Fluorescence detector

A fluorescence detector method and apparatus for detecting biological activities in a fluid specimen, such as blood, urine or sputum, where the specimen and a culture medium are introduced into sealable containers and exposed to conditions enabling a variety of metabolic, physical, and chemical changes to take place in the presence of microorganisms in the sample. In operation, the detector method and apparatus illuminate a chemical sensor material in the sealable container with excitation light which is turned on and off periodically according to a symmetric square wave, split the measured fluorescence photocurrent into two components that represent different harmonics of the symmetric square wave signal, measure the amplitudes of the two components, generate the ratio of the two components, and use that ratio as the sensor output signal to indicate biological activity.

BACKGROUND OF THE INVENTION 
The present invention relates to a wide variety of chemical sensors that 
are based on changes in fluorescence lifetime. As an example, the present 
invention is applied to non-invasive apparatus and method for detecting 
biological activities in a fluid specimen, such as blood, urine or sputum, 
where the specimen and a culture medium are introduced into sealable 
containers and exposed to conditions enabling a variety of metabolic, 
physical, and chemical changes to take place in the presence of 
microorganisms in the sample. The biological activity being detected by a 
chemical sensor based on changes in fluorescence lifetime. 
Usually, the presence of microorganisms such as bacteria or mycobacteria in 
a patient's body fluid, particularly blood, is determined using culture 
vials. A small quantity of body fluid is injected through a sealing robber 
septum into a sterile vial containing a culture medium. The vial is 
incubated at a temperature conducive to bacterial growth, e.g., 37.degree. 
C., and monitored for bacterial growth. 
Known instrumental methods detect changes in the CO.sub.2 content in 
culture bottles, which is a metabolic by-product of the bacterial growth. 
Recently, automated blood culture systems have been developed which use 
chemical sensors disposed inside a vial. Such sensors often respond to 
changes in the CO.sub.2 concentration by changing their color or by 
changing their fluorescence intensity (see, e.g., U.S. Pat. No. 
4,945,060). The outputs from these sensors are based upon light intensity 
measurements. This means that errors may occur, particularly if the light 
sources used to excite the sensors, or the photodetectors used to monitor 
intensities, exhibit aging effects over time. 
In known automated non-invasive blood culture systems, individual light 
sources, individual spectral excitation and emission filters, and 
individual photodetectors are arranged adjacent to each vial. Such 
arrangements result in certain station sensitivity variations from one 
vial to the next. Due to the fact that most known blood culture sensors 
generate only a moderate contrast ratio in the measured photocurrent 
during bacterial growth, extensive and time-consuming calibration 
procedures and sophisticated detection algorithms are required to operate 
these systems. Moreover, light sources, spectral filters, and 
photodetectors with extreme narrow specification tolerances must be 
utilized. It is also possible to use so-called source monitor photodiodes 
at each light source, but each of these measures result in increased cost. 
However, even if it would be possible to equalize all vial stations, 
certain lot-to-lot variations in the sensor composition and certain 
vial-to-vial geometry variations would remain. 
The disadvantage of such intensity-based sensor arrangements can be 
overcome by utilizing fluorescent sensors that change their fluorescence 
lifetime, wherein intensity measurement is replaced with time parameter 
measurement and intensity changes have no impact on the sensor output 
signal. Many chemical sensor materials are known that change their 
fluorescence lifetime with changing oxygen concentration, pH, carbon 
dioxide concentration,. or other chemical parameters (see, e.g., G.B. 
Patent No. 2,132,348). 
A change in sensor fluorescence lifetime is commonly monitored by applying 
a well-known phase shift method (see, e.g., U.S. Pat. No. 5,030,420), 
wherein the excitation light is sinusoidally intensity-modulated. That 
method remits in a sinusoidally intensity-modulated fluorescence emission 
that is phase-shifted relative to the excitation phase. Phase shift angle, 
.theta., is dependent on the fluorescence lifetime, .tau., according to 
the equation: 
EQU tan .theta.=.omega..tau. (1) 
where .omega.=2.pi.f, is the circular light modulation frequency. 
