Fluorometer with high sensitivity and stability

The present inventive fluorometer provides a rapid means for accurate sample quantitative measurements by making instrumental calibration measurements during sample equilibration time. Increased speed of equilibration and calibration and simultaneously performing these two functions increases the throughput speed with which individual measurements can be accomplished, thus increasing the number of samples which can be routinely processed. This decreases the cost per test and more importantly renders new treatment protocols possible which require the doctor and patient to be able to know within minutes the level of certain drugs in various body fluids.

FIELD OF THE INVENTION 
This invention relates to fluorometer systems for rapidly equilibrating the 
temperature of a sample and for measuring its fluorescence. More 
particularly, the invention is directed to methods and apparatus providing 
highly stable and highly sensitive fluorometry measurements. 
DESCRIPTION OF THE PRIOR ART 
Prior art fluorometers are known employing both single beam or double beam 
optical paths. When using a single beam instrument, to calibrate the 
instrument for instrumental drifts and offsets, it is customary to place a 
sample material having a known response into the beam. This is frequently 
done prior to and after taking a measurement with an unknown material. 
While single beam instruments exhibit high sensitivity due to their high 
optical throughput, they suffer from drift and gain instability. Factors 
influencing the stability include characteristics of the light source, the 
photodetector, and the measuring system. It is not uncommon for such 
systems to exhibit 10 percent (10%) variations in the output signal for 
the same "known" sample during the course of a few hours. Therefore, it is 
necessary when using such single beam systems to perform very frequent 
"calibrations" of the instrument using standard samples to assure accurate 
quantitative measurements. Clinical application would require calibration 
at the time of each measurement. 
In double beam instruments, a fraction of the exciting radiation is 
channeled through a separate optical path to provide a reference signal 
while the bulk of the exciting radiation impinges upon the sample 
providing a sample signal. Double beam, dual detector systems typically 
automatically compensate their measurements for variations in the light 
intensity from the lamp, but since two detectors have dissimilar 
characteristics, double beam, dual detector instruments exhibit a residual 
uncompensated drift of several percent during the course of a few hours. 
Another uncompensated factor in such double beam, dual detector systems 
results from optical/physical changes in the reference and sample channels 
relative to one another. 
Double beam, single detector fluorometers overcome the problems of detector 
mismatch and in general provide excellent compensation for drift and gain 
instability. In double beam systems, either single or dual detector, a 
chopper is typically used to sequentially select the photodetector dark 
current, the reference channel or the sample channel to be observed. 
Typically a chopper is rotated at high constant angular speeds, about 1800 
rpm, and the chopper position is monitored to signal sample, reference, 
and dark measurements intervals. This information is transmitted to three 
corresponding digitizers or sample and hold networks in order to derive a 
corrected signal in a well-known manner. Higher speed chopper operation 
improves stability by providing more frequent calibration measurements. 
Variations in lamp intensity, detector response and drift of measurement 
electronics are correctable in these double beam chopper schemes since 
these variations contribute equally to expressions in both the numerator 
and denominator of an expression of the form of S*=(S-D)/(R-D) where 
S*=corrected signal 
S=measured sample signal 
R=measured reference signal 
D=measured dark signal 
Hence, double beam fluorometers provide considerably better stability and 
compensation than single beam fluorometers. However, we have determined 
that the double beam-constant speed chopper approach has disadvantages for 
measurement of rapidly completing fluorescent reactions. A double beam 
fluorometer cycle is typically divided equally between sample, reference, 
and dark interval measurements. Thus, the actual sample time/unit time 
spent on monitoring the sample is reduced by two-thirds from the single 
beam configuration which monitors the sample signal continuously. This 
time sharing situation in double beam systems results in a corresponding 
loss in sensitivity for dynamic reaction measurements for samples in which 
the fluorescence intensity is changing as a function of time. The 
measurement precision becomes increasingly inferior for fast reactions. 
This is particularly true for observation of small sample quantities where 
emission intensity is very low and where the instrument stability is 
critical. The inventive fluorometry system provides a double beam, single 
detector configuration wherein the chopper or shutter is stationary in the 
sample measurement position during a long period of time so that the 
instrument is configured as a single beam instrument during sample 
measurement and where the instrument calibration measurements using the 
other channel (reference) are made during the period of sample temperature 
equilibration immediately prior to measurement. 
