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Claims  |
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The embodiments of the invention in which an exclusive property or
privilege is claimed are defined as follows:
1. In a cross-correlation, frequency domain spectrofluorometer operable to
measure the phase and modulation response of a fluorescent sample, the
combination comprising:
means for producing amplitude modulated light having multiple, harmonically
related, phase-locked, frequencies,
means for exciting a fluorescent sample with said light to produce
harmonically related, phase-locked, multiple frequency light emission
signals;
means for transforming said emission signals into low level frequency
electrical detector signals having the modulation and phase
characteristics of said multiple frequency emission signals, and
means for simultaneously extracting the multi-harmonic phase and modulation
data of the detector signals.
2. The combination of claim 1, wherein said light is produced by external
harmonic modulation of a collimated coherent or incoherent DC light
source.
3. The combination of claim 1, wherein said light is produced from
intrinsically modulated laser pulse trains.
4. The combination of claim 1, and means for detecting said emission
signals.
5. The combination of claim 1, and means for visually displaying said
multi-harmonic phase and modulation data of said detector signals
simultaneously in selected form.
6. The combination of claim 1, wherein said emission signals are high
frequency light and said detector signals are relatively low frequency
electrical signals.
7. The combination of claim 1, wherein said means for transforming said
emission signals into said detector signals comprises a photomultiplier
tube.
8. In a frequency domain fluorometer, the combination comprising: means for
generating phase-locked ratio frequency signals containing a fundamental
frequency and its integer harmonics; means for exciting a test sample with
amplitude modulated light containing said fundamental frequency and
integer harmonics to produce harmonically related, multiple frequency,
phase-locked light signals; means for cross-correlating said light signals
with said radio frequency signals simultaneously at said multiple,
harmonically related, phase-locked frequencies to produce resultant
cross-correlated signals having the same phase and modulation
characteristics as said light signals; and means for simultaneously
extracting and measuring the multi-harmonic phase and modulation responses
of each frequency of the cross-correlated signals.
9. The combination of claim 8, and means for visually presenting said phase
and modulation responses simultaneously in selected form.
10. The combination of claim 8, wherein the last named means thereof
comprises means for digitizing said cross-correlated signals, and means
for transforming the digitized data thereof into the frequency domain.
11. The combination of claim 10, wherein said means for transforming
comprises an array processor operable to perform fourier transforms on
said data.
12. The combination of claim 8, and means for generating said amplitude
modulated light by external harmonic modulation of a coherent or
incoherent D.C. light source.
13. In a frequency domain fluorometer, the combination comprising: means
for providing amplitude modulated light containing multiple harmonically
related phase-locked frequencies; means for exciting a test sample with
said light to produce harmonically related, multiple frequency,
phase-locked emission signals; means for detecting said emission signals,
means for generating phase-locked, radio frequency signals containing a
fundamental frequency and its integer harmonics, means for
cross-correlating said emission signals with said radio frequency signals
simultaneously at multiple harmonically related phase-locked frequencies
to produce a resultant cross-correlated signal of like characteristics
with said emission signals, and means for simultaneously extracting the
phase and modulation data of each individual frequency of the
multiple-frequency cross-correlated signal for analysis and simultaneously
visual presentation of the sample's frequency response.
14. The combination of claim 13, wherein said means for generating said
radio frequency signals and integer harmonics comprise a comb generator.
15. The combination of claim 13, and means for generating first and second
sinusoidal R-F signals of different fundamental frequency, comb generator
means receptive of said first R-F signal for producing said harmonically
related, phase-locked frequencies, and means for extrinsically modulating
a DC light source with said frequencies to effect said amplitude modulated
light.
16. The combination of claim 15, and additional comb generator means
receptive of said second R-F signal and productive of the integer
harmonics of said second fundamental frequency; and said means for
detecting said emission signals comprises a photomultiplier detector
receptive of the signal output of said additional comb generator means.
17. A frequency domain spectrofluorometer comprising:
means for generating a pair of phase-coherent, sinusoidal signals of
different fundamental frequency,
means for generating phase-coherent, multi-frequency integer harmonic
signals from each of said fundamental frequency signals,
a source of collimated coherent or incoherent DC light,
means for extrinsically modulating said light with one of said
multi-frequency harmonic signals to produce multi-frequency,
phase-coherent amplitude modulated light signals,
means for exciting a fluorophore with said light signals to effect
multi-frequency emission signals which are phase-coherent with said light
signals;
means for simultaneously cross-correlating the harmonics of said emission
signals and the harmonics of said other fundamental frequency signal to
produce multi-frequency resultant signals having a repetition rate equal
to the difference between the cross-correlated signals, and
means for simultaneously extracting the phase and modulation data for each
frequency of said multi-frequency resultant signals.