From equation (1), the disadvantage of the phase shift method becomes 
evident. If the product .omega..tau. is small, then the resulting phase 
shift .theta. is also small. This limits the resolution of the chemical 
sensor arrangement with regard to the analyte that has to be measured. To 
overcome this resolution problem, one could increase the modulation 
frequency. Doing so results in another limitation, the resulting phase 
shift angles are compressed because they are approaching the 70-90 degree 
range. The maximum possible phase shift angle for a single exponentially 
decaying fluorophore is 90 degrees, even for an infinitely long 
fluorescence lifetime. Due to these limitations, the dynamic range of a 
chemical sensor based on the phase shift method is limited. 
A second disadvantage of the phase shift method is related to the fact that 
the electronic circuitry introduces an additional phase shift, which 
depends on the light modulation frequency. It is therefore common practice 
to use a non-fluorescent scattering medium in order to determine the 
electronic phase shift, and to subtract this value from the one which is 
observed while monitoring fluorescence. Unfortunately, the electronic 
phase shift can change over time. Therefore, the scatter measurement has 
to be repeated, or the system switches periodically between fluorescence 
and scatter measurement. This, however, results in a higher complexity of 
the sensor arrangement. 
Another disadvantage of the phase shift method is caused by the fact that 
the phase of the excitation light may show a drift relative to the phase 
of the modulation driving signal. This artifact is known for internally 
modulated lasers, and also for light-emitting diodes, acousto-optic 
modulators and for electrooptic modulators. The consequence is that the 
phase information for the excitation modulation can not be derived from 
the electronic driving signal, but has to be measured via an auxiliary 
photodetector. Again, this represents an increase in the complexity of the 
sensor arrangement. 
Still another disadvantage of the phase shift method results, if 
fluorophores are utilized that react via lifetime quenching. In this case, 
the relationship between the measured phase shift and the analyte 
concentration is a non-linear one. In other words, the sensor resolution 
for a low analyte concentration is high, but it decreases with increasing 
analyte concentration. This limits the dynamic range of the sensor, and is 
not acceptable for many practical applications. 
It would also be possible to monitor the fluorescence lifetime by measuring 
the modulation degree of the fluorescence which is emitted by the 
fluorophore. In this case, the excitation light is, as in the phase shift 
method, modulated sinusoidally. This results in a sinusoidally modulated 
fluorescence emission, where the modulation degree, m.sub.F, depends on 
the lifetime according to the equation 
##EQU1## 
In equation (2), m.sub.EX is the modulation degree of the excitation light 
source. 
The modulation degree, m, of a sinusoidally modulated signal is defined as 
##EQU2## 
where AC means haft of the peak-to-peak amplitude of the time-varying 
component, and DC means the component obtained by averaging over at least 
one sinusoidal period. In practice, the two components are separated by 
splitting the photodetector signal into two channels. One channel 
comprises a high pass filter and measures the AC component. The other 
channel comprises a low pass filter and measures only the DC component. A 
ratio device is employed to generate a sensor output signal AC/DC=m. 
Such a modulation method has not found practical application in 
fluorescence sensor apparatus, since any change in the photodetector dark 
current or any daylight leaking into the sensing apparatus, would cause a 
change in the measured DC component. This, of course, would generate an 
error in the sensor output signal. It has been proposed already to 
overcome this problem by turning the excitation fight source periodically 
on and off, and subtracting the signal DC.sub.dark, which is measured 
while the source is off, from the signal DC, which is measured while the 
source is on, to calculate a corrected signal DC.sub.corr which is then 
used to calculate the true modulation degree. This correction procedure 
requires additional electronic modules such as a lock-in amplifier, and 
results in an increased complexity of the apparatus. 