The object of the present invention is to provide a fluorometer system 
which exhibits an extremely high degree of stability as well as very high 
signal sensitivity. 
It is a further object of this invention to provide a fluorometer system 
which can accurately measure fluorescence as a function of time on fast 
reactions or small sample quantities wherein reference and dark current 
measurements are automatically made, after a cycle has started, during the 
same time that rapid thermal equilibration of the sample is accomplished 
and where the sample fluorescence signal monitoring, once started, can be 
monitored continuously without interruption until stopped. Another object 
of the present invention is to provide the equivalent of the electronic 
compensation available in a double beam system and the sensitivity of a 
single beam system.

DESCRIPTION OF THE PREFERRED EMBODIMENT 
In FIG. 1 herein a simplified block diagram appears setting forth the key 
elements forming part of the present fluorometer. In system 1, excitation 
light emitted by an appropriate source lamp 21 is collimated using lens 2 
and is filtered to select an appropriate wavelength band using filter 3. 
The output of filter 3 forms an exciting beam which is split into 
reference and sample channel beams by beam splitter 4. During sample 
measurement the portion of the light beam directed into the sample channel 
through shutter 14 is focused via lens 2' on sample cell 5. The sample 
cell 5 is a tiny fluorescence flow cell enabling rapid temperature 
equilibration which cell is described in copending application, Ser. No. 
864,137, filed Dec. 23, 1977, by the same inventor now U.S. Pat. No. 
4,180,739. 
Light emitted by the fluorescing sample in cell 5 is collimated by lens 6 
and passes through filter 7 for focusing onto semitransparent mirror 8. 
Light passes through the semitransparent mirror 8 and is then focused by 
lens 9 on photodetector 10. 
The light signal detected by photodetector 10 is input to amplifier 11, the 
output of which is passed to control and computation system 12 for 
processing. The corrected output for each measurement is then provided at 
output means 13. 
Test parameters which are input to the test parameter entry and storage 
means 40 are provided to the control and computation system 12, which 
control the times for T1 and T2 period (FIG. 3) for a particular 
designated type of measurement. For rate measurements in quickly 
saturating reactions, T1, the time to temperature equilibrate the sample, 
is as short as possible, i.e., 5 sec. For reactions in which the 
stabilized fluorescence intensity level is to be measured, T1 may be set 
to end and T2 commence when the fluorescence or reaction rate of the 
mixture is expected to be stabilized. The test parameter entry and storage 
means 40 can be a keyboard, an optical card reader, a magnetic card reader 
or any other appropriate means for inputing the test parameters to the 
control and computation system 12. 
When a sample is loaded into cell 5, for system calibration, shutter 14 is 
positioned by shutter indexing driver 15 such that the excitation beam is 
blocked so that it does not fall onto semitransparent mirror 8 or onto the 
sample cell. This permits a calibration of the photocell 10 output current 
when no light is incident thereto. This current is called the "dark 
current." The shutter 14 is disclosed in FIG. 2A with appropriate aperture 
30 which can be adjusted to simultaneously pass one path and block the 
other path as well as block both paths simultaneously. Alternately the 
configuration of FIG. 2B could also be employed as a shutter or mask to 
effect the same result. 
Reference channel measurements for calibration purposes are also made 
during the same time that the temperature of the sample is being 
equilibrated. The shutter driver 15 is commanded by the control and 
computation system 12 so that the portion of the excitation beam directed 
into the reference channel by the beam splitter 4 falls onto mirror 16 and 
is directed towards semitransparent mirror 8. Shutter 14 is positioned to 
permit this light to pass to mirror 8. The light beam striking 
semitransparent mirror 8 is deflected to lens 9 and focused onto 
photodetector 10. This signal is amplified and processed to provide an 
electronics drift calibration signal. The reference channel calibration 
preferably continues up to the instant that sample measurements are 
started. Preferably the dark current measurement is a fixed time interval, 
and the remainder of the T1 interval is determined by the control 12 in 
response to test parameter input information. 
