DD205522A1 - ARRANGEMENT FOR MEASURING LUMINESCENCE ABCILLATION FUNCTIONS BY TIME CORRELIZED INDIVIDUAL PHOTO NUMBERING - Google Patents

Abstract

The invention relates to the field of short-time luminescence spectroscopy. The object of the invention is to have an arrangement available for measuring Lumineszenzabklingfunktionen or characteristic parameters thereof according to the principle of time-correlated Einzelphotonenzaehlung with continuous mode-locked lasers as the excitation light source. In this case, sufficient sensitivity and time resolution should be achieved for extreme applications. This is achieved by improving the synchronization of the measuring process with the pulse sequence of the laser light, increasing the maximum possible counting rate of the luminescence photons, compensating for the dark current of the light receiver and the scattered light. A possibility for the registration of the luminescence decay function over several excitation periods as well as the direct digital data processing for the determination of extremely short luminescence decay times down to the range below 10 high minus 11 s is described. The arrangement is thus used for research in the fields of photochemistry and physics, condensed phase physics, molecular biology and the like. a. used.

Description

2AO20 1

Dr. Wolfgang Becker Berlin, 17. 05. 1982

ZuBtellungsbevollmächtigt:

Academy of Sciences of the GDR Central Institute of Optics and Spectroscopy - Patent Office

1199 Berlin-Adlershof, Rudower Chaussee 6

Arrangement for measuring luminescence decay functions by time-correlated single-photon counting

Field of application of the invention

The invention relates to an arrangement for measuring luminescence decay functions or luminescence decay times. It ensures very high sensitivity with high time resolution and is therefore particularly suitable for measurements at extremely low concentrations of the substance under investigation and for the investigation of substances with low Lumineszenzquantenausbeute. This concerns in particular problems of photochemistry and physics and molecular biology.

28.MA! 1982 * 012 447

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Characteristic of the known technical solutions

All known methods for measuring Lumineszensabklingfunktionen based on the irradiation of the Meßob.jek tes with a time-varying excitation light and the measurement of the then radiated from the Meßob, project luminescence light. The signal-to-noise ratio in the registration of the measuring light is limited even in an ideal, free from own noise sources by the quantum crystal receiver of the light and averaging the signal over several excitation periods in the measuring time T.

In this case, I is the time-averaged intensity of the measurement light in photons per second, At the time resolution, X is the luminescence decay time and K is a factor which indicates which portion of the photons falling on the receiver is actually processed by the measurement system. In order to achieve a high signal-to-noise ratio, even at high time resolution, ie, at low Δt, it is obviously necessary to provide large values of I and E. "I can, above all, increase the intensity of the excitation light be increased by an optimized measurement method. All known hosing methods can be considered from this common point of view.

In the required Ze itauflösungen in ns- and ps-Bereicn come to register the measuring light following electronic methods into consideration

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1. Sampling or boxing methods

The sample is periodically excited with a short burst of light. The signal supplied by the light receiver is scanned exactly once with a gate in each period. The sampling duration is small compared to the characteristic times of the signal, so that the signal course can be reconstructed from the sampled values obtained when the sampling point is shifted in time (W, Becker et al., Sxp. Techn. Phys. 2 ^ O975) / 3, 297-306). , By the sampling method, the utilization factor K becomes very small, so that such measurement methods for luminance measurements are only suitable in conjunction with the high excitation intensity of pulse lasers.

The achievable time resolution is determined by the width of the impulse response of the light receiver and is at 10 "° s for photovoltaic photomultipliers (SET) and between 10 *" and 10 * "s for semiconductor podiododes.