A second disadvantage of a possible modulation method is the fact that 
equation (2) is highly non-linear. Optimum sensor resolution is only 
obtained in a relatively narrow range for the so-called frequency lifetime 
product, .omega..tau.. It has already been proposed to overcome this 
limitation by automatically tuning .omega. in such a way that the 
frequency lifetime product coy is kept constant while .tau. is changing. 
This, however, would also result in a significant increase in complexity. 
Therefore, a need exists to overcome the disadvantages of the known 
sensing methods. 
SUMMARY OF THE INVENTION 
It is an objective of the present invention to overcome the above problems 
of the prior art by providing an arrangement and the operational principle 
for a fluorescence detector that is based on changes in fluorescence 
lifetime, where a high dynamic range with regard to the analyte is 
achieved, where the sensor resolution is almost independent of the analyte 
concentration, where changes in the photodetector dark current or changes 
in the intensity of light leaking into the apparatus have no impact on the 
sensor output signal, and which is simple in construction so that it can 
be produced at low cost. 
According to the present invention, the above objective is achieved by an 
apparatus and method for illuminating a chemical sensor material with 
excitation light which is turned on and off periodically according to a 
symmetric square wave, splitting the measured fluorescence photocurrent 
into two AC components that represent different harmonics of the 
square-wave signal, measuring the amplitudes of the two components, 
generating the ratio of the two components, and using this ratio as the 
sensor output signal. 
By using chemical sensors that are based on changes in fluorescence 
lifetime, production-related lot-to-lot variations in the sensor 
composition, small changes in the sensor position, changes in the light 
source intensity, changes in optical filter characteristics, and 
sensitivity changes in the photodetector have no impact on the sensor 
output signal. Therefore, the present invention allows for simplified 
sensing algorithms and for an excellent long-time stability of the 
instrument. Finally, the number of optical and electronic parts that are 
required to operate the sensor is reduced to a minimum, which has a cost 
reduction effect compared to known sensor apparatus based on fluorescence 
lifetime. 
These and other features, objects, benefits and advantages of the present 
invention will become more apparent upon reading the following detailed 
description of the preferred embodiments, along with the appended claims 
in conjunction with the drawings, wherein reference numerals identify 
corresponding components.

DETAILED DESCRIPTION 
A fluorescence detector arrangement embodying the principles and concepts 
of the present invention is depicted schematically in FIG. 1. In this 
arrangement, the specimen and a culture medium 4 are introduced into an 
optically transparent container 1 that is sealed up by a cap 2. A 
fluorescent chemical sensor material 3 is disposed to the inner wall or to 
the inner bottom of container 1, and is illuminated by an excitation light 
source 5, preferably by a blue or green light-emitting diode ("LED"). 
Light source 5 is connected to an electronic signal source 6 that provides 
a symmetric square-wave signal that is switching between the states off 
("ZERO") and on ("HIGH"). 
Fluorescence light reemerging from sensor material 3 is detected by means 
of a photodetector 8 such as a photomultiplier. An emission filter 7 is 
arranged between sensor material 3 and photodetector 8 in order to reject 
back-scattered excitation light. The signal output of photodetector 8 is 
fed to a power splitter 9. One output of power splitter 9 is connected to 
the input of a first band pass filter 10. The output of filter 10 is 
connected via a first high-frequency voltmeter 11 to the B-input of an A/B 
ratio unit 12. The first band pass filter 10 is tuned to one harmonic of 
the square-wave frequency sent by signal source 6. The other output of 
power splitter 9 is connected to the input of a second band pass falter 
13. The output of filter 13 is connected via a second high-frequency 
voltmeter 14 with the A-input of the A/B ratio unit 12. The second band 
pass falter 13 is tuned to another harmonic of the square-wave frequency 
sent by signal source 6. Finally, the output channel of ratio unit 12 is 
connected to a signal recorder 15. 