Control and computation system 12 commands the shutter 14 positioning such 
that the sample measurement period starts at the correct time and is long 
enough for the signal-to-noise ratio to be high even though the reaction 
to be measured is very quick, i.e., completed in 20 seconds. For low level 
signals or for rapidly changing signals the sample measurement time is 
long in comparison to the time during which source lamp and dark current 
measurements are performed. This provides for uninterrupted integration of 
the signal and hence maximum signal-to-noise ratio capabilities. 
Mixer and cell loader 22 controls the introduction of sample fluid into the 
sample cell 5 through conduit 29. In auto position of switch 32, upon 
initiation of start switch 33, a control signal from control 12 on line 19 
causes a measured amount of sample fluid from the sample reservoir 26 and 
reagent from the reservoir 25 to mix together and to be introduced into 
the cell 5. Alternately, mixing and loading can be manually carried out 
with switch 32 in manual, or the mixer and loader can be initiated when 
switch 32 is in manual position by closing switch 33'. Mixing can also be 
accomplished in the cell. Control 12 also initiates a signal on line 18' 
to activate and close valve 20 in the drain line 28 from sample cell in 
order to retain fluids in the cell during the equilibration and 
measurement periods. In auto position of switch 32, after initiation of 
switch 33, the start commands 18 and 18', respectively, are synchronized 
closely to the loading of mixed fluids of the cell 5 so that the 
calibration and correction measurements can take place immediately prior 
to sample measurement during the period that the sample is being brought 
to proper temperature, i.e., approximately 5 seconds. 
This system provides an optimum time utilization because drifts present in 
the electronics and optics just at the instant of the start of measurement 
are recorded for compensation and correction of the immediately following 
measurement. With this system, the maximum sensitivity is possible because 
no interruption of the data takes place during measurement on the sample, 
especially during the critical early seconds in a measurement of quick 
reaction fluorescent experiments. Temperature controller 30 is connected 
to a thermoelectric device and to a thermocouple in heater and sensor 27 
for rapid temperature equilibration at a selectable temperature. 
With reference to FIG. 3, the preferred timing relationship for a typical 
experiment using the present inventive fluorometer is described. The 
length of the measurement period during which the sample is observed is 
selected by the test parameters entry and storage means 40 to start at the 
correct time in the reaction of the reagents employed and to be long 
enough to maximize the signal-to-noise ratio for the selected experiment. 
Use of a comparatively long period of time for the integration of the 
sample measurement provides sensitivity to sample concentrations in the 
picogram (10.sup.-12 gm) per milliliter range. The configuration of the 
system during sample monitoring corresponds to equivalent arrangements 
employed by single beam fluorometers which afford five to ten fold 
improvement in detection limits relative to chopped double beam systems. 
The inventive fluorometer system whereby the reference and dark current 
measurements are made during rapid temperature equilibration of the sample 
enables a study of a variety of sample types which could not be accurately 
handled with prior fluorometer systems. These include: 
(1) Small sample quantities where the signal-to-noise ratio is very low. In 
the present system, measurement of the reference beam just prior to the 
sample measurement compensates for drifts in the instrument, without 
interrupting the integration during sample measuring period. 
(2) Fast reaction rate experiments where the important measurement time 
window is short and which commence as soon as chemicals are brought 
together in the sample cell. This system provides the rapid temperature 
equilibration of the sample which permits measurement before the reaction 
has completed and which at the same time accomplishes the calibration 
measurements so that as soon as the sample reaches the specified 
temperature, sample measurements can commence uninterrupted by the need to 
calibrate. 
(3) Large numbers of samples. This device has a high throughput measurement 
capability matching the clinical need which requires fast turn around time 
for large numbers of samples. The present inventive fluorometer provides a 
rapid means for accurate sample quantitative measurements by making 
instrumental calibration measurements during sample equilibration time. 
Increased speed of equilibration and calibration and simultaneously 
performing these two functions increases the throughput speed with which 
individual measurements can be accomplished, thus increasing the number of 
samples which can be routinely processed. This decreases the cost per test 
and more importantly renders new treatment protocols possible which 
require the doctor and patient to be able to know within minutes the level 
of certain drugs in various body fluids.