III
Fluorescence is one of the most important contrasting
methods in biological confocal microscopy.
Cellular structures can be specifically labeled with dyes
(fluorescent dyes = fluorochromes or fluorophores) in vari-
ous ways. Let the mechanisms involved in confocal fluores-
cence microscopy be explained by taking fluorescein as an
example of a fluorochrome. Fluorescein has its absorption
maximum at 490 nm. It is common to equip a confocal LSM
with an argon laser with an output of 15 – 20 mW at the
488 nm line. Let the system be adjusted to provide a laser
power of 500 µW in the pupil of the microscope objective.
Let us assume that the microscope objective has the ideal
transmittance of 100 %.
With a C-Apochromat 63 x/1.2W, the power density at
the focus, referred to the diameter of the Airy disk, then is
2.58 ·10
5
W/cm
2
. This corresponds to an excitation photon
flux of 6.34 ·10
23
photons/cm
2
sec. In conventional fluores-
cence microscopy, with the same objective, comparable
lighting power (xenon lamp with 2 mW at 488 nm) and a
visual field diameter of 20 mm, the excitation photon flux is
only 2.48 ·10
18
photons/cm
2
sec, i.e. lower by about five
powers of ten.
This is understandable by the fact that the laser beam in a
confocal LSM is focused into the specimen, whereas the
specimen in a conventional microscope is illuminated by
parallel light.
The point of main interest, however, is the fluorescence (F)
emitted.
The emission from a single molecule (F) depends on the
molecular cross-section (
σ
), the fluorescence quantum
yield (Qe) and the excitation photon flux (I) as follows:
Fluorescence
Details
F =
σ
· Qe · I
[photons/sec]
In principle, the number of photons emitted increases with
the intensity of excitation. However, the limiting parameter
is the maximum emission rate of the fluorochrome mole-
cule, i.e. the number of photons emittable per unit of time.
The maximum emission rate is determined by the lifetime
(= radiation time) of the excited state. For fluorescein this is
about 4.4 nsec (subject to variation according to the ambi-
ent conditions). On average, the maximum emission rate of
fluorescein is 2.27·10
8
photons/sec. This corresponds to an
excitation photon flux of 1.26·10
24
photons/cm
2
sec.
At rates greater than 1.26 ·10
24
photons/cm
2
sec, the fluo-
rescein molecule becomes saturated. An increase in the
excitation photon flux will then no longer cause an increase
in the emission rate ; the number of photons absorbed
remains constant. In our example, this case occurs if the
laser power in the pupil is increased from 500 µW to rough-
ly 1 mW. Figure 22 (top) shows the relationship between
the excitation photon flux and the laser power in the
pupil of the stated objective for a wavelength of
488 nm. Figure 22 (bottom) illustrates the excited-state
saturation of fluorescein molecules. The number of photons
absorbed is approximately proportional to the number of
photons emitted (logarithmic scaling).
Absorpt.
σ
/10
–16
Qe
σ
*Q/10
–16
max.(nm)
Rhodamine
554
3.25
0.78
0.91
Fluorescein
490
2.55
0.71
1.81
Texas Red
596
3.3
0.51
1.68
Cy 3.18
550
4.97
0.14
0.69
Cy 5.18
650
7.66
0.18
1.37
The table below lists the characteristics of some important
fluorochromes:
Source:
Handbook of Biological Confocal Microscopy, p. 268/Waggoner
In the example chosen,
F = 1.15 ·10
8
photons/sec or 115 photons/µsec
337_Grundlagen_Infoboxen_e 25.09.2003 16:17 Uhr Seite 3