JP2694200B2 - Multi-channel fluorescence decay waveform measuring device - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial field of application] The present invention relates to an excitation light pulse applied to a sample,
The photons of fluorescence emitted from the sample are converted into photoelectron pulses, the converted photoelectron pulses are supplied to a shift register that is shifted by a clock pulse, and a photoelectron pulse train having information corresponding to the time of occurrence of the photons is generated. The present invention relates to a multi-channel fluorescence decay waveform measuring device that measures a fluorescence decay waveform by creating a pattern in the shift register, reading the pattern, and performing statistical integration processing.

[Prior Art] As a fluorescence decay waveform measuring method, a single photon delay matching method (hereinafter abbreviated as TAC method) is widely used from the viewpoint of sensitivity and decomposition time when the fluorescence intensity is weak. Although the TAC method can achieve a decomposition time of a few picoseconds, the signal utilization rate is extremely low due to its measurement principle. In order to obtain a signal without waveform distortion, only one photon can be generated at most for one excitation of the sample, and normally, the amount of light is such that one photon is generated on average for several tens of excitations. I have to Therefore, the undesirable means of dimming must be used for some "bright" samples.

In contrast to the TAC method, the photoelectron pulse train simultaneous detection method aims to improve the signal utilization rate even if the decomposition time is sacrificed to some extent. In the photoelectron pulse train simultaneous detection method,
When multiple photons are generated for one excitation,
Measure them all. The principle is shown in FIG.
(A) is the excitation light pulse to the sample, and (B) is the fluorescence decay waveform when the light intensity from the sample is very strong. When the amount of light becomes weak, the output from the photomultiplier that photoelectrically converts this becomes a photoelectron pulse corresponding to each photon, and the probability density function of the pulse generation time was proportional to the intensity of (b). It becomes a thing. The photoelectron pulse generation state after each excitation is different every time, and this is shown in (c), (d), (e), and (f). Therefore, as shown in (G) (each time-divided region corresponds to a counter, and ○ indicates one photon), a counter corresponding to the photon generation time is prepared, and the photoelectron pulse 1 If only 1 is added to the content of the corresponding counter for each generation, the waveforms similar to the waveform of (B) are finally obtained by performing integration by multiple excitations. The decomposition time in this case is Δ shown in (g).
t.

The simplest multi-channel fluorescence decay waveform measuring device based on this principle is shown in FIG. In this multi-channel fluorescence decay waveform measuring device, the pulse train from the photomultiplier tube 10 after the sample excitation by one excitation light pulse is amplified and
It is supplied to the N-bit shift register 14 which can operate at high speed through the discriminator 12, and the clock pulse generator 16
Create a bit pattern on the shift register 14 that is shifted by 1 bit per Δt by the clock pulse from
After Δt, the control pulse is received from the timing controller 18, the contents of the shift register 14 are read into the buffer register 20, and the bit data is counted by the counter 22 provided corresponding to each bit of the buffer register 20.
The bit pattern that occurs each time the sample is excited
To be added. The decomposition time in this case is the period Δt of the clock pulse from the clock pulse generator 16.

The multi-channel fluorescence decay waveform measuring apparatus of this configuration is ideal from the viewpoint of integration processing of bit patterns, but requires many counters, and when trying to read the contents of the counters for data processing, It requires many multiplexers and complicated wiring.

The present inventor has devised and manufactured a multi-channel fluorescence decay waveform measuring apparatus shown in FIG. 5 in which the decomposition time is doubled and the configuration is simplified. Two-stage shift register 14A, 14B
Is output from the clock pulse generator 16 ', the phase is
The bit patterns of the photoelectron pulses are created alternately on the shift registers 14A and 14B by alternately shifting every Δt with clock pulses different by 180 degrees. On the other hand, under the control of the timing controller 18 ', the contents of the RAMs 24A, 24B having the addresses corresponding to the respective bits of the shift registers 14A, 14B for each .DELTA.t are countered by the counters 28A, 28B via the buffer registers 26A, 26B. The data is sequentially read out, and the bit data serially taken out from the most significant bits of the shift registers 14A and 14B are added and stored at the address. The histogram of the fluorescence decay waveform can be created by such a series of time-series processing.

The performance of the manufactured device is a decomposition time of 2.5 nsec, a measurable time span of 100 nsec, and a repetition frequency of 25 KHZ.

The present inventor has devised and produced another multi-channel fluorescence decay waveform measuring device that realizes the photoelectron pulse train synchronous detection method, using a vernier chronotron.

