Biomedical Engineering Reference
In-Depth Information
sample. The fluorescence efficiency can be described as the combination of three factors.
First is the quantum yield, which is the probability that an excited molecule will decay by
emitting a photon rather than by losing its energy nonradiatively. This parameter varies
from 1.00 to 0.05 and varies with time on the order of nanoseconds. Thus, in addition to
intensity measurements, time-resolved fluorescence measurements are possible using
pulsed light sources and fast detectors. The second parameter is the geometrical factor,
which is the solid angle of fluorescence radiation subtended by the detector and that
depends on your probe design. Last is the efficiency of the detector itself for the emitted
fluorescence wavelength.
Since fluorescence is an absorption/reemission technique, it can be described in terms of
Beer's law as
I
f
¼ F
f
I
o
½
1
exp
ðe
Cl
Þ
ð
17
:
66
Þ
in which
is the molar absorp-
tivity. Equation (17.66) can be described in terms of a power series, and for weakly absorb-
ing species (
C
is the concentration of the analyte,
l
is the path length, and
e
0.05) only the first term in the series is significant. Therefore, under these
conditions, the response of the fluorescence sensor becomes linear with analyte concentra-
tion and can be described as
e
Cl
<
I
f
¼ F
f
I
o
e
Cl
ð
17
:
67
Þ
The primary fluorescent sensors are based on the measurement of intensity, but life-
time measurements in the time or frequency domain are also possible. To gain the most
information, particularly in a research or teaching setting, dual monochromators
(grating-based wavelength separation devices) are used with either a photomultiplier
tube as the detector or a CCD array detector. In a typical benchtop fluorimeter, a broad,
primarily ultraviolet/visible xenon bulb is used as the light source. The light is coupled
first through a monochromator, which is a wavelength separator that can be set for any
excitation wavelength within the range of the source. The light then passes through the
sample and is collected by a second monochromator. The light reflected from the grating
within the second monochromator can be scanned so a photomultiplier tube (PMT)
receives the different wavelengths of light as a function of time. Alternatively, all the
wavelengths from the grating can be collected simultaneously on a CCD detector array.
The advantage of the CCD array is that it provides for real-time collection of the fluores-
cence spectrum. The advantage of the PMT is that it is typically a more sensitive detector. In
many systems a small portion of the beam is split at the input and sent to a reference detec-
tor to allow for correction of fluctuations in the light source. Once the optimal configuration
for a particular biomedical application, such as cervical cancer detection or glucose sensing,
has been investigated using the benchtop machine, an intensity measurement system can be
designed with a simpler, more robust configuration. Such a system can be designed with
lasers and/or wavelength-specific filters instead of monochromators and be made to work
at two or more discrete wavelengths. In addition, optical fibers can be used for delivery and
collection of the light to the remote area. Since the excitation wavelength and fluorescent
emission wavelengths are different, the same fiber or fibers can be used to both deliver and
collect the light. In any configuration it is important to match the spectral characteristics of
