Zeiss LSM 880 Operating Manual page 683

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 . Therefore, a fluorescence molecule
–5
can, on average, be excited n = 26.000 times (n = Q/Q
before it disintegrates.
n
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) experiments,
it is definitely too high for normal fluorescence applications.
Therefore it is highly important that the excitation power
can be controlled to fine increments in the low-intensity
range.
A rise in the emission rate through an increased fluorophore
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,
therefore, is the number of dye molecules contained in
the sampling volume at a particular dye concentration. In
the following considerations, diffusion processes of fluoro-
phore molecules are neglected. The computed numbers of
photoelectrons are based on the parameters listed above.
Details
1.5 . 10
24
1.29 . 10 24
1.07 . 10 24
)
b
8.57 . 10
24
6.43 . 10 24
4.29 . 10 24
2.14 . 10
24
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8
10 21
10
20
10 19
10 18
10
17
10 16
10 15
10
14
10 10
17
Fig. B Excitation photon flux at different laser powers (top)
and excited-state saturation behavior (absorbed photons) of
fluorescein molecules (bottom).
With λ = 488 nm and NA = 1.2 the sampling volume can be
calculated to be V=12.7·10
tion 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
expected 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.
Laser power [mW]
10 10 10 10 10
18 19 20 21 22 23
Incident photons [1/s . cm ]
2
–18 l. Assuming a dye concentra-
PART 3
0.9 1
10 10
24 25
IV