Biology Reference
In-Depth Information
scanning). Also, series of holographic or curved mirrors in the scanhead have also
been utilized to scan more than one pixel at a time, but this has remained more of a
rarity compared to the abovementionedmicroscopy modifications (
Callamaras and
Parker, 1999; Tsien and Bacskai, 1995
).
The aforementioned confocal scanning approaches depend on the use of single-
photon lasers as the source of illumination and excitation. These are based on the
principle that a single photon provides enough energy to excite a single fluorescent
molecule, that is, to ''lift'' it from a ground state to the ''excited'' state. The phase
where the fluorophore is lifted to the excited state lasts for femtoseconds (10
15
s),
whereas the fluorophore remains in the higher-energy excited state for picoseconds
(10
12
s) where it undergoes internal conversion and starts to vibrate, which e
ec-
tively leads to dissipation of energy, such that it drops back to the ground state;
measurable to a time scale of nanoseconds (10
9
s). When this happens, the
fluorophore releases a photon that due of the loss of energy has a longer wavelength
(less energy), and this is what creates the fluorescence emission that may be
measured by signal detectors such as PMTs or CCD cameras. The di
V
erence
between the excitation and emission spectra (emission wavelengths being longer
than excitation wavelengths) is called the Stokes shift. The process of excitation and
subsequent relaxation with photon release and fluorescence emission can be illu-
strated by a Jablonski diagram (
Fig. 2
), and is not restricted to confocal microsco-
py, but is in fact the basis for all fluorescence techniques including epifluorescence
microscopy and spectroscopy. As detailed above, it is the volume of the recorded
fluorescent emission that di
V
ers between confocal and epifluorescence microscopy
modalities, though the volume of excitation may also di
V
er, but this has to do with
how much of the specimen is subjected to the illumination light. However,
although the laser excites fluorophores along the entire Z-axis of the specimen
(see also above and
Fig. 3
), peak excitation and as such peak brightness occurs at
the focal plane, whereas out-of-focus excitation decreases with the square of the
distance from the focal plane. This is because the laser excitation beam presents
with an hourglass shape, with the ''waist'' of the hourglass coinciding exactly with
the focal plane.
Several laser lines have been developed that allow single-photon excitation
of fluorescent Ca
2
þ
indicators (fluorophores), in particular, the multiline
argon ion (Ar-ion) laser that provides high-intensity light from the ultraviolet
(UV) to the green spectrum (
V
250-514 nm wavelengths), the single-line helium-
neon (He-Ne) lasers that extend the covered spectrum to
633 nm, and argon-
krypton (Ar-Kr) lasers that provide high-intensity light fromblue to redwavelengths.
Thus, these lasers are well suited for exciting the common Ca
2
þ
indicators dyes and
are also as such much used in Ca
2
þ
signaling research. Recent developments in solid
state and diode lasers have also added more choices for the microscopist. However,
lasers will not be covered in detail here, but the interested reader will find a wealth of
literature on this topic by searching the appropriate literature databases or microsco-
py textbooks,
literature that covers the topic from both physics and biology
perspectives.