In operation, light source 5 illuminates chemical sensor material 3 with 
square-wave modulated excitation light having a time-dependent excitation 
light intensity, E(t), in the form 
##EQU3## 
where t is time, a is the square-wave amplitude, c is describing the duty 
cycle, and .omega. is the square-wave frequency. If we use a symmetrical 
square wave, c=.pi./2, all even harmonics are equal to zero and equation 
(4) reads 
##EQU4## 
The re-emitted fluorescence intensity, F(t), has a rather complex course in 
the time domain. However, by tuning the first and the second band pass 
filters to different harmonics of the square-wave signal, two AC 
photocurrent components are generated that are sinusoidally modulated and 
do not contain a DC bias. If the first band pass filter is tuned to the 
first harmonic, and the second band pass filter is tuned to the third 
harmonic, the output signal, R, of ratio unit 12 is given by the following 
equation (6) 
##EQU5## 
As an example, we assume a fluorophore with a lifetime, .tau., that 
depends on the concentration of oxygen, O, according to the Stem-Volmer 
law 
##EQU6## 
where q is a quenching constant. 
FIG. 2 depicts the ratio R31 of the third and the first harmonic versus 
oxygen concentration for a fluorophore with .tau.o=4.74 .mu.s and with 
q=0.29/%, the fluorophore being illuminated with a square-wave modulated 
light intensity and quenched according to the Stem-Volmer relationship. 
The three curves correspond to square-wave frequencies of 30, 100, and 300 
kHz, respectively. As can be seen from this figure, an almost linear 
relationship between R31 and O can be established by selecting an optimum 
.omega.. 
FIG. 3 illustrates the relative amplitudes of the first, third and fifth 
harmonics for a 100 kHz symmetrical square wave signal. The figure shows 
that even the fifth harmonic has a sufficiently high amplitude. The figure 
also shows that the band pass filters are not required to have a high Q 
value, because the second and forth harmonics are missing. 
FIG. 4 depicts the ratio R51 of the fifth and the fh'st harmonic versus 
oxygen concentration for the same fluorophore as in FIG. 2. The three 
curves correspond to square-wave frequencies of 20, 80 and 240 kHz, 
respectively. As in FIG. 2, an almost linear relationship between R51 and 
O can be established by selecting an optimum .omega.. Using the fifth and 
the first harmonic results in a higher contrast between low and high 
oxygen concentration. 
As has been mentioned already, most known blood culture systems detect 
changes in the carbon dioxide content of the culture bottles, which is a 
metabolic by-product of the bacterial growth. For historical reasons, 
growth curves are preferred that show a signal which increases over time. 
Also, many sophisticated detection algorithms have been developed that are 
oriented towards positive-going growth curves. 
If oxygen consumption is used to detect the presence of microorgansims, 
then the ratio R51 in FIG. 4 would start at a high level, and would then 
decrease to a lower level. Therefore, it may be more practical to 
calculate the ratio R15 which shows an increase over time as consequence 
of oxygen consumption. FIG. 5 depicts R15 for square-wave frequencies of 
80, 120 and 160 kHz, respectively, assuming the same fluorescent sensor as 
in FIGS. 2 and 4. 
It should be appreciated that the scope of the present invention is not 
limited to fluorophores that are quenched by an analyte according to the 
Stem-Volmer law shown in Equation (7). FIG. 6 depicts the ratio R15 of the 
first and the fifth harmonic versus fluorescence lifetime for a 
fluorophore that is illuminated with a square-wave modulated light 
intensity and has a fluorescence lifetime that depends on any sensor 
input. The three curves correspond to square-wave frequencies of 10, 40 
and 200 kHz, respectively. However, the same curves would still apply if 
the fluorescence lifetime was in the. nanosecond range. In that case, the 
indicated square-wave frequencies would be in the MHz range. As in FIGS. 2 
and 4, an almost linear relationship can be established between the sensor 
output signal and the input. In FIG. 6, we assume that the fluorescence 
lifetime is linearly dependent on the analyte. 
A fluorescence detector according to the present invention has some 
important advantages over conventional apparatus in the prior art. In 
measuring two different AC components, instead of measuring an AC 
component and the DC component, the effect of a change in the dark current 
of the photodetector, and the effect of daylight leaking into the 
apparatus are eliminated. Moreover, by selecting an optimum square-wave 
frequency, the relationship between the sensor output signal and the 
analyte concentration can be "tailored" to be almost a linear one, so that 
the sensor resolution becomes independent of the analyte concentration. 
