CN113701879A

CN113701879A - Time-correlated single photon counting circuit and system and method thereof

Info

Publication number: CN113701879A
Application number: CN202110994568.XA
Authority: CN
Inventors: 约翰·麦金托什; 托尼·莱纳斯; 德克·纳特
Original assignee: Tianmei Yituo Laboratory Equipment Shanghai Co ltd; EDINBURGH INSTRUMENTS Ltd
Current assignee: Tianmei Yituo Laboratory Equipment Shanghai Co ltd; EDINBURGH INSTRUMENTS Ltd
Priority date: 2021-08-27
Filing date: 2021-08-27
Publication date: 2021-11-26
Anticipated expiration: 2041-08-27
Also published as: CN113701879B

Abstract

The embodiment of the invention relates to a time-correlated single photon counting circuit, a system and a method thereof, which comprise a constant ratio phase discriminator, a time-amplitude converter and a voltage amplifier which are connected in sequence, and are characterized in that a first signal selector is arranged between the constant ratio phase discriminator and the time-amplitude converter, and a starting circuit channel and a stopping circuit channel are arranged between the first signal selector and the time-amplitude converter; a delay element is arranged on the stop circuit channel; the first signal selector is used for selecting switching between a start pulse and a stop pulse; the delay element is used for adjusting the delay time of the signal. The invention can accurately position the attenuation signal and select the forward/reverse mode operation by adding the signal selector and the time delay element without changing the length of a cable or signal input, thereby improving the simplicity of acquiring the measurement data.

Description

Time-correlated single photon counting circuit and system and method thereof

Technical Field

The invention relates to the field of fluorescence delay testing, in particular to a time-correlated single photon counting circuit, a system and a method thereof.

Background

The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.

Time-dependent single photon counting (TCSPC) is widely used to capture fluorescence decay kinetics of organic and inorganic samples from picoseconds to microseconds. Modern technical solutions come in the form of acquisition modules comprising: a single electronic printed circuit board containing all analog and digital functions, a power supply, a computer interface (USB) and two 50 ohm inputs. One of which is used for synchronization and the other of which receives electrical pulses from a single photon detector, such as a photomultiplier tube.

The sample is excited with a high repetition rate, short pulse light source (typically a laser). The sample responds to repeated pulsed excitations by emitting photons. If the signal remains low, only a single photon will reach the detector. The function of the TCSPC electronics module is to measure the arrival time of a single photon relative to a reference pulse signal derived from a high repetitive pulse excitation source. These two pulses trigger a start-stop cycle, resulting in a single time measurement being recorded. The arrival times of thousands to millions of start-stop cycles were measured in TCSPC experiments. The results of all independent measurements are displayed in the form of a histogram of arrival times. The histogram is typically in the shape of a single or multiple exponential decay, representing the fluorescence decay of the sample. The test schematic is shown in fig. 1.

In order to adequately match the attenuation characteristics of the sample, the time scale of the histogram needs to be set. A typical full time range is ten nanoseconds to ten microseconds. The width of a single channel of the histogram is typically 1/1000 to 1/4000 of the entire time range.

One particular difficulty in setting up TCSPC experiments is finding conditions that properly display a complete histogram. The position of the histogram is influenced by the time the photons travel through the air, through different transparent materials (filters, tube walls, lenses) and the electrical pulse travels through the cable. The latter means that a cable of exact length has to be used so that the histogram appears at a specific location on the time axis (fig. 2). This means that cables of different lengths have to be used if the time range (complete time scale) is to be changed. Empirically, photons travel through air at a velocity of 30 cm/sec and electrons travel through coaxial cable at a velocity of 20 cm/sec. Therefore, it is very difficult to adjust the commercial equipment by changing the length of the cable.

The above arrangement generally refers to a forward mode of operation, i.e. the excitation pulse as the start pulse and the electronic part detecting a photon as the stop pulse. There is also a mode where the electronic part operates in reverse mode (fig. 3), which is generally recommended by tests for ultrafast attenuation, since the effects of dead time can be avoided. In the reverse mode, the detection of the first photon provides a start pulse for the electronic part and an electrical delay pulse from the laser provides a stop pulse. Typically, switching between forward and reverse modes is accomplished by replacing the start and stop signal input cables.

