CN113701879B

CN113701879B - Time-dependent single photon counting circuit and system and method thereof

Info

Publication number: CN113701879B
Application number: CN202110994568.XA
Authority: CN
Inventors: 约翰·麦金托什; 托尼·莱纳斯; 德克·纳特
Original assignee: EDINBURGH INSTRUMENTS Ltd; Tianmei Yituo Laboratory Equipment Shanghai Co ltd
Current assignee: EDINBURGH INSTRUMENTS Ltd; Tianmei Yituo Laboratory Equipment Shanghai Co ltd
Priority date: 2021-08-27
Filing date: 2021-08-27
Publication date: 2024-01-30
Anticipated expiration: 2041-08-27
Also published as: CN113701879A

Abstract

The embodiment of the invention relates to a time-dependent single photon counting circuit, a system and a method thereof, comprising a constant ratio phase detector, a time amplitude converter and a voltage amplifier which are sequentially connected, 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. The invention can accurately position the attenuation signal and select the forward/reverse mode operation by adding the signal selector and the delay element without changing the cable length or signal input, thereby improving the easiness of acquiring the measurement data.

Description

Time-dependent 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-dependent 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 of ordinary skill in the art.

Time-dependent single photon counting Technology (TCSPC) is widely used to obtain fluorescence decay kinetics for organic and inorganic samples ranging 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 the inputs is for synchronization and the other receives an electrical pulse 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 excitation 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 individual photons relative to a reference pulse signal derived from a high repetition pulse excitation source. These two pulses trigger a start-stop cycle, resulting in a recorded single time measurement. 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 time of arrival histogram. The histogram is typically in the shape of a single or multiple exponential decay, representing the decay of fluorescence of the sample. The test schematic is shown in fig. 1.

In order to sufficiently match the decay characteristics of the sample, the time scale of the histogram needs to be set. Typical full time ranges are 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 establishing TCSPC experiments is finding conditions that properly display a complete histogram. The position of the histogram is affected by the time the photons pass through the air, through different transparent materials (filters, cuvette walls, lenses) and the electrical pulse through the cable. The latter means that an exact length of cable must be used so that the histogram appears at a specific location on the time axis (fig. 2). This means that cables of different lengths must be used if the time frame (complete time scale) is to be changed. Empirically, photons travel through air at 30 cm/sec and electrons travel through the coaxial cable at 20 cm/sec. In commercial instruments, it is therefore very difficult to adjust by changing the cable length.

The above arrangement is generally referred to as a forward mode of operation, i.e. with an excitation pulse as a start pulse and one photon as a stop pulse detected by the electronics. Yet another mode, which is generally recommended for ultra-fast decay testing, is where the electronics are operated in reverse mode (fig. 3), since dead time effects can be avoided. In the reverse mode, the first photon detected provides a start pulse for the electronic portion 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, a long cable must be used to stop the channel in order to allow a sufficient delay of the synchronisation signal from the excitation source so that it can be reached after the photons emitted by the sample reach the electronic part (figures 4 and 5). The length of this cable may be as long as several tens of meters, the total length requiring accurate calculation in units of 1 cm. In addition, the length of such a cable for delay is also very dependent on the time frame chosen. The situation shown in fig. 6 is actually the best case. Typically, the signal is lost entirely, and "finding" the histogram requires time and experience, and also requires changing cables of different lengths.

Disclosure of Invention

Technical problem

In view of this, 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 using a conventional manner (by changing a cable length or replacing a cable) and improve the easiness of acquiring measurement data, and which employs a single electronic board having an integrated delay function, which can control switching between a forward mode and a reverse mode, and which can freely adjust a delay in both modes.

Solution scheme

In order to solve the technical problems, the embodiment of the invention provides a time-dependent single photon counting circuit, which comprises a constant ratio phase detector, a time-amplitude converter and a voltage amplifier which are sequentially connected, 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.

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

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

Further, the long-delay element is an element that time-shifts the histogram in a sub-microsecond or microsecond time range.

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

Further, the short-time delay element may be formed by connecting a plurality of independent LC delay packets in series.

Further, the short-time delay element is provided with a plurality of connection points for adjusting different delay time, and the connection points are used for selecting and connecting corresponding connection points according to the input delay value to carry out short-time delay.

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

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

Further, the circuit also comprises an amplifier compensation connected with the voltage amplifier and used for fine tuning the delay time through variable offset.

Further, the time-frame 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 test system is provided, including the time-dependent 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 incoming pulse signal and sets its time position according to the slope, so that the return pulse has a standard height and shape;

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

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

Further, the method further comprises the following steps: 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 frame.

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

Further, the short delay is a short delay within 1ns to 300ns, controlled by a plurality of individual LC delay packets connected in series, and a corresponding delay value is selected by being connected to a specific point.

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

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 delay element without changing the cable length or signal input, thereby improving the easiness of acquiring the measurement data.