18. A cross-correlation, frequency domain spectrofluorometer comprising:
means for generating a pair of phase-locked sinusoidal radio frequency
signals of different fundamental frequency,
means for generating the integer harmonics of each of said fundamental
frequency signals,
a source of collimated coherent or incoherent DC light,
means for modulating the intensity of said light with the harmonics of one
of said pair of fundamental frequency signals to produce corresponding
phase-coherent, multi-frequency, amplitude modulated light signals;
means for selectively exciting zero lifetime and fluorescent samples with
said light signals to effect phase-coherent, multi-frequency emission
signals therefrom;
photomultiplier detector means receptive of said emission and light
signals,
means for cross-correlating said light and emission signals with the
harmonics of said other of said fundamental frequency signals to produce
beat note detector signals which have a repetition rate equal to the
difference between the cross-correlated signal frequencies and contain the
phase and modulation data for said other fundamental harmonic frequencies;
means for simultaneously extracting the phase and modulation data of each
individual frequency of said detector signals; and
means for visually presenting said phase and modulation data simultaneously
in selected form.
19. The combination of claim 18, wherein said source of light comprises a
CW laser.
20. The combination of claim 18, wherein said source of light comprises an
intrinsically modulated, coherent or incoherent light source.
21. A cross-correlation, frequency domain spectrofluorometer comprising:
means for generating a pair of phase-locked sinusoidal radio frequency
signals of different fundamental frequency,
means for generating the integer harmonics of each of said fundamental
frequency signals,
a source of collimated coherent or incoherent DC light,
means for modulating the intensity of said light with the harmonics of one
of said pair of fundamental frequency signals to produce corresponding
phase-coherent, multi-frequency, amplitude modulated light signals;
means for selectively exciting zero lifetime and fluorescent samples with
said light signals to effect phase-coherent, multi-frequency emission
signals therefrom;
means for transforming said high frequency emission signals into workable
low level frequency detector signals while preserving the phase and
modulation characteristics of said emission signals,
means for simultaneously extracting the phase and modulation data of each
individual frequency of said detector signals; and
means for visually presenting said phase and modulation data simultaneously
in selected form.
22. In a device for measuring the frequency response of a system
comprising:
means for generating two harmonic phase coherent, amplitude modulated,
frequency combs, nf and n(f+.DELTA.), respectively;
first mixer means for providing a synchronizing, cross-correlated,
reference frequency N(.DELTA.) by mixing said two frequency combs, nf and
n(f+.DELTA.);
means for supplying energy at frequency comb nf to the system,
detector means for selectively detecting the input and output frequencies
of the system,
second mixer means for providing a cross-correlated, measurement frequency
N(.DELTA.) by mixing said output frequency and said frequency comb
n(f+.DELTA.), and
means for acquiring, transforming, and analyzing said reference frequency
and said measurement frequency to determine the system's frequency
response. |
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Claims |
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Description |
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This invention relates to spectrofluorometers and more particularly to an
improved type of frequency domain spectrofluorometer which is operable to
acquire phase and modulation fluorescence lifetime data simultaneously at
multiple frequencies of excitation.
Fluorescence emission from a fluorophore excited by a sinusoidal, amplitude
modulated light is also sinusoidally modulated. However, the emission
frequency is shifted and demodulated with respect to the phase and
modulation of the exciting light by an amount related to the lifetime of
the fluorophore's excited state. Instruments for measuring fluorescence
lifetime by phase and modulation techniques are known as phase or
frequency domain fluorometers.
According to heretofore known practice, frequency domain fluorescence
lifetime analysis is dependant on the acquisition of high resolution phase
shift and demodulation values of a test sample's emission at a number of
single frequencies of excitation. Typically, in practice, a dozen or more
measurements are required at different excitation frequencies in order to
adequately define a sample's response curve. Consequently, inasmuch as all
known current phase/modulation fluorometers operate at selected single
detection frequencies, the construction of a sample's response curve is
not only tedious, but time consuming-usually requiring one-half to one and
one-half hours to complete.
The present invention overcomes the aforedescribed difficulty by capturing
the excited fluorophore's response curve in a single measurement through
simultaneous acquisition of phase and modulation data at multiple
frequencies. This capability affords acquisition of the samples response
curve in a matter of seconds rather than hours.