In the reverse mode, the stop channel must use a long cable in order to allow sufficient delay of the synchronization signal from the excitation source to arrive after the photons emitted by the sample reach the electronic part (fig. 4 and 5). The length of this cable can be as long as several tens of meters and the total length needs to be accurately calculated in units of 1 cm. In addition, the length of such a cable for delay is also strongly dependent on the selected time range. The situation shown in fig. 6 is actually the best situation. Often, the signal is lost altogether, and it takes time and experience to "find" the histogram, and also requires replacement of cables of different lengths.

Disclosure of Invention

Technical problem

In view of the above, the present invention provides a time-dependent single photon counting circuit (TCSPC) and a system and method thereof, which can adjust a delay time without a conventional method (by changing a cable length or replacing a cable) to improve the ease of acquiring measurement data, and the TCSPC of the present invention employs a single electronic board with an integrated delay function, which can control the switching between a forward mode and a reverse mode, and can freely adjust a delay in both modes.

Solution scheme

In order to solve the above technical problems, an embodiment of the present invention provides a time-dependent single photon counting circuit, including a constant ratio phase detector, a time-amplitude converter and a voltage amplifier, which are connected in sequence, wherein a first signal selector is arranged between the constant ratio phase detector and the time-amplitude converter, and a start circuit channel and a stop circuit channel are arranged between the first signal selector and the time-amplitude converter; a delay element is arranged on the stop circuit channel; the first signal selector is used for selecting switching between a start pulse and a stop pulse; the delay element is used for adjusting the delay time of the signal.

Furthermore, a second signal selector connected with the time delay element is also arranged on the stop circuit channel; the second selector is used for selecting and switching the short-time delay or the long-time delay according to the input delay value.

Further, the short-delay element is an element that temporally shifts the histogram in the picosecond or low nanosecond time range.

Further, the long delay elements are elements that time shift the histogram in the sub-microsecond or microsecond time range.

Further, the short-time delay element is an element capable of adjusting the delay time of 0ns-300 ns.

Further, the short delay elements may be formed by a plurality of independent LC delay packets connected in series.

Furthermore, the short delay element is provided with a plurality of connection points for adjusting different delay times, and is used for selecting and connecting corresponding connection points according to the input delay value to perform short delay.

Further, the long time delay element is an element capable of adjusting the delay time of 0.8-8 mus.

Further, the long delay element comprises a ramp function generator and a comparator for selecting different points in the ramp to adjust the delay time after the stop pulse triggers the ramp function generator.

Further, the device also comprises an amplifier compensation connected with the voltage amplifier, and the amplifier compensation is used for finely adjusting the delay time through variable deviation.

Further, the time-amplitude converter is a converter capable of selecting a test time range.

Further, the delay element is a delay element capable of selecting a delay value.

In another aspect, a fluorescence delay testing system is provided, which comprises the time-correlated single photon counting circuit.

In yet another aspect, a method of obtaining a TCSPC decay kinetics histogram is provided, comprising:

1) the constant ratio phase discriminator analyzes the transmitted pulse signal and sets the time position according to the slope, so that the return pulse has standard height and shape;

2) the first signal selector selects two input pulse signals to switch into a forward mode or a reverse mode;

3) and inputting a selected delay value into the delay element, adjusting the delay time and establishing a histogram.

Further, still include: applying a variable compensation to the analog-to-digital converter to effect fine tuning of the delay value; optionally, the resolution of the fine tuning is better than 0.1% of the time range.

Further, a short-time delay value or a long-time delay value is selected by the second signal selector.

Further, the short-time delay is a short delay within 1ns to 300ns, controlled by a plurality of individual LC delay packets in series, the respective delay values being selected by being connected to specific points.

Furthermore, the long delay is in the range of 0.8-8 mus and is controlled by a ramp function generator and a comparator; optionally, the stop pulse triggers a ramp function generator, and the comparator selects a particular point in the ramp to generate the stop pulse, thereby providing a longer delay range; optionally, the delay time is adjusted by selecting a different point.

Advantageous effects

(1) The invention can accurately position the attenuation signal and select the forward/reverse mode operation by adding the signal selector and the time delay element without changing the length of a cable or signal input, thereby improving the simplicity of acquiring the measurement data.

(2) The invention also applies a variable compensation to the ADC to achieve fine tuning of the delay value, as previously described, the ADC value obtained for each individual TAC defines the time channel on the histogram, and the corresponding count value is incremented. Applying an offset to the ADC will change the time channel for which the count value is incremented. The resolution of the fine delay adjustment is better than 0.1% of the time range, which is sufficient to allow the histogram attenuation to be adjusted to the optimum position. The combination of fixed delay and variable offset enables accurate and fine tunable delay over a large time range, covers all the requirements of histogram panning, and is applicable to both forward and reverse modes.