2nd phase fluorometer

Hie :? the phase shift between the luminescence and a periodic excitation light is measured (S * '', Resewitz, E. Lippert, 3rd, Bunsenges, Phys, Chesi, J8 (1974) 1227) *

A disadvantage of the method is that assumptions about the shape of these rna.'xtipn are needed to determine characteristic parameters of the desired decay function · from the phase shift. made - have to verden. Above all, several superimposed effects (for example, 3 * already by scattered excitation light) can therefore easily lead to malformations. enter "Phasenfluorometer are therefore usually only used for measurements in the field very kurser decay, which is not yet accessible by other methods d.en"

The photon utilization factor K in the phase fluorometer is close to 1. With excitation frequencies of the order of magnitude of sound 100 MHs, as can be easily achieved with mode-synchronized continuous lasers, time resolutions of up to 10 "° s can be achieved (K · Berndt, K. Junge, E, Klose - Proc « XH International Congr. High Speed Photogr. And Photonics - Moscow, USSR, Oct. 80).

The limit is that the transfer function of all light receivers is slightly dependent on the wavelength of the light, so that systematic errors of the order of 10 ps occur.

3 * single photon enumeration

These methods are based on the pulsed excitation of luminescence and the registration of single photons with a SBV * The photons are summed in a digital memory sorted according to their temporal position with respect to the excitation pulse sequence. Under the condition that the probability of the arrival of two photons per excitation pulse is sufficiently small, one obtains in the memory the temporal density function of the photons and thus the sought time function of the light (S. Cova, M. Bertalaccini, C. Bussolati, Phys * stat , sol. (a) 18 (1973), 11 - 62) »

Technically, the method is solved in the following way. The timing of the registration of a photon in the SFV is first determined as accurately as possible from the single photon pulse delivered by the SIV. Pulses derived from the light pulses of the excitation light source serve as temporal reference points for the synchronization of the measuring process The time difference between the occurrence of the Photons and a synchronization pulse is converted into a proportional voltage by a time-amplitude converter. Out of this tension is in one. Analog-to-digital converter generates a number that is used to address a memory location in

a digital memory is used. On the addressed memory space, a 1 is added, so that after the registration of enough photons in the memory, the temporal distribution of the photon density wins, liach finished measurement, this puncture can either read directly from digital or via a digital-to-analog converter as an analog signal from the memory become. The method allows a high photon utilization factor, which approaches asymptotically to the value 1 with weak light signals. The time resolution is determined by transit time scattering of the electrons in the SSV and by the electronically achievable trigger accuracy and is 100 to 300 ps.

The method is well-controlled in connection with ns flash lamps as excitation light source. However, the excitation intensity is so low that luminescence measurements with Boscarmethoden and Pulul3lasera.nregung provide better sensitivities. In addition, pulse durations below 1 .mu.s can hardly be achieved with flash lamps, so that the high time resolution of the measurement procedure is not utilized.

In contrast, the combination of single-photon counting with mode-locked continuous lasers is extremely attractive

(A. Muenter, J. Phys, Chem. 80 (1976), 2178). Depending on the type, these lasers allow pulse widths of 10 "". 10 "° s and achieve on the equilateral of all usable excitation light sources, the highest average power, the product of excitation intensity.I and photon utilization factor K is extremely large. It is therefore an extremely high sensitivity with high time resolution to expect.

The pulse sequence sequence of the mode-locked continuous laser leads to difficulties on the detection side, which have hitherto prevented the widespread use of photon counting in connection with these lasers. "

Known realizations of the measuring method have the following!

1. Due to the limited count rate of the detection electronics (typical values are 10 ^ s "), the registered luminescence photons can be completely detected only in extremely weakly luminescent samples · At higher light intensities, the photon utilization factor K decreases and the measurement time is unnecessarily prolonged.

2 · At low light intensities, which still allow a factor Ϊ? Ώ1, where the sensitivity of the method is fully effective, the unavoidable darkness of the SEV already disturbs. These lead to a shift of the baseline of the measurement result and thus to evaluation problems. The achievable sensitivities are further limited by scattered excitation light.

3 · Ss only the representation of less than one excitation period is possible. Luminescences with a longer cooldown can thus be poorly examined, as well as those with extremely short cooldowns.