BRIEF DESCRIPTION OF THE INVENTION
In brief, this invention comprises a cross-correlation, frequency domain
spectrofluorometer operable to measure simultaneously the phase and
modulation responses of a fluorescent sample at multiple frequencies of
excitation. Amplitude modulated light is produced from extrinsically
modulated D.C. light sources or intrinsically modulated laser pulse trains
containing multiple, harmonically related, phase-locked frequencies for
exciting the sample. Means are provided for detecting and
cross-correlating test sample emission signals and radio frequency signals
simultaneously at the multiple harmonically related frequencies thereof to
provide detector signals having the phase and modulation characteristics
of the emission signals. Additional means serve to extract the phase and
modulation data of each individual frequency of the detector signal for
analysis and visual presentation in the form of frequency response curves,
excited state reactions, dynamic rotational rates or energy transfer
curves of the test sample.
It is an important object of this invention to provide a new class of
frequency domain fluorometer which is operable to measure the phase and
modulation responses of an excited fluorescent sample simultaneously at
multiple frequencies.
It is another important object of this invention to provide a fluorometer
of the order set out in the preceding object which employs
cross-correlation detection and fourier transform techniques.
It is another object of this invention to provide a frequency domain
fluorometer capable of capturing the frequency response curves, excited
state reactions, dynamic rotational rates or energy transfer curves of a
fluorescent sample in a single measurement which fully satisfies the
requirements of lifetime analysis and which is obtained at millisecond
time intervals for kinetic recording.
It is still another object of this invention to provide a frequency domain
fluorometer which is capable of exciting a sample fluorophore and
detecting its emission response simultaneously and at multiple frequencies
of excitation.
Another important object of this invention is to provide a frequency domain
fluorometer which employs amplitude modulated light from DC light sources
containing multiple harmonically related, phase-locked frequencies used
for exciting the sample fluorophore.
A still further object of this invention is to provide a fluorometer
employing any collimated incoherent light source, a CW laser, a
synchronously pumped dye laser or any intrinsically modulated coherent or
incoherent light source for exciting the test sample.
A further important object of this invention is to provide a
multi-harmonic, fourier fluorometer for collecting phase/modulation
lifetime data simultaneously at multiple frequencies of excitation.
Another important object of this invention is to provide a fluorometer of
the nature set out in the next preceding object which employs phase
coherent, harmonic generators and photomultipliers which simultaneously
cross-correlate and detect high-frequency phase and amplitude information
at a fundamental frequency and its integer harmonics.
Having described this invention, the above and further objects, features
and advantages thereof will appear from the following detailed description
of preferred and modified embodiments thereof, illustrated in the
accompanying drawings and representative of the best mode presently
contemplated for carrying out its concepts and teachings so as to enable
one of skill in the art to practice this invention.
IN THE DRAWINGS:
FIG. 1 is a schematic block diagram of a frequency domain fluorometer
employing a CW laser light source in accordance with this invention;
FIGS. 2-6, are sample phase shift and demodulation response curves for
samples of known characteristics;
FIG. 7 (broken into parts 7C-7E) is a schematic diagram of the circuitry
employed for implementing a fluorometer of the order illustrated in FIG.
1;
FIGS. 7A and 7B are subcircuit diagrams of the FIG. 7 schematic circuit;
and
FIG. 8 is a schematic block diagram of a frequency domain fluorometer of
this invention employing a pulsed dye laser light source.
In order to facilitate understanding of the features and scope of this
invention, two implementations of the fluorometer will be described; one
employing an inexpensive CW laser as the light source and one using a
synchronously pumped dye laser for that purpose. The two instruments
differ principally in the fact that the dye laser source is inherently
modulated whereas the CW laser requires external modulation.
DESCRIPTION OF THE PREFERRED EMBODIMENT
With initial reference to FIG. 1 of the drawings, a block diagram of a
preferred form of fluorometer according to this invention is set forth to
illustrate the fundamental or the basic components of a multi-harmonic
fourier transform fluorometer employing a continuous wave (CW) laser as a
light source (L). Any collimated, coherent or incoherent DC light source
may be substituted for the CW laser.
As there shown, a frequency generator (FG) generates a pair of phase locked
sinusoidal radio frequencies (f) and (f+.DELTA.f). For illustration
purposes, the fundamental frequency (f) may be considered to have a value
of 5 MHz and the second frequency (f+.DELTA.f) a value of 5 MHz plus 3 Hz.
The fundamental 5 MHz frequency is fed to a buffer amplifier (A1) and
serves to drive a harmonic comb generator (HCG-1) whose output contains
all integer harmonics of the 5 MHz fundamental frequency i.e., (5, 10, 15,
20, 25 . . . MHz). This signal is used to modulate the intensity of the CW
laser (L) via a pockels cell (PC) and its associated driver (D). The
modulated laser output contains the 5 MHz fundamental frequency (f) and
its harmonics and is utilized to excite samples located in a rotatable
turret (T) which carries a zero lifetime solution (SC) and a test sample
fluorophore (SA). The turret (T) may be manually indexed or power driven
according to known practice.