(2) The invention also applies a variable compensation to the ADC to achieve fine tuning of the delay values, as previously described, the ADC values obtained for each individual TAC define a time channel on the histogram whose corresponding count value is incremented. Applying an offset to the ADC will change the time channel in which the count value increases. The resolution of the fine delay adjustment is better than 0.1% of the time range, which is sufficient to allow the histogram decay to be adjusted to the optimal position. The combination of fixed delay and variable offset achieves a precise and fine adjustable delay over a large time range, covering all the requirements of histogram shifting, applicable to both forward and reverse modes.

(3) The invention is used for obtaining TCSPC decay kinetics histogram, the time-dependent single photon counting circuit can be a single electronic board with integrated variable delay function, the electronic board can control the switching of the forward mode and the reverse mode, and the delay can be freely regulated in both modes. The traditional mode of adjusting through cable length and replacement is avoided, so that the technology can be applied to commercial instruments.

(4) The delay function of the present invention controls the time shift, which allows the histogram to be time shifted in small and medium time ranges (picoseconds and low nanoseconds) during "forward mode" operation. In the reverse mode, the histogram may be time-shifted over a long time range (sub-microsecond or microsecond time range). The integrated delay function is adopted, so that the setting of TCSPC experiments is facilitated, and manual adjustment when different TCSPC time ranges are selected is avoided.

(5) The invention combines discrete passive propagation delay, LC delay and quasi-continuous variable bias delay to realize large-scale, quasi-continuous and adjustable delay. The dashed arrow represents a parameter that can be adjusted by software.

The foregoing description is only an overview of the present invention, and it is to be understood that it is intended to provide a more clear understanding of the technical means of the present invention and to enable the technical means to be carried out in accordance with the contents of the specification, while at the same time providing a more complete understanding of the above and other objects, features and advantages of the present invention, and one or more preferred embodiments thereof are set forth below, together with the detailed description given below, along with the accompanying drawings.

Drawings

One or more embodiments are illustrated by way of example and not limitation in the figures of the accompanying drawings, in which like references indicate similar elements, and in which the figures of the drawings are not to be taken in a limiting sense, unless otherwise indicated.

FIG. 1 is a general schematic diagram of TCSPC forward mode fluorescence decay histogram acquisition;

FIG. 2 illustrates 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 early arrival of a photon pulse: decreasing the length of cable a or increasing the length of cable B; b represents a suitable cable length; c represents that the photon pulse arrives too late: increasing the length of cable a or decreasing the length of cable length B;

FIG. 3 is a general schematic diagram of TCSPC inversion mode obtaining fluorescence decay histograms;

FIG. 4 is a graph illustrating forward and reverse modes with three photon counts, which can form a histogram of arrival times after accumulating millions of photon counts;

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

FIG. 6 illustrates the effect of three different lengths of cable A and cable B on the histogram in the reverse mode in the prior art; wherein a represents that the photon pulse arrives too early, either increasing the length of cable A or decreasing the length of cable length B; b represents a suitable cable length; c represents that the photon pulse arrives too late, either shortening the length of cable a or increasing the length of cable B;

FIG. 7 is a prior art TCSPC electronic schematic circuit diagram; CFD-constant ratio phase detector; a TAC-time-frame converter; AMP-voltage amplifier; an ADC-analog-to-digital converter; MEM-memory. The working flow 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 period between the start signal and the stop signal is converted into a voltage by a time-to-amplitude converter (TAC), a voltage ramp is started by a start pulse and stopped by a stop pulse. Therefore, the later the stop pulse arrives, the greater 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 into 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. The process is repeated millions of times during the measurement, creating an attenuation histogram;

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

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

FIG. 10 is a diagram of an exemplary test of the present invention in reverse mode; wherein: a represents the measurement shift due to TR (in the stop channel) going from 50.0ns to 20.0 ns; b represents the TR value of 50.0ns, the histogram is correctly displayed; c represents the measurement shift due to TR from 50.0ns to 72.0 ns.

Detailed Description

The following detailed description of embodiments of the invention is, therefore, to be taken in conjunction with the accompanying drawings, and it is to be understood that the scope of the invention is not limited to the specific embodiments.

Throughout the specification and claims, unless explicitly stated otherwise, the term "comprise" or variations thereof such as "comprises" or "comprising", etc. will be understood to include the stated element or component without excluding other elements or other components.

Spatially relative terms, such as "below," "beneath," "lower," "above," "upper," and the like, may be used herein for ease of description to describe one element's or feature's relationship to another element's or feature's in the figures. It will be understood that the spatially relative terms are intended to encompass different orientations of the article in use or operation in addition to the orientation depicted in the figures. For example, if the article in the figures is 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" may encompass both a direction of below and a direction of above. The article may have other orientations (rotated 90 degrees or other orientations) and the spatially relative descriptors used herein interpreted accordingly.