FIG. 6 shows a vernier chronotron, and two coaxial cables 30A and 30B having propagation delay times τ and τ + Δτ,
Two refresh amplifiers 32A and 32B for waveform shaping
And a coincidence circuit that detects the coincidence of two pulses
It consists of 34 and a scaler 36 that counts the number of pulse turns. The principle is as follows. When two pulses A and B having a time difference t are input as shown in the figure, each pulse circulates in each path with a period of τ + Δτ and τ. Here, since the pulse B has a cycle shorter than the pulse A by Δτ, the pulse B relatively approaches the pulse A by Δτ for each circulation. Therefore, the pulse B catches up with the pulse A at the t / Δτth time, and a match is detected. On the other hand, the number of rounds of the pulse B is counted by the scaler 36, and this counting is stopped when a match is detected. Since Δτ is known, the number of turns n
, The first pulse interval t is t = n
Calculated by Δτ. This vernier chronotron has extremely stable differential linearity, and the decomposition time is determined by Δτ which is proportional to the difference in cable length.

Vernier chronotron can only measure the time difference between two pulses. In order to make it multi-channel,
The configuration shown in FIG. 7 devised by the inventor may be used. If pulse A and pulse train B are input as shown in the figure, A
Indicates a pulse synchronized with the excitation light pulse to the sample, and B corresponds to the photoelectron pulse train from the photomultiplier tube. A shift register 14 is prepared in place of the scaler 36 in FIG. 6, and the shift operation of the shift register 14 is performed by the round pulse counted by the scaler 36, and the coincidence detection pulse from the coincidence circuit 34 is sent to the shift register 14. Assume serial data input pulse. In this way, the pulse train B is the coaxial cable 30
While circulating B, this pulse train B is converted into a bit pattern on the shift register 14.

The processing of the bit pattern on the shift register 14 that occurs each time the sample is excited can be performed by using the circuit shown in FIG. 4 or FIG. 5, and the present inventor manufactured the one using the circuit shown in FIG. did.

The dynamic range of the multi-channel fluorescence decay waveform measuring device described above depends on the amplifier / discriminator.
Limited to 12 pulse-pair resolutions. On the other hand, the decomposition time is
In the case of the shift register system, it is determined by the clock frequency to the shift register, and in the case of Vernier Chronotron, it is determined by the cable length difference.

Therefore, if the clock frequency is raised in the former case and the cable length difference is shortened in the latter case, the disassembly time should be improved to the extent of the electrical system jitter.

However, in reality, since the photoelectron pulse from the photomultiplier tube 10 has a width of about 1.5 to 3.0 nsec and one pulse is counted over a plurality of channels,
It was generally considered that the lower limit of the decomposition time was about the output pulse width of the photomultiplier tube 10.

Therefore, the present inventor has devised a multi-channel fluorescence decay waveform measuring device that solves this problem (2, 1988).
Patent application dated March 29).

This will be described with reference to FIG. The same components as those in FIG. 5 are designated by the same reference numerals and the description thereof will be omitted.

After setting the switch 38 to the clock pulse generator 16 side, the controller 18 supplies the excitation pulse to the excitation light source (not shown) and irradiates the sample with the excitation light pulse. This excitation light pulse is detected by an optical sensor (not shown), and the clock pulse generator 16 starts generating a clock pulse in synchronization with this detection signal. The controller 18 counts this clock pulse. The clock pulse is also supplied to the shift register 14 as a shift pulse, and the clock pulse period Δt ₁
Each time, the shift register 14 is shifted by 1 bit to the right in FIG. Fluorescent photons emitted from the sample are converted into photoelectron pulses by the photomultiplier tube 10 and supplied to the shift register 14 through the amplifier / discriminator 12. Therefore, when the time NΔt ₁ elapses after the sample is excited,
A pattern of a photoelectron pulse train indicating the fluorescence lifetime is created in the shift register 14.

9A shows a photoelectron pulse train output from the amplifier / discriminator 12, and FIG. 9B shows a bit pattern created in the shift register 14 by this pulse train. In the figure, the shaded area is a bit corresponding to the photoelectron pulse, "1" is stored, and "0" is stored in the other areas.

When the controller 18 counts N clock pulses after outputting the sample excitation pulse, the switch 38 switches the switch 38 to the frequency divider 40 side, and shifts the clock pulse from the clock pulse generator 16 divided by the frequency divider 40. The pulse is supplied to the shift register 14. That is, the serial data is read relatively slowly from the shift register 14 in relation to the subsequent data processing speed.