The secondary frequency output signal (f+.DELTA.f) of the frequency
generator (FG), i.e. 5 MHz plus 3 Hz, is amplified by (A2) and fed to a
power splitter (PS) which produces a pair of equal strength signals that
are used to drive a pair of harmonic comb generators (HCG-2 and HCG-3).
The outputs of the comb generators contain the fundamental and harmonic
frequencies of N(f+.DELTA.f) i.e., 5 MHz plus 3 Hz, 10 MHz plug 6 Hz, 15
MHz plus 9 Hz . . . etc. These generator output signals are utilized to
modulate the gain of two photomultiplier detectors (PMr and PMm), as will
now be described.
Reference photomultiplier (PMr) receives a small portion (approximately 3%)
of the intensity modulated, exciting light signal N (f).sub.r from the CW
laser via a beam splitter (BS). This signal is cross-correlated with the
input signal N(f+.DELTA.f) from the comb generator (HCG-2), by hetrodyning
or mixing the harmonics of the two signals to produce a low frequency beat
note or reference signal N(.DELTA.f).sub.r which has a repetition rate
equal to the difference between the mixed signals; in this case 3 Hz.
In a similar fashion the measuring photomultiplier (PMm) detects the
signals N(f).sub.m from either the scattering (zero lifetime) solution
(SC) or the test sample (SA), (which it observes alternately) and mixes
the harmonics of such detected signals with the respective harmonics of
the gain modulating signal N(f+.DELTA.f) input from HCG3. This produces a
low frequency beat note N(.DELTA.f).sub.m having a repetition rate equal
to the difference between the frequency pair created by generator (FG),
i.e., 3 Hz. This signal contains the cross-correlated phase and
demodulation information for all the radio frequency harmonics. Thus the 3
Hz component of the photomultiplier tube output signal contains the
information for 5 MHz; the 6 Hz component, the information for 10 MHz; the
9 Hz component, the information for 15 MHz . . . and so forth.
The output signals from the photomultipliers (PMr) and (PMm) are fed to a
computer (C) which digitizes and stores the signals which are then
transformed into the frequency domain by means of an array processor (AP)
that performs complex fast fourier transforms on the data. This latter
operation results in arrays of real and imaginary values used to calculate
the amplitudes and phases of the time domain wave which is transformed
into frequency domain.
The array processor also implements a mathematical low pass filter which
produces the average DC values of the photomultiplier signals (DC av).
Phases and amplitudes for the harmonics are extracted from the fourier
transforms by conventional operations.
The results are visually displayed on the screen of a personal computer in
the form of response curves (phase and amplitude vs. frequency) for the
test sample (SA).
In order to measure a test sample the following general procedure is
employed:
The turret (T) is positioned so that (PMm) is observing the zero lifetime
scattering solution (SC). Under these conditions the amplitudes of the
(PMm) harmonics divided by the common (DC av) value creates a set of
normalizing modulation values which are stored for later use, as will
appear presently.
Phase angles are computed and set to a common reference by taking the phase
differences between the harmonics of the reference photomultiplier (PMr)
signals and the respective harmonics of the measuring photomultiplier
(PMm) signals when observing the scattering solution (SC). For example,
assume that this difference is 20.degree. at the frequency for harmonic
(n). Corresponding differences are measured for each of the harmonic
frequencies and these measurements create a set of normalizing phases
which are analogous to the set of reference or normalizing modulation
values.
The turret is next positioned so that (PMm) is observing the sample (SA).
The amplitudes of the (PMm) harmonics are measured, their modulations
calculated by ratioing them against their common (DC av) value and those
values are normalized by ratioing them against the corresponding
normalizing modulation values previously noted. This creates a set of
sample demodulations for the harmonics of the measured sample (SA).
A set of sample phase shifts for the measuring photomultiplier (PMm)
harmonics is obtained by subtracting the normalizing phases from the
measurement phases.
A plot and printout of phase shifts and demodulations vs. frequency of
excitation constitute the basic output of the instrument which is
presented as a sample response curve visually presented on the personal
computer screen. Noise may be reduced by repetitive, interleaved
measurements of the scatter and sample observations and averaging of the
phase shifts and demodulations.
The response curves set out in FIGS. 2-6 are examples of data produced by a
fluorometer instrument in accordance with this invention and demonstrate
the comparative ability thereof to accurately resolve single and
multi-component lifetimes as well as dynamic rotational rates on samples
of well known characteristics.