4 · The page resolution is affected by inaccurate synchronization of the measurement with the laser pulse train. Due to the high repetition rate of the Laserispuise the energy of a pulse is relatively low, despite the high average light output, and a z. B. obtained with a photodiode synchronization signal has a certain noise component, which leads to a temporal jitter of the synchronization »D & au error come from unavoidable Amplitudanschwankungen the Laaerimpülse.

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5. For some lasers, stable mode-locking is difficult to achieve. PaIIs the mode synchronization fails briefly, ac that no clean pulse train more, it comes to significant measurement errors. For the measurement of extremely short luminescence decay times, the principle of photon counting must be abandoned and the phase fluoro ethers used. "However, the phase inversion using ice phase fluorescence to determine cooldowns in the ps range is also included in the single photon counting result without limitation (assuming equal excitation conditions). , e3 must be able to obtain all measurement results accessible via phase fluorometry, also by single-photon counting za. Such an extension of the field of application of single photon counting would have the advantage of standardizing the measuring methods and thus devices of short-range spectroscopy.

Object of the invention

The object of the invention is to have an arrangement available for measuring luminescence decay functions or characteristic parameters thereof according to the principle of "side-corrected single photon counting with continuous field-synchronized lasers as excitation light source." For extreme applications, sufficient sensitivity and time resolution should be achieved.

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Explanation of the essence of the invention

The invention has for its object to provide at Zeitaüflösten luminescence measurements on the principle of time-correlated single photon counting using mode-locked continuous lasers an arrangement with much greater accuracy, increased signal-to-noise ratio and increased Zeitauflöeung.

For this purpose, an exact synchronization of the measuring process with the Laaerimpulsfolge be guaranteed even with noisy synchronization signal and short-term Amplitudenachwanlcungen the laser pulses to represent more ale an excitation period to increase the maximum count rate of the detection electronics, the influence of the dark pulses of the SEV and the scattered Anreßßlichtes To compensate and provide opportunities for direct signal processing in the measuring system and thus allow the determination of parameters of the decay function with the same time resolution and accuracy as phase fluorometers.

This object is achieved according to the invention by the following measures:

An exact synchronization of the measuring process even with disturbed synchronization signal is achieved by noise immunity by means of a phase locked loop (PLL). The phase locked loop consists of a voltage controlled oscillator, a phase detector and a low pass filter. The phase of the oscillation generated by the oscillator is compared in the iPhasendetektor with the phase of the input signal. The phase detector supplies a control voltage, which re-adjusts the oscillator via the low-pass filter until the phase difference between the input and oscillator signal becomes constant or zero for an integrating component of the low-pass function. As short-term disturbances of

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Low pass be suppressed, the phase jitter of the oscillator oscillation is much smaller than that of the input signal. For a good synchronization of the measuring process is guaranteed even with disturbed EingangB synchronization signal.

A representation of several signal periods is achieved by a frequency division of the synchronization signal. This frequency division can take place either by the PLL itself or by a downstream frequency divider. Between the analog-to-digital converter and memory, an address register is inserted for buffering the address. This ensures that during processing of a registered photon in memory, i. during the reading of the memory location, the + 1 addition and during the subsequent writing already for the next registered photon the A-D conversion can proceed. Since the analog-to-digital conversion and the memory access are the most time-consuming steps of the signal processing, results from this temporally interleaved operation, an increase in the maximum registration rate.

The registration rate is further increased by monitoring the output signal of the time-amplitude converter by a window discriminator with adjustable thresholds. The further processing of a photon is only carried out when the output voltage of the time-amplitude converter is in the desired range between the two thresholds of the discriminator. As a result, only those photons are processed, which are within a certain, predetermined for the measurement tent interval. All other photons do not trigger A-D conversion and memory access, thus saving these time-consuming steps for "uninteresting" photons *

In order to eliminate the dark pulses of the SEV and of the scattered excitation light, the sample to be measured and a reference substance (eg a cuvette which is only

