What has been said so far is valid only as long as the mol-
ecule is not affected by photobleaching. In an oxygen-rich
environment, fluorescein bleaches with a quantum efficien-
cy of about 2.7·10
–5
. Therefore, a fluorescence molecule
can, on average, be excited n = 26,000 times (n = Q/Q
b
)
before it disintegrates.
With t= , and referred to the maximum emission rate,
F
max
this corresponds to a lifetime of the fluorescein molecule of
about 115 µs.
It becomes obvious that an increase in excitation power
can bring about only a very limited gain in the emission
rate. While the power provided by the laser is useful for
FRAP (fluorescence recovery after photobleaching) experi-
ments, it is definitely too high for normal fluorescence
applications. Therefore it is highly important that the exci-
tation power can be controlled to fine increments in the
low-intensity range.
A rise in the emission rate through an increased fluo-
rophore concentration is not sensible either, except within
certain limits. As soon as a certain molecule packing density
is exceeded, other effects (e.g. quenching) drastically
reduce the quantum yield despite higher dye concentration.
Another problem to be considered is the system’s detection
sensitivity. As the fluorescence radiated by the molecule
goes to every spatial direction with the same probability,
about 80% of the photons will not be captured by the
objective aperture (NA = 1.2).
With the reflectance and transmittance properties of the
subsequent optical elements and the quantum efficiency of
the PMT taken into account, less than 10 % of the photons
emitted are detected and converted into photoelectrons
(photoelectron = detected photon).
In case of fluorescein (NA = 1.2, 100 µW excitation power,
λ
= 488 nm), a photon flux of F~23 photons/µsec results.
In combination with a sampling time of 4 µsec/pixel this
means 3 – 4 photoelectrons/molecule and pixel.
In practice, however, the object observed will be a labeled
cell. As a rule, the cell volume is distinctly greater than the
volume of the sampling point. What is really interesting,
IV
therefore, is the number of dye molecules contained in the
sampling volume at a particular dye concentration. In the
following considerations, diffusion processes of fluo-
rophore molecules are neglected. The computed numbers
of photoelectrons are based on the parameters listed
above.
With
λ
= 488 nm and NA = 1.2 the sampling volume can
be calculated to be V = 12.7 ·10
–18
l. Assuming a dye con-
centration of 0.01 µMol/l, the sampling volume contains
about 80 dye molecules. This corresponds to a number of
about 260 photoelectrons/pixel. With the concentration
reduced to 1 nMol/l, the number of dye molecules drops to
8 and the number of photoelectrons to 26/pixel.
Finally it can be said that the number of photons to be ex-
pected in many applications of confocal fluorescence
microscopy is rather small (<1000). If measures are taken
to increase the number of photons, dye-specific properties
such as photobleaching have to be taken into account.
Fig. 22 Excitation photon flux at different laser powers (top)
and excited-state saturation behavior (absorbed photons) of
fluorescein molecules (bottom).
Incident photons
Absorbed photons
1.5
.
10
24
1.29
.
10
24
1.07
.
10
24
8.57
.
10
24
6.43
.
10
24
4.29
.
10
24
2.14
.
10
24
10
21
10
20
10
19
10
18
10
17
10
16
10
15
10
14
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
Laser power [mW]
10
17
10
18
10
19
10
20
10
21
10
22
10
23
10
24
10
25
Incident photons [1/s
.
cm
2
]
n
0
337_Grundlagen_Infoboxen_e 25.09.2003 16:17 Uhr Seite 4