(3) The invention is used for obtaining TCSPC decay dynamics histogram, the time-correlated single photon counting circuit can be a single electronic board with integrated variable delay function, the electronic board can control the switch of the forward mode and the reverse mode, and the delay can be freely adjusted in the two modes. The traditional mode of adjusting by cable length and replacement is avoided, so that the technology can be applied to commercialized instruments.

(4) The delay function of the invention controls time shifting, which can shift the histogram in small and medium time ranges (picoseconds and low nanoseconds) during the 'forward mode' operation. In the reverse mode, the histogram can be time-shifted over a long time range (sub-microsecond or microsecond time range). The integrated time delay function is adopted, the TCSPC experiment can be set conveniently, and manual adjustment in selection of different TCSPC time ranges is avoided.

(5) The invention realizes large-range, quasi-continuous and adjustable delay by combining discrete passive propagation delay, LC delay and quasi-continuous variable bias delay. The dashed arrows indicate parameters that can be adjusted by software.

The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical means of the present invention more clearly understood and to make the technical means implementable in accordance with the contents of the description, and to make the above and other objects, technical features, and advantages of the present invention more comprehensible, one or more preferred embodiments are described below in detail with reference to the accompanying drawings.

Drawings

One or more embodiments are illustrated by way of example in the accompanying drawings, which correspond to the figures in which like reference numerals refer to similar elements and which are not to scale unless otherwise specified.

FIG. 1 is a generalized schematic of TCSPC forward mode to obtain fluorescence decay histograms;

FIG. 2 is the effect of three different lengths of cable A and cable B on the histogram in the forward mode of the prior art; where a represents the premature arrival of the photon pulse: decreasing the length of cable a or increasing the length of cable B; b represents that the cable length is appropriate; c represents the photon pulse arriving too late: increasing the length of cable a or decreasing the length of cable length B;

FIG. 3 is a generalized schematic of TCSPC inversion mode to obtain fluorescence decay histograms;

FIG. 4 is a histogram of arrival times that can be formed after accumulating millions of photon counts, using three photon counts to illustrate the forward and reverse modes;

FIG. 5 is the effect of the dead time of TCSPC when operating in forward (up) and reverse (down) modes with high repetition frequency light sources; (dead time may be much longer than the time between excitation pulses shown in the figure, which is schematically indicated for clarity in the schematic).

FIG. 6 prior art effect on the histogram of three different lengths of cable A and cable B in reverse mode; wherein a represents the premature arrival of the photon pulse by increasing the length of cable A or decreasing the length of cable length B; b represents that the cable length is appropriate; c represents the photon pulse arriving too late, shortening the length of cable a or increasing the length of cable B;

FIG. 7 is a prior art TCSPC electronic schematic circuit diagram; a CFD-constant ratio phase discriminator; a TAC-time-amplitude converter; an AMP-voltage amplifier; an ADC-analog-to-digital converter; MEM-memory. The working process is as follows: a constant ratio phase detector (CFD) analyzes the incoming pulse and sets its time position according to the slope, the return pulse having a standard height and shape. The time-amplitude converter (TAC) converts the time period between the start signal and the stop signal into a voltage, starts a voltage ramp by a start pulse, and is stopped by a stop pulse. Therefore, the later the stop pulse arrives, the larger the TAC voltage. The TAC output voltage is connected to an Amplifier (AMP) that appropriately scales the TAC voltage. An analog-to-digital converter (ADC) converts the output of the amplifier to a digital value, which is then transferred to a memory (MEM) corresponding to a particular channel on the histogram time axis, such that the count value at that channel is incremented by 1. This process is repeated millions of times during the measurement, creating a histogram of attenuation;

FIG. 8 is a circuit diagram of the TCSPC electronics portion of the present invention;

FIG. 9 is a diagram of an example of the testing of the present invention in the forward mode; wherein: a represents the histogram shift by a start signal increment of 25 ns; b represents the correct display form of the histogram; c represents the histogram shift resulting from a stop signal increase of 37.0 ns;

FIG. 10 is a diagram of an example of the testing of the present invention in reverse mode; wherein: a represents the measured displacement due to the change in TR (in the stop channel) from 50.0ns to 20.0 ns; b represents the correct display of the histogram using a TR value of 50.0 ns; c represents the measured displacement due to the TR change from 50.0ns to 72.0 ns.