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contains the solvent) irradiated by the excitation light. The luminescent light + scattered light of the sample and the scattered light of the comparison substance are alternately applied to the receiver. The SEV then alternately supplies pulses which are generated by the measuring light + scattered light or by the scattered light. In addition there are dark pulses whose frequency is the same in both cases. An elimination of the interference signal is possible in that the address range of the memory is switched synchronously with the light switching. Measuring and interfering signal thus reach different memory areas and can then be subtracted. Another possibility of the compensation is to switch during memory access between an addition and a subtraction, so that the registrations generated by the interference signal are subtracted directly. If only the dark pulses of the SEV are to be eliminated, the excitation light is interrupted only periodically. The registrations received during the interruption - i. the dark impulses - become after one of the o.g. Subtracting methods, so that subtract the dark pulses on average for the same period of irradiation and interruption of light.

To determine the parameters of extremely fast decay processes, the luminescence signal and the excitation light itself are recorded one after the other and stored in different memory areas. To determine the desired puncture parameters, a microcomputer system included in the measuring system is used. The microprocessor can access directly to the memory of the measuring system via a predetermined address range. This makes it possible to process the measured values according to the programs stored in the ROM.

24020 1

AuafUhrungsbeispiel

The invention will be explained below in an Aueführungsbeispiel. The electronic principle of the measuring system is shown in FIG.

The exciting laser beam L is split into two parts. A part stimulates the luminescence in the measurement object M, with the other part a synchronization signal is generated in the photodiode FD, which is used for the synchronization of the phase locked loop (PLL). The oscillator signal of the phase-locked loop controls a frequency divider, at whose output either the pulse repetition frequency of the laser itself or an integral fraction thereof is available. The selected divider ratio determines the number of detected signal periods. Although a PLL with a separate frequency divider is more complicated than a direct frequency division in the PLL which is possible via the phase comparison of harmonics; But ** has the advantage that the loop filter of the PLL can be dimensioned for optimal JitterunterdrUckung.

The luminescence photons generated in the test object normally arrive with the interposition of an optical system OS - the photomultiplier (SEV), which supplies a usable pulse at the registration of each photon. Since this pulse varies greatly in amplitude, a so-called constant-fraction trigger follows, which provides a defined pulse at the output, which correlates exactly with the time of the photon registration independently of the amplitude of the SEV pulse.

By this impulse the tent amplitude converter (TAC) is started at the registration of a Photoi, which generates a zej proportional increasing voltage until the frequency plate FT arrives at a stop pulse. From this moment on, the output voltage of the time-amplitude converter remains constant. It is thus linear with the temporal position of the

240201 Q.

registered photons linked to the laser pulse train.

The generated voltage is monitored by a window discriminator DIS. This module consists of two comparators with a subsequent logic link and outputs a signal whenever the applied voltage is not between the adjustable switching thresholds of the two comparators. This means that the registered photon is not in the time interval of interest. Therefore, such a photon is not further processed by the ADC disabled and the TAG immediately reset. The measuring system is thus ready to process the next photon.

For photons which are in the required time interval, the analog-to-digital converter (ADC) is started after the stop of the time-amplitude converter. After the AD conversion has ended, the generated binary number is transferred to the address register AR whose output signal in the memory MEM selects a memory location which corresponds to the time of the photon registration. The address is formed from the binary number generated by the AD converter and one or more additional bits, via these bits a changeover of the address range is possible, in which the measurement curve is stored. Thus, the signals from the actual measurement and from the comparison registration and, if necessary, the recording of the excitation light can be stored in parallel. The content of the addressed memory location is read via the memory output O and supplied to an addition / subtraction circuit AS, which simultaneously has memory properties. The memory contents are stored here first, then either a 1 is added or a 1 subtracted in response to an applied control signal S2. The result is fed to the input I of the memory and re-stored. The circuit A / S can

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be very favorably formed by a forward / reverse counter. The counter is first loaded with the memory contents, then counted forward or backward once and thus the addition or subtraction is achieved. If the circuit A / S is switched to addition by S2, then the temporal distribution of the luminescence photons is obtained in the memory, still "contaminated" by stray light components and dark pulses of the SEV.