FIG. 2 graphically illustrates simultaneous multifrequency phase shift and
demodulation measurements for the organic scintillator, POPOP, p-bis
[2-(5-phenyloxazolyl) benzene] dissolved in methyl alcohol. Literature
values of the fluorescence lifetime for POPOP range from 1.32 to 1.369
nanoseconds. Non-linear least squares fitting of the instrument's measured
data to a single exponential decay model recovered a lifetime for POPOP of
1.34 nanoseconds.
FIG. 3 graphs simultaneous multifrequency phase shift and demodulation
measurements for the organic dye, rose bengal dissolved in ethyl alcohol.
Literature values for the fluorescence lifetime of rose bengal range from
500 to 830 picoseconds. Non-linear least squares fitting of the measured
data to a single exponential decay model recovered a lifetime for rose
bengal of 750 picoseconds.
A demonstration of the ability of the fluorometer of this invention to
resolve multiple lifetime components in a fluorescent sample is set out in
FIG. 4. The molecule TNS, 2-p-Toluidinyl-6-napthalenesulfonic acid,
undergoes solvent relaxation which results in the observation of multiple
decay rates. Non-linear least squares fitting of the measured data to a 3
decay model recovered: 2.3% of the decay at 360 picoseconds; 57% of the
decay at 4.01 nanoseconds and 40.7% at 6.54 nanoseconds. This result is
consistent with values reported in the literature.
Simultaneous multifrequency measurements of the differential polarized
phase and ratio of the modulated amplitudes in order to recover the
dynamic rotational rate of the molecule fluorescein dissolved in propylene
glycol is shown in FIG. 5. Non-linear least squares fitting of the
measured data to a single rotational rate (isotropic)-single decay model
recovered a rotational correlation time of 2.7 nanoseconds; a decay rate
of 4.48 nanoseconds and a limiting anisotropy of 0.364. Comparable values
are reported in the literature.
FIG. 6 demonstrates the kinetics measuring capability of the fluorometer of
this invention. The inorganic salt, potassium iodide (KI) is a quencher of
fluorescence emission causing a decrease in emission intensity and a
shortening of the fluorescence lifetime. The instrument was set to make a
multifrequency phase/modulation measurement every 33 milliseconds and
observe the emission from a solution of fluorescein in 0.1 N sodium
Hydroxide contained in a stirred cuvette. Twenty (20) microliters of a
concentrated solution of KI was injected into the cuvette at a point on
the figure notated "KI addition".
The fluorometer clearly resolved the time course of fluorescein/KI mixing
as a decrease in fluorescence lifetime from 4.3 nanoseconds to a final
value of 1.8-1.9 nanoseconds. Each point on the main graph represents the
analysis of a simultaneous multifrequency data set from the fluorometer.
Typical data sets at various times and their residual "goodness of fit" to
the model are shown in insets A and B, respectively. Complete frequency
response curves were acquired every 30 milliseconds.
IMPLEMENTATION
The implementation of a multi-harmonic fourier fluorometer in accordance
with the teachings of this invention is graphically illustrated by the
schematic circuit diagram shown in FIG. 7 (Parts 7C-7E) of the drawings.
As there set out, (FIG. 7C) a direct frequency synthesizer 20 is used as
the frequency source or generator; (FG of FIG. 1). A synthesizer for this
purpose is commercially available from Programmed Test Sources, Inc. of
Littletown, Mass., Model PTS-250. The 10 MHz reference oscillator output
of the PTS-250 is capacitively coupled to the clock input of a
transistor-transistor logic (TTL), J-K flip-flop 21 with appropriate level
shifting, or resistor network, on the clock input, necessary to trigger
the flip-flop to toggle and divide the 10 MHz input frequency in half,
i.e., 5 MHz. Model 74S112 TTL Schottky flip-flop is available for this
purpose from Texas Instruments, Inc., Dallas, Tex. Since the output (Q) of
the flip-flop 21 is a square wave, it is passed through a low pass filter
22 (PLP-10.7, Mini-Circuits Laboratory, Brooklyn, N.Y.) which removes the
harmonics of the 5 MHz fundamental frequency.
The 5 MHz output of filter 22 is fed to a hybrid amplifier 23 (Model
MHW-592 Motorola, Inc. Semi-Conductor Products, Phoenix, Ariz.) and
boosted to approximately 1 watt after which it is impedance matched to a
first impulse network comprising a variable capacitor 24 (Arco
Electronics, Chatsworth, Calif. Model 4215); a 560 pf silver mica
capacitor 25; a seven turn, one-half inch, I.D. inductance 26; and a 5,000
pf ceramic capacitor 27. This impedance matching network feeds an impulse
generator network composed of a five turn, one-half inch ID storage coil
28 and a step recovery diode (SRD) 29 (Alpha Industries, Woburn, Mass.,
Model RA 6180-1). The impulse network comprising the coil 28 and SRD 29 is
rematched to 50 ohms by high-pass filtering using an output network
comprising a single turn hairpin loaded with a binocular core indicated at
30; a variable capacitor 31 and an impedance 32, formed as a four turn
one-half inch ID coil. The impedance matching, impulse circuit and the
output circuit comprising items 24-32 constitute the comb generator HCG-1
of FIG. 1.