The terms "first," "second," and the like herein are used for distinguishing between two different elements or regions and are not intended to limit a particular position or relative relationship. In other words, in some embodiments, the terms "first," "second," etc. may also be interchanged with one another.

The invention provides an embodiment of a time-dependent single photon counting circuit, which comprises a constant ratio phase detector CFD, a time-amplitude converter TAC and a voltage amplifier AMP which are sequentially connected, wherein a first signal selector is arranged between the constant ratio phase detector CFD and the time-amplitude converter TAC, and a start circuit channel and a stop circuit channel are arranged between the first signal selector and the time-amplitude converter TAC; 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.

In use of the present invention, the start or stop channel may simply be assigned to the signal source or detector by the first signal selector as shown in fig. 8 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 assigns pulse 1 as a start pulse and pulse 2 as a stop pulse; 2) In the reverse mode, the first signal selector assigns pulse 1 to the stop pulse and pulse 2 to the start pulse.

Further, a second signal selector connected with the delay element is also arranged on the stop circuit channel; the second selector is used for selecting to switch to use short-time delay or 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" applied to the stop pulse, depending on the delay value entered by the user in the software.

Further, the short-time delay element may be formed by serially connecting a plurality of independent LC delay packets, for example, 11 independent LC delay packets.

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.

An example of a short delay may be: the short time delay element is capable of controlling a short delay within about 0ns-300ns (alternatively 1ns-300 ns), and can be controlled by 11 individual packets of LC delays in series, 11 LC delays being 1,2.5,10, 30,60 ns, respectively. By connecting to the 11 specific points described above, the available delay values are respectively: 0 (i.e., no connection delay), 1,2,4.5,14.5, 24.5, 54.5, 84.5, 114.5,174.5,234.5,294.5ns.

An embodiment of a long delay may be: the long delay element can control a long delay in the range of 0.8 mus to 8 mus, 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.

Since the short and long delays of the delay element are some specific 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 previously described, the ADC value obtained by each individual TAC defines a time channel on the histogram, whose corresponding count value is incremented. Applying an offset to the ADC will change the time channel in which the count value increases. The resolution of the fine delay adjustment is better than 0.1% of the time range, which is sufficient to allow the histogram decay to be adjusted to the optimal position. The combination of fixed delay values and variable offsets enables accurate and fine adjustable delays over a large time range, covering all the requirements of histogram shifting, applicable to both forward and reverse modes.

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

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

The forward and reverse modes of the first signal selector may be selected based on fluorescence decay lifetime, e.g., the test ultrashort lifetime may be used in reverse mode.

The constant ratio phase detector is a common element of a time-dependent single photon counting circuit, when an electronic part has an input signal, the constant ratio phase detector can evaluate an arriving pulse signal, only a signal with a pulse height higher than a certain threshold value can be accepted for further signal processing, a noise signal with small amplitude can be shielded, and the steepest slope part of the 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 may input an adjustment threshold, the first signal selector may input a selection of forward and reverse modes, the delay element may input a selection of a fixed delay value, the TAC may input a time frame of the selection test, and the variable offset compensation software may input a fine adjustment delay value.

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

The foregoing descriptions of specific exemplary embodiments of the present invention are 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 the specific principles of the invention and its practical application to thereby enable one skilled in the art to make and utilize the invention in various exemplary embodiments and with various modifications as are suited to the particular use contemplated. Any simple modifications, equivalent variations and modifications of the above-described exemplary embodiments should fall within the scope of the present invention.

Claims

1. The time-dependent single photon counting circuit comprises a constant ratio phase detector, a time amplitude converter and a voltage amplifier which are sequentially connected, and is characterized in that 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.

2. The time-dependent single photon counting circuit according to claim 1, wherein the stop circuit channel is further provided with a second signal selector connected with a delay element;

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

3. The time-dependent single photon counting circuit according to claim 2, wherein the short-time delay element is an element that time-shifts the histogram in a picosecond or low nanosecond time range;

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

4. A time-dependent single photon counting circuit according to claim 2 or 3, wherein the short-time delay 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-time delay element is provided with a plurality of connection points for adjusting different delay time, and the connection points are used for selecting and connecting corresponding connection points according to the input delay value to carry out short-time delay;

and/or the long-time delay element is an element capable of adjusting a delay time of 0.8 mu s to 8 mu s;

and/or 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.

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

6. The time-dependent single photon counting circuit according to any one of claims 1 to 5, wherein the time-frame 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 testing system comprising the time-dependent single photon counting circuit of claims 1-6.

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

9. The method of obtaining a TCSPC decay kinetics histogram in accordance with 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 frame.

10. The method of obtaining TCSPC decay kinetics histogram in accordance with claim 9, wherein either the short delay value or the long delay value is selected by a second signal selector;

optionally, the short delay is a short delay within 1ns to 300ns, optionally controlled by a plurality of individual LC delay packets connected in series, the corresponding delay value being selected by connection to a specific point;

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