Detailed Description

The following detailed description of the present invention is provided in conjunction with the accompanying drawings, but it should be understood that the scope of the present invention is not limited to the specific embodiments.

Throughout the specification and claims, unless explicitly stated otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or component but not the exclusion of any other element or component.

Spatially relative terms, such as "below," "lower," "upper," "above," "upper," and the like, may be used herein for ease of description to describe one element or feature's relationship to another element or feature in the figures. It will be understood that the spatially relative terms are intended to encompass different orientations of the object in use or operation in addition to the orientation depicted in the figures. For example, if the items in the figures are turned over, elements described as "below" or "beneath" other elements or features would then be oriented "above" the elements or features. Thus, the exemplary term "below" can encompass both an orientation of below and above. The article may be otherwise oriented (rotated 90 degrees or at other orientations) and the spatially relative terms used herein should be interpreted accordingly.

In this document, the terms "first", "second", etc. are used to distinguish two different elements or portions, and are not used to define a particular position or relative relationship. In other words, the terms "first," "second," and the like may also be interchanged with one another in some embodiments.

The invention provides an embodiment of a time-correlated single photon counting circuit, which comprises a constant ratio phase discriminator CFD, a time-amplitude converter TAC and a voltage amplifier AMP which are sequentially connected as shown in figure 8, wherein a first signal selector is arranged between the constant ratio phase discriminator CFD and the time-amplitude converter TAC, and a starting circuit channel and a stopping circuit channel are arranged between the first signal selector and the time-amplitude converter TAC; the stopping circuit channel is provided with a time delay element; a first signal selector for selecting switching between a start pulse and a stop pulse; the delay element is used to adjust the delay time of the signal.

In use, the invention can simply assign a start or stop channel to the signal source or detector as shown in fig. 8 by the first signal selector to select either the forward mode or the reverse mode, for example, assuming pulse 1 from the light source and pulse 2 from the detector: 1) in the forward mode, the first signal selector distributes pulse 1 as a starting pulse and pulse 2 as a stopping pulse; 2) in the reverse mode, the first signal selector assigns pulse 1 as a stop pulse and pulse 2 as a start pulse.

Furthermore, a second signal selector connected with the time delay element is also arranged on the stop circuit channel; the second selector is used for selecting to switch to use the short-time delay or the long-time delay according to the delay value input by the software. The first signal selector or the second signal selector may be a dual multiplexer.

The second signal selector selects either a "short delay" or a "long delay" to be applied to the stop pulse, depending on the delay value entered by the user in the software.

Further, the short delay element may be formed by connecting a plurality of independent LC retardation packets in series, for example, 11 independent LC retardation packets in series.

An example of a short delay may be: the short delay element can control short delay within 0ns-300ns (1 ns-300ns is optional), and can be controlled by 11 independent series LC delay packets, and 11 LC delays are 1,1,2.5,10,10,30,30,30,60,60,60ns respectively. By connecting to the above-mentioned 11 specific points, the available delay values are respectively: 0 (i.e., no delay connected), 1,2,4.5,14.5, 24.5, 54.5, 84.5, 114.5,174.5,234.5,294.5 ns.

An example of a long delay may be: the long delay element can control the long delay in the range of 0.8 mu s to 8 mu s, and is controlled by a ramp function generator and a comparator. The stop pulse triggers a ramp function generator and the comparator selects a particular point in the ramp to generate the stop pulse, thereby providing a longer delay range. The delay time is adjusted by selecting different points. Useful delay values are 0.8, 1,2,4, 8 mus.

The short delay and the long delay due to the delay elements are of a certain value. To achieve fine tuning of the delay value, a variable compensation is applied to the ADC to achieve fine tuning of the delay value (fig. 8). As mentioned before, the ADC value obtained for each individual TAC defines a time channel on the histogram, and its corresponding count value is incremented. Applying an offset to the ADC will change the time channel for which the count value is incremented. The resolution of the fine delay adjustment is better than 0.1% of the time range, which is sufficient to allow the histogram attenuation to be adjusted to the optimum position. The combination of fixed delay values and variable offsets enables accurate and fine tunable delay over a large time range, covers all the requirements of histogram panning, and is applicable in both forward and reverse modes.

Further, the delay element is a delay element that can select a fixed delay value.

Further, a short-term delay value or a long-term delay value is selected by the second selector.

