Zeiss LSM 880 Operating Manual page 682

Fluorescence
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 63x/1.2W, the power density at the
focus, referred to the diameter of the Airy disk, then is
2.58·10 W/cm . This corresponds to an excitation photon
5 2
flux of 6.34·10 23 photons/cm
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
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:
F = σ · Qe · I [photons/sec]
III
2 sec. In conventional fluores-
2 sec, i.e. lower by about five
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
ambient 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
At rates greater than 1.26·10
fluorescein 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 ab-
sorbed remains constant. In our example, this case occurs
if the laser power in the pupil is increased from 500 µW
to roughly 1mW. Figure B (top) shows the relationship be-
tween the excitation photon flux and the laser power in the
pupil of the stated objective for a wavelength of 488 nm.
Figure B (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).
The table below lists the characteristics of some important
fluorochromes:
Absorpt.
σ/10
max.(nm)
Rhodamine 554 3.25
Fluorescein 490 2.55
Texas Red 596 3.3
Cy 3.18 550 4.97
Cy 5.18 650 7.66
Source:
Handbook of Biological Confocal Microscopy, p. 268/Waggoner
In the example chosen,
F = 1.15·10 8 photons/sec or 115 photons/µsec
24 photons/cm 2 sec.
photons/cm sec, the
24 2
Qe
–16 σ*Q/10 –16
0.78 0.91
0.71 1.81
0.51 1.68
0.14 0.69
0.18 1.37