This linearization can be achieved for sensor materials that follow a 
typical Stern-Volmer quenching relationship, but also for other sensor 
materials. 
A fluorescence detector according to the present invention is not effected 
by changes in the excitation light source intensity, small changes in the 
sample container position, changes in the emission filter characteristics, 
or changes in the photodetector sensitivity. This is because all these 
artifacts have the same influence on the two harmonics that are selected 
and are, therefore, canceled out by ratio unit 12. 
It is well-known that lot-to-lot variations in the fluorescence sensor 
production process can have a major impact on the fluorescence intensity. 
One major reason are the variations in dye concentration within the sensor 
material. However, this is not the case with regard to fluorescence 
lifetime since lifetime is much less sensitive to concentration 
variations. Consequently, an optical sensor according to the present 
invention provides an excellent opportunity for high reliability, absolute 
calibration, and long-time stability. This is especially important if the 
sensor is used to monitor tuberculosis ("TB") samples that require 
extraordinary long observation periods covering many days. It would even 
be possible to arrange a very large number of sample containers on a few 
portable racks. Removal and subsequent re-entry of these racks into a 
fluorescence reader in a slightly different position would not cause 
signal variations. 
The scope of the present invention is also not limited to a symmetric 
square-wave signal. Other asymmetric square-waves with c smaller or larger 
than .pi./2, and periodic non-square-wave signals could also be applied. 
However, a symmetric square-wave signal is a preferred option because it 
offers the following advantages: 
First, a square-wave signal can be generated most easily with high 
precision, and with a minimum of electronic circuitry. 
Secondly, for a symmetric square wave, all even harmonics are equal to 
zero. This results in a large frequency difference between neighboring 
harmonics and allows use of band pass filters of low Q values, which are 
more stable over extended periods of time. 
Thirdly, the modulation degree of the excitation light is well-defined and 
stable over time. Therefore, no source monitor is required to make sure 
that the excitation light source is still well modulated. This second 
aspect would have even more importance for a conventional modulation 
sensor arrangement with AC and DC measurement, where the actual source 
modulation has a direct impact on the sensor output signal. If a biased 
sinusoidal signal is utilized to modulate the light source, any drift in 
the AC/DC ratio will cause an error in the sensor output information. A 
fluorescence detector according to the present invention, however, is free 
of such problems. 
Fourthly, a symmetric square wave provides a reasonable ratio between the 
fifth, third and first harmonic, while representing a non-dangerous mode 
of driving. If one would use shorter pulses, the amplitude ratio between 
the three harmonics could be better equalized. However, a decreasing c in 
equation (4) results in lower signal amplitudes for all the harmonics. The 
decrease in signal amplitude could be compensated by pulsing an LED to 
much higher forward currents, i.e., by increasing the quantity a in 
equation (4). By doing so, the danger of damaging the LED increases 
rapidly and has a negative effect on the life expectancy for the whole 
detector arrangement. 
Finally, the scope of the present invention is not limited to the use of 
LED's. The invention can also be applied while using diode lasers, 
internally or externally modulated lasers or other light sources. 
A modification of the present invention is given, if a chemical sensor 
material is illuminated with excitation light which is turned on and off 
periodically according to a symmetric square wave, and the measured 
fluorescence photocurrent is analyzed within a computer, which separates 
digitally the received signal into two AC components that are representing 
different harmonics of the square-wave signal, calculates the amplitudes 
of the two components, and generates the ratio of the two components, 
whereby this ratio is utilized as the sensor output signal. 
It should be understood that the above-described embodiment is simply 
illustrative of an apparatus embodying the principles and concepts of the 
present invention. Of course, other suitable variations and modifications 
could also be made to the apparatus and method described and still remain 
within the scope of the present invention.