A suppression of the scattered light and the dark pulses is possible via one of the two control signals S1 or S2 in cooperation with an optiochen system according to Pig «2, the two mirrors Sp3 and Sp4 are mounted on a common shaft and are rotated stepwise by 180 °. This can z. B. happen via a suitably controlled stepper motor SM. The laser beam L thereby alternately hits the cuvette K1, which contains the substance to be measured, and the comparison cuvette K2. The light emanating from the respectively irradiated cuvette passes through the lenses L1 or L2 and the mirrors Sp1 and Sp2 onto the mirror Sp3, which directs the light onto the entrance slit of the monochromator MC, at whose exit the SEV is arranged. (For pur measurements with reduced spectral resolution, the monochromator can also be omitted or replaced by filters.) In this arrangement, if one of the two signals S1 or S2 is switched in synchronism with the rotation of the mirrors Sp3 and Sp4, one obtains measuring and interfering signal either ( by S1) stored in different memory areas or (by S2) directly the difference between the two signals in the same memory area. This contains after completion of the measurement then the interference-free Luraineszenzsignal. The speed of the mirror rotation can be relatively low, since the alternating measurement of useful and interference signal is necessary only at low intensity, ie at a long measuring time. In the case of sufficient light intensity, the mirrors remain firmly aligned with the measuring cuvette.

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The optical system can be simplified if the compensation of the scattered light is dispensed with and only the dark pulses of the SBV are to be suppressed. In this case, a rotating aperture in the excitation beam and e.in synchronous switching of the A / S circuit via S2 is sufficient. In order to determine the parameters of extremely fast decay processes, the excitation signal is also stored after the measurement signal in a special memory area. For this purpose, a special cuvette is used in the measuring system, the storage area switched and otherwise proceed as in the usual measurements.

The measured value memory is assigned a microcomputer with direct accessibility.

To determine extremely short cooldowns, two methods come into consideration:

1. Laser and luminescence light pulse train are each decomposed into a Pourierreihe. In the case of the individual harmonics (in the case of exponential decay, the fundamental wave is sufficient), the phase shifts between the luminescence light and the excitation light are determined. From this the desired parameters of the decay function can be determined. The operations performed by the microcomputer are related to the electronic processes occurring in the phase fluorometer. Consequently, the same accuracies are achieved.

2. A second possibility is to simulate a specific decay function. This function is folded in the computer with the measured laser pulse sequence. The resulting function is compared with the luminescence pulse sequence obtained, the parameters of the simulated function being changed until the deviations are minimal.

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One possibility for integrating a microcomputer in the measuring system is shown in FIG. The computer itself consists of the central processing unit (CPU), a read-write memory (RAM), the read-only memory (ROM) and an input / output unit (I / O) and the associated bus system (control bus SB, address bus AB , Data bus DB).

For coupling to the computer, the address register AR and the addition / subtraction circuit A / S are provided with tri-state outputs, via the inputs DS, the outputs of these circuits can be switched by an applied Η signal in the inactive (tri-state ) State, when an access of the microcomputer system to the measuring memory is desired. The access of the microcomputer to the measuring memory takes place after the memory request signal MREQ has assumed the value L on the microcomputer control bus SB and at the same time the decoder DEC 1 detects that the address lying on the microcomputer address bus AB is allocated to a memory location in the memory MEM belongs to the measuring system. If the memory MEM is currently required by the measuring system at this time, then the computer is put into the WAIT state by the control circuit of the measuring system via the signal WAIT until the current operation is completed.

Subsequently, the control circuit switches the signal MS / MP to H and outputs the read-write signal RD from the microcomputer control bus to the measurement memory. The outputs of the address register AR and the addition-subtraction circuit are thereby disabled and the address lying on the microcomputer address bus is given to the measurement memory MEM by the address buffer TP-A.