This serial data is as shown in FIG. 9 (B), but when it passes through the effective data extraction circuit 42, as shown in (E), the first 1 bit of the continuous plural bits corresponding to the photoelectron pulse is shown. The data is extracted as valid data.

That is, the pulse shown in (C) is the on-delay circuit 46.
After passing through, as shown in (D), a waveform whose rising edge is delayed by a time equal to one cycle Δt ₂ of the output pulse of the frequency divider 40 is formed, which is then inverted by the inverter 48, and the AND gate 44
Is supplied to the AND gate 44 and the logical product of the pulse from the shift register 14 is output from the AND gate 44, as shown in (E). The on-delay circuit 46 can use an integrating circuit including a resistor R and a capacitor C as shown in the figure.

On the other hand, every time one pulse is output from the frequency divider 40, the following series of processing is performed.

That is, the data D _A of the address A of the RAM 24 is supplied to the counter 28 via the buffer register 26, and the bit data output from the AND gate 44 is counted by the counter 28 (1 is set only when the bit data is “1”). Added)
Then, it is stored in the original address A of the RAM 24 via the buffer register 26. Then, the address of the RAM 24 is incremented.

When such a series of processing is repeated N times,
The process of writing data to the RAM 24 is temporarily stopped, the switch 38 is switched to the clock pulse generator 16 side, and the photoelectron pulse train pattern for the shift register 14 is created.

[Problems to be Solved by the Invention] However, if the excitation light pulse is increased in order to shorten the measurement time, as shown in FIG. 10 (A), a plurality of photoelectron pulses output from the amplifier discriminator 12 are overloaded. The frequency of appearance in the wrapped state increases, and a pattern in which a large number of bits are continuous is formed in the shift register 14 as shown in FIG.

In this case, in the device shown in FIG. 8, since only the first bit is extracted from 42 as the effective bit as shown in FIG. 10 (C), only the first photoelectron pulse P ₁ in time is counted, The subsequent photoelectron pulse P ₂ is not counted. Therefore, the range of the incident light quantity in which the average count of photons is proportional to the light quantity of the incident fluorescence, that is, the dynamic range is determined by the amplifier / discriminator 12
It was limited by the pulse-pair resolution of and could not be expanded further.

In view of the above problems, an object of the present invention is to provide a multi-channel fluorescence decay waveform measuring device capable of improving the decomposition time to be equal to or shorter than the photoelectron pulse width and expanding the dynamic range with respect to the amount of incident light.

[Means for Solving the Problem] In order to achieve this object, in the multi-channel fluorescence decay waveform measuring apparatus according to the present invention, the fluorescence emitted from the sample in response to the excitation light pulse applied to the sample The photon is converted into a photoelectron pulse, and the converted photoelectron pulse is supplied to a shift register which is shifted by a clock pulse to create a pattern of a photoelectron pulse train having information corresponding to the time of occurrence of the photon in the shift register. In a multi-channel fluorescence decay waveform measuring device for measuring a fluorescence decay waveform by reading out the pattern and performing a statistical integration process, a continuous multi-bit data corresponding to a photoelectron pulse stored in the shift register Of these, a valid data extraction method that extracts bit data at regular intervals as valid bit data It is characterized by the provision of steps.

Hereinafter, an embodiment of the present invention will be described with reference to the drawings.

FIG. 1 shows a block circuit of the multi-channel fluorescence decay waveform measuring apparatus. The same components as those in FIG. 8 are designated by the same reference numerals and the description thereof will be omitted.

After the sample is excited in the state shown by the solid line of the switch 38, the time NΔt ₁
After the lapse of time, the pattern of the photoelectron pulse train showing the fluorescence lifetime is formed in the shift register 14 as in the above. When the photoelectron pulses P ₁ and P ₂ as shown in FIG. 2A are obtained from the amplifier discriminator 12, the pulse train shown in FIG. 2B is formed in the shift register 14.

After the elapse of this time NΔt ₁ , the switch 38 is switched to the state shown by the dotted line by the command from the controller 18, and the serial data is read from the shift register 14 at a relatively slow speed.

This serial data is supplied to one input terminal of the AND gate 50. The other input terminal of the AND gate 50 is always at a high level, as described later, and the serial data is input to the monostable multivibrator (hereinafter referred to as monomulti) 52 through the AND gate 50 and is triggered. . The non-stable (ON) time of the monomulti 52 is selected to be equal to one cycle Δt ₂ of the output pulse of the frequency divider 40. Therefore, the Q output of the monomulti 52 is turned on for a time corresponding to the first bit data regarding the photoelectron pulse P ₁ , as shown in FIG. 2 (C), and its output is supplied to the counter 28.