Inasmuch as success of a multi-harmonic fluorometer in accordance with this
invention is largely dependent on a circuit which generates an output
consisting of many integer harmonics of a single input frequency, the
workings of the harmonic comb generator circuit just described is primary
to an understanding of this invention.
In accordance with the embodiment illustrated in FIG. 7C, the important
comb generator function is carried out by appropriate operation of a
special semi-conductor, step recovery diode SRD indicated at 29. Under the
parameters of the FIG. 7C circuit, it is necessary that SRD 29 be capable
of working at a low input frequency, in this case 5 MHz, supplied by the
bi-polar sinewave frequency produced by amplifier 23 which is impedance
matched to the inductance/SRD combination to form the impulse generator.
More specifically, amplifier 23 supplies one to one and one-half watt
sinewave input power and its 50 ohm output is matched to the impulse
circuit impedance (item 28) by an adjustable pi network comprising items
24-27, of the described circuit. Output network (items 30-32) combines
impedance matching back to 50 ohms with a simple high pass filter to
reject the fundamental sinewave feed-through from amplifier 23. During the
positive part of the input cycle, the SRD 29 conducts current, building
minority carriers and storing charge in coil 28. The diode continues to
conduct for a short period (delay time) after the input swings negative
due to the presence of the minority carriers. When the carriers are
depleted, the diode suddenly stops conducting current and energy stored in
coil 28 is transferred to the load as an impulse which contains integer
harmonics of the input frequency up to a limit determined by the
transition time of the diode. Spectrum analysis of the output appears as
an envelope of evenly spaced frequencies and hence the name "comb" or
"picket fence" generator.
In the illustrated circuit of FIG. 7C the output of the harmonic comb
generator HCG-1 is fed to a pre-driving amplifier 35 of the pockel's cell
driver (D in FIG. 1) where it is pre-amplified to approximately +80 volts
peak. Amplifier 35 in the illustrated circuit constitutes an ultra-linear,
1-500 MHz broadband amplifier, (commercially available as Model 525LA
Broad Band, 25 watt, class A amplifier, ENI Power Systems, Rochester,
N.Y.). The pre-amplified impulse emanating from amplifier 35 contains the
5 MHz fundamental frequency and all of its integer harmonic frequencies,
up to approximately 350 MHz.
The output generated by the pre-driving amplifier 35 is capacitively
coupled to the grids of a pair of planar triodes 36 and 37 (3CX100A5,
Varian Eimac, Salt Lake City, Utah) over a capacitor network 38,
comprising three 1000 pf silver mica capacitors. The triodes are normally
kept from conducting by the application of a small negative bias voltage
(-V bias) of 6 to 8 volts DC, by means of a resistor-inductor network 39,
40 and 41. The application of the driving impulse from 35 causes the tubes
36 and 37 to conduct heavily. The plate current passes through an
inverting transformer 42, formed in this instance from a 10" length of
coaxial cable (10T, RG58, 50 ohms) loaded with a high frequency ferrite
toroid (Arnold Engineering Co., Marengo, Ill.). The amplified impulse from
transformer 42 is capacitively coupled to the grid of a final stage triode
43 (3CX400A7, Varian Eimac) through a "bridged-T" network having a
variable capacitor 44 and a center tap, four turn 1/2-inch I.D., 0.6 inch
long coil 45 adjusted to cancel the grid capacitance of the triode 43.
The final stage triode 43 is a zero-bias triode which is normally
non-conducting until the arrival of an impulse at its grid which causes it
to suddenly conduct and transfer energy stored in inductor 46 through
capacitor 47 to an ultra-low inductance, resistive load 48, while
impressing a voltage transient on the center terminal of a double
crystalline pockel's effect, electro-optic light modulator 50, having an
integral back reflecting mirror 51 (Lasermetrics 1042). This activity
conditions the pockel's cell to pass a pulse of harmonically modulated
light, as will be amplified hereinafter.
After inductor 46 is discharged it isolates the triode 43 from the power
source (+v 1-1.5 kv) and then recharges for the next pulse. The pockel's
cell pulse driver circuit or amplifier described (items 35-48 and related
circuit components) can deliver peak power levels of at least 3,000 watts
at high repetition rates and with harmonic phase coherence. This circuit
arrangement is also relatively inexpensive, compact and very rugged and
dependable in operation.