Since most microprocessors work with data word widths of 8 bits, but a larger word width (16 or 24 bits) is more favorable in the measuring system, the transmission of the data between the measuring memory and the microcomputer system takes place

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Multiplexer M and a data selector S with tri-state outputs necessary. These circuits are also activated via the signal MS / MP, the signal RD present on the control bus SB ensuring that the data selector S is only switched on during a memory read command. The decoder DEC2 ensures that the 16 or 24-bit word of the measuring memory is divided into 2 or 3 8-bit words, which are accessible from the microcomputer via different addresses.

The microcomputer can thus work with the measurement memory in the manner of the other, the central unit (CPU) permanently assigned read-write memories (RAM) or read-write (ROM). In general, however, you will make the program of the computer so that accesses to the measuring memory are not necessary during the current measurement program, because this would reduce the registration rate of the measuring system.

It should be noted that the address registers AR and in the addition-subtraction circuit expiring functions could in principle be taken over by the microcomputer. With such a measuring arrangement, however, a high registration rate would only be achievable if a very fast microcomputer were used. Such a computer, which would have to contain not only an extremely fast central processing unit (CPU), but also very fast memory, but is not necessary for the actual processing of the measured values. Of course, in order to avoid overlapping of individual puncture sequences at higher registration rates, the operation must of course be monitored by a control circuit ST. The controller ensures that the time-amplitude converter remains locked after registration of a photon until the A / D converter (ADC) has given its result to the address register AR and is ready for the next conversion.

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On the other hand, the transfer of the address to the register AR must be delayed if the memory is still busy processing the last photon. Such a control of the workflow ensures that during a memory access already the A / D conversion for the next photon can be performed.

Claims (8)

24 0 20 1 Invention claim 1. Arrangement for measuring Lumineszenzabklingfunktionen by time-correlated single photon counting with a mode-locked continuous laser as an excitation light source,; comprising an optical system for detecting the luminescent light, a photo secondary electron multiplier, a trigger circuit, a time-amplitude converter, a fast analog-to-digital converter, a measured value memory, a module for synchronizing the measurement process with the laser pulse train and a circuit for controlling the sequence of operations, characterized in that the receiver of the synchronization signal, a phase locked loop for interference suppression of this signal and optionally for frequency division or a phase locked loop and a special frequency divider are arranged downstream and that at the output of the time-amplitude converter, a window discriminator with adjustable thresholds is arranged with the Help a time interval for the photon registration can be specified and that an address register is connected between the analog-to-digital converter and measured value memory and that in the areas for separate storage of measurement, comparison and excitation subdivided measured value memory a circuit for on-line correction of the measured values and optionally a microcomputer for their on-line processing are permanently assigned and contains the optical system between laser and SEV elements for signal type switching and for deriving control signals. 24020 2. Arrangement according to 1, characterized in that the measured value memory is geachaltet in a loop with an addition / subtraction circuit with memory properties, in accordance with a aua the trained for alternately receiving a measuring and a comparison signal optical system, derived control signal at registration a photon performs an increment or decrement of the content of the addressed memory location. 3. arrangement according to 1 and 2, characterized in that the addition-subtraction circuit is a forward / backward counter. 4. Arrangement according to 1, characterized in that the main element of the optical arrangement for alternately receiving a measuring and a comparison signal is a rotating aperture in the excitation beam. 5. Arrangement according to 1, characterized in that the controllably movable optical system for receiving the measuring and comparison signal elements for alternately guiding the excitation beam to the measuring or a comparison sample and for synchronous feeding either the measuring or the comparison signal to the SEV about a mirror pair switched by a stepper motor. 24020 Arrangement according to 1, characterized in that the separate measuring or Comparison signal ranges of the measured value memory is followed by a subtraction circuit « 7 «arrangement according to 1, characterized in that the optionally connected microcomputer takes over the subtraction of the memory area contents. 8. Arrangement according to the first and second, characterized in that the microcomputer is connected via a decoder of known design with the control circuit of the measuring system and this with to be provided for tri-state outputs of address register and addition-subtraction circuit. For this 3 pages drawings