On the other hand, this output is output to the
Supplied to 56. Monomulti 56 is triggered by the falling edge (trailing edge) of the output pulse of monomulti 52. The output of the mono-multi 56 constantly outputs "1" as shown in (D), and the output becomes "0" by the trigger. This non-stable (off) time Δt ₃ is Δt ₃ = nΔt ₂ where n is the number of consecutive bits corresponding to one photoelectron pulse.
Selected for −Δt ₂ . This off time Δ of Mono Multi 56
At t ₃ , since the AND gate 50 is closed, the data from the shift register 14 is not supplied to the monomulti 52.

After the on time Δt ₃ described above, the output of the monomulti 56 turns on and the AND gate 50 is opened.
The bit data from is supplied to the monomulti 52. This bit data relates to the photoelectron pulse P ₂ shown in (A). This causes the monomulti 52 to be re-triggered, turned on for the time Δt _{2 as} described above, and its output supplied to the counter 28. The falling edge of the output pulse of mono-multi 52 causes mono-multi 56 to be re-triggered, and AND gate 50 is now closed for a time Δt ₃ and then re-opened.

These AND gate 50, Mono-multi 52, Inverter 5
4. The data extraction circuit 58 is composed of the mono-multi 56.

It should be noted that after the binary data is input from the monomulti 52 to the counter 28, the operation is similar to that described with reference to FIG.

By the above operation, by irradiating the sample with a relatively strong excitation light pulse, two overlapping photoelectron pulses P ₁ and P ₂ are input to the shift register 14, and a continuous “1” pattern is generated in the shift register 14. Even if formed, each photoelectron pulse P ₁ and P ₂ can be accurately counted.

The degree of overlap in which the pulses P ₁ and P ₂ can be distinguished is such that the peak-to-peak distance between both pulses is approximately Δt ₂ or more. Therefore, even if n = 3, most of the overlaps can be separated and identified, and the dynamic range can be greatly expanded.

Further, the device of this embodiment also has the function of the device shown in FIG. 8, and the decomposition time is improved to the width of one photoelectron pulse or less.

Furthermore, the apparatus of this embodiment can count each pulse even when three or more photoelectron pulses are overlapped and input. However, in order to obtain an accurate count value, it is preferable to adjust the intensity of the excitation light so that the maximum number is three.

(2) Expansion Note that the present invention can of course be applied to the various multi-channel fluorescence decay waveform measuring devices described in FIGS. 4, 5, and 7.

Further, depending on the software configuration of the microcomputer, it is possible to extract, as valid bit data, the bit data at constant intervals from the continuous multiple bit data stored in the shift register 14 and corresponding to the photoelectron pulse.

[Effect of the Invention] In the multi-channel fluorescence decay waveform measuring apparatus according to the present invention, among the continuous multiple bit data stored in the shift register and corresponding to the photoelectron pulse, the bit data at regular intervals is converted into valid bit data. Since it is possible to improve the decomposition time to the photoelectron pulse width or less, it is possible to significantly widen the range of the incident light amount in which the average count of photons is proportional to the amount of incident fluorescence light, that is, the dynamic range. It has an excellent effect.

[Brief description of the drawings]

1 is a block circuit diagram showing the configuration of a multi-channel fluorescence decay waveform measuring apparatus according to the embodiment of the present invention, and FIG. 2 is an operation explanatory diagram of the data extraction circuit 58 shown in FIG. 3 to 7 relate to a conventional example, FIG. 3 is an explanatory view of the principle of the photoelectron pulse train simultaneous detection method, and FIG. 4 is a block circuit diagram of a first multi-channel fluorescence decay waveform measuring apparatus which realizes this principle. FIG. 5 is a block circuit diagram of the second multi-channel fluorescence decay waveform measuring device, FIG. 6 is a principle configuration diagram of the Bernian chronotron, and FIG. 7 is a third multi-channel using the circuit of FIG. It is a circuit diagram of a channel fluorescence decay waveform measuring device. FIG. 8 is a block circuit diagram showing the configuration of the previously proposed fluorescence decay waveform measuring apparatus, FIG. 9 is an operation explanatory diagram of the effective data extraction circuit 42 shown in FIG. 8, and FIG.
It is explanatory drawing in the case where the photoelectron pulse shown to (A) is