It will be noted that the pockel's cell 50 is coupled to an optic system
comprising a depolarizing quartz wedge 52 and a side exit polarizer 53
arranged to transmit polarized light to the pockel's cell in response to
operation of the CW light source 55 (L of FIG. 1) which in this case may
be a commercially available helium-cadmium laser (Model 4240NB from
Liconix, Inc. of Sunnyvale, Calif.). The laser 55 is operable to provide
CW light at 325 or 442 nanometers as input to the pockel's cell light
modulator.
As indicated in FIG. 7C, the light source 55 produces a vertically oriented
polarized output (crossed circle symbol) which passes through the
depolarizing quartz wedge 52 and enters a side exit calcite polarizer 53.
This polarizer rejects vertically polarized light and allows horizontally
polarized light, as indicated by the vertical double ended arrow symbol,
to pass into the light modulating pockel's cell 50. The latter cell
operably rotates the electric vector of the horizontal polarized light
toward the vertical; rotating the same to a degree which is proportional
to the electric field generated by the initiating impulse received from
the driver circuit (D of FIG. 1).
A back mirror 51 of the pockel's cell modulator reflects the laser beam
light through the KDP crystals of the pockel's cell where the polarization
undergoes further rotation and reenters the side exit polarizer 53 which
reflects the vertical component of the polarized light laterally and
directs the same into an optical module 60, (see FIG. 7E) such as an Model
OP-450, available from SLM Instruments, Inc., Urbana, Ill.
Upon entering module 60 the polarized light excites the appropriate sample
(SC or SA) in the turret 61 (T) after passing through beam splitter 62
(BS), the latter of which reflects a portion of the light signal
(approximately 3%) to the reference photo tube (PMr) indicated at 63. The
remaining light which passes through the sample in turret 61 enters the
measuring photo tube 64 (PMm) via filter 65.
In brief summary of the foregoing operation, it is to be noted that no
light will be present at the sample in turret 61 until the arrival of an
electric impulse at the center terminal of the pockel's cell 50. Such
impulse generates an electric field across a pair of electro-optic
crystals of the pockel's cell which rotate the electric vector of the
input light in a direction that causes the light to reflect out the side
of the calcite polarizer 53 and onto the sample (SA) or (SC) in the turret
61 (T). The light applied to the sample is therefore an intensity
modulated replica of the electrical impulse and as such contains the
fundamental 5 MHz and its integer harmonics up to a practical limit
dictated primarily by the frequency response of the particular pockel's
cell (250 MHz in this circuit example).
Turning now to the operation of the photomultiplier cross correlating
generators, attention is again directed to the synthesizer or frequency
generator 20 (FG) of FIG. 7D which is set to produce a main frequency
output of 5.000,003 MHz, in the case herein of illustration. This
frequency corresponds to (f+.DELTA.f) of the FIG. 1 illustration and is
amplified to 2 watts by a radio frequency amplifier 70 (A.sub.2) for which
purpose a broad band RF, amplifier Model 102L is available from RF Power
Labs, Inc., Bothell, Wash. The 2 watt output of amplifier 70 is fed to a
power splitter 71 (PS), such as a commercially available Model HH-108
hybrid power splitter from Adams Russell - Anzac Division, Burlington,
Mass. Power splitter 71 divides the 2 watt signal equally in order to
drive a pair of impulse generators 72 and 73 which correspond to the
generators (HCG-2) and (HCG-3) of the FIG. 1 illustration. Inasmuch as
these generators are identical to the above described (HCG-1) generator
used for the light modulator, no further description thereof is deemed
necessary.
The outputs of the two generators 72 and 73 will be 5.000,003 MHz and it's
integer harmonics up to approximately 350 MHz. Outputs of the two
generators HCG-2 and HCG-3 are fed to the photomultiplier tubes 63 (PMr)
and 64 (PMm), respectively, and serve to modulate the gain of such tubes
by varying the voltage bias of their second dynode D.sub.2 (see the
subcircuit FIG. 7A, labeled PMT Circuit). It will be understood in this
respect that each of the photomultiplier tubes 63 and 64 has a circuit in
accordance with FIG. 7A. The second dynode D.sub.2 of each of the
photomultiplier tubes is biased by a potentiometer (P) so that the gain of
the photomultiplier tube is low in the absence of an impulse from it's
generator. An impulse received from a generator (HCG-2) or (HCG-3) to its
respective photomultiplier tube 63 or 64, as the case may be, serves to
increase the gain of the photomultiplier tube approximately 10 fold.
