimen (given molecules able to be excited are present along
the Z-axis). The latter point presents often considerable problems because the
photodamage generated along the entire Z-axis may kill live specimens. Though
this may be mitigated by lowering the laser power, the restricted light capturing
from the small volume accommodated by the pinhole tends to drive toward
microscopists increasing laser power. This point is further accentuated by excita-
tion at short wavelengths (300-500 nm) including UV light, as those are severely
damaging to live specimens due to the associated high energy. Finally, higher
purchase and maintenance costs relative to conventional epifluorescence and wide-
field microscopy may also represent a disadvantage to the researcher.
Adi
V
ectively limiting the penetration depth to
erent excitation approach with many of the same advantages as confocal
laser scanning microscopes as described above, while also substantially limiting the
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