The forward and reverse mode selection of the first signal selector may be based on fluorescence decay lifetime, e.g., the reverse mode may be used for testing ultra-short lifetime.

The constant ratio phase detector is a common element of a time correlation single photon counting circuit, when an electronic part has an input signal, the constant ratio phase detector can evaluate an arriving pulse signal, only the signal with the pulse height higher than a certain threshold value can be received for further signal processing, a small-amplitude noise signal is shielded, and the steepest slope part of an initial edge of the input pulse is generally selected as a time position when the time position is selected.

The CFD of the present invention can input an adjustment threshold, the first signal selector can input a selection of forward and reverse modes, the delay element can input a selection of a fixed delay value, the TAC can input a selection of a time range for testing, and the variable offset compensation software inputs a fine adjustment delay value.

The test results of obtaining histograms by TCSPC of the present invention are shown in fig. 9 and 10. The invention can well select the proper delay time.

The foregoing descriptions of specific exemplary embodiments of the present invention have been presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and its practical application to enable one skilled in the art to make and use various exemplary embodiments of the invention and various alternatives and modifications as are suited to the particular use contemplated. Any simple modifications, equivalent changes and modifications made to the above exemplary embodiments shall fall within the scope of the present invention.

Claims

1. A time correlation single photon counting circuit comprises a constant ratio phase discriminator, a time-amplitude converter and a voltage amplifier which are connected in sequence, and is characterized in that a first signal selector is arranged between the constant ratio phase discriminator and the time-amplitude converter, and a starting circuit channel and a stopping circuit channel are arranged between the first signal selector and the time-amplitude converter; a delay element is arranged on the stop circuit channel;

the first signal selector is used for selecting switching between a start pulse and a stop pulse;

the delay element is used for adjusting the delay time of the signal.

2. The time-correlated single photon counting circuit of claim 1, wherein said stopping circuit path is further provided with a second signal selector connected to a delay element;

the delay element comprises a short delay element and a long delay element which are respectively used for carrying out short delay time and long delay time, and the second signal selector is used for selecting and switching short delay time or long delay time according to an input delay value.

3. The time-correlated single photon counting circuit of claim 2, wherein said short time delay elements are elements that time shift the histogram in picoseconds or in low nanoseconds;

and/or the long delay element is an element that shifts the histogram in time in the sub-microsecond or microsecond time range.

4. The time-correlated single photon counting circuit according to claim 2 or 3, wherein said short delay time element is an element capable of adjusting a delay time of 0ns-300 ns;

and/or the short time delay element is formed by connecting a plurality of independent LC delay packets in series;

and/or the short delay element is provided with a plurality of connection points for adjusting different delay times, and the connection points are selected to be connected with corresponding connection points according to the input delay value to perform short delay;

and/or the long time delay element is an element capable of adjusting the delay time of 0.8-8 mus;

and/or the long-time delay element comprises a ramp function generator and a comparator, and the comparator is used for selecting different points in the ramp to adjust the delay time after the stop pulse triggers the ramp function generator.

5. The time correlated single photon counting circuit of any one of claims 1 to 4 further comprising an amplifier compensation coupled to the voltage amplifier for fine tuning the delay time by a variable offset.

6. The time-correlated single photon counting circuit according to any of the claims 1 to 5, wherein said time-amplitude converter is a converter capable of selecting a test time range;

and/or the delay element is a delay element capable of selecting a delay value.

7. A fluorescence delay test system comprising the time-correlated single photon counting circuit of claims 1 to 6.

8. A method of obtaining a histogram of TCSPC decay kinetics, comprising:

9. The method for obtaining a histogram of TCSPC decay kinetics of claim 8 further comprising: applying a variable compensation to the analog-to-digital converter to effect fine tuning of the delay value; optionally, the resolution of the fine tuning is better than 0.1% of the time range.

10. The method of deriving a histogram of TCSPC decay kinetics of claim 9 wherein the short time delay value or the long time delay value is selected by a second signal selector;

optionally, the short-time delay is a short delay within 1ns-300ns, optionally controlled by a plurality of individual LC delay packets in series, the respective delay value being selected by connection to a particular point;

optionally, the long delay is a long delay in the range of 0.8 μ s to 8 μ s, and is controlled by a ramp function generator and a comparator; optionally, the stop pulse triggers a ramp function generator, and the comparator selects a particular point in the ramp to generate the stop pulse, thereby providing a longer delay range; optionally, the delay time is adjusted by selecting a different point.