The photomultipliers observe harmonically modulated light and
simultaneously are gain modulated by a comb of phase-locked harmonics,
offset from the modulated light signal by (.DELTA.f), which in this
illustrated case is 3 Hz. In this latter regard the 3 Hz value is
arbitrary and in kinetic operation the (.DELTA.f) is selected at 100-300
Hz in order to increase time resolution for that mode of operation of the
described fluorometer of this invention.
The light impulse and gain impulse are mixed in the photomultiplier tubes
at dynode D.sub.2, creating a beat note at a 3 Hz repetition rate which
contains simultaneously the phase and amplitude information for all
detected radio frequency harmonics, viz. 3 Hz=5 MHz; 6 Hz=10 MHz; 9 Hz=15
MHz and so on, up to a limit for this specific instrument of about 250
MHz. The beat note emanating from dynode D.sub.2 is amplified by D.sub.3
and succeeding dynodes D.sub.4 -D.sub.9 to produce a photocurrent which is
sent to a command module (see sub FIG. 7B) indicated generally by 75. In
this instance, the command module illustrated is of a type commercially
available as an SLM 48000 Translator Command Module, SLM Instruments,
Inc., Urbana, Ill. (see FIG. 7E).
The command module provides high voltage bias for the photomultipliers 63
and 64 and converts the photocurrents received from the photomultiplier
tubes into proportional voltage outputs which serve as inputs for a high
speed, high accuracy digitizer 76 (Model DT2847 Data Translation, Inc.,
Marlboro, Mass.) comprising a circuit board resident in a personal
computer 77 marked (PCOMP) in FIG. 7E. In practice a commercially
available Model PC-286, Dell Computer Company, Austin, Tex. has been
employed for the computer 77.
The photomultiplier tube wave forms are digitized and contain the phase and
amplitude information of the radio frequency harmonics. This information
is extracted from the wave forms through fourier transformation by an
array processor 78 (AP) (see FIG. 7E) for which purpose a Model DT7020
Array Processor circuit board, available from Data Translation, Inc.,
Marlboro, Mass. has been found satisfactory.
The fourier transformed wave forms are then displayed as phase and
amplitude vs. frequency curves on the presentation screen of the personal
computer 77 and constitute the end product of the fluorometer of this
invention. The fourier transforms on the array processor are accomplished
by using a commercial library of subroutines and other appropriate
software in computer 77.
PULSED DYE LASER FLUOROMETER
While the CW fluorometer hereinabove described constitutes one embodiment
of the present invention, in FIG. 8 a fluorometer employing a pulsed dye
laser as the light source is illustrated in diagrammatic form
corresponding to the showing of the CW laser fluorometer of FIG. 1. The
fluorometer of FIG. 8 while functionally equivalent to the CW laser
instrument described, is capable of higher frequency operation since the
laser pulse harmonics extend into the gigahertz region of the spectrum.
As shown laser L' in the FIG. 8 embodiment is typically a synchronously
pumped, cavity-dumped dye laser driven by a mode-locked, Argon-ion or
Neodymium: YAG pump laser. Any intrinsically modulated coherent or
incoherent light source may be used for L'. The frequency generator (FG)
is phase locked to the laser pulsed repetition rate, either by supplying a
reference frequency (fref) to the laser or being supplied with such a
reference frequency from the mode-locking frequency generator in the laser
control electronics (LC). The frequency generator (FG) also creates
(fcd+.DELTA.f) a frequency equal to the laser cavity dumper repetition
rate (fcd) plus a small offset (.DELTA.f), again 3 Hz. This frequency is
amplified by (A.sub.2) and split by power splitter (PS) to generate the
cross-correlating combs produced by comb generators (HCG-2) and (HCG-3) to
supply n(fcd+.DELTA.f) to the two photomultipliers (PMr) and (PMm) all as
in the described FIG. 1 CW embodiment.
Measurement of the sample (SA) held in turret (T) as well as analysis and
display is carried out in the same manner as in the aforedescribed CW
laser instrument of FIG. 1.
The present major drawback of a pulsed dye laser fluorometer version of
this invention resides in the relatively high cost of the laser light
source as compared to the CW laser of the first described version. A
further limitation of operating frequencies is encountered by the
frequency response capability of currently available photomultiplier
detectors.
Having described this invention it is believed that those familiar with the
art will readily understand and appreciate that the preferred and modified
embodiments thereof illustrated in the accompanying drawings and described
above are susceptible to modification and substitution of equivalents
without departing from the spirit and scope of the invention which is
intended to be unlimited by the foregoing except as may appear in the
following appended claims.
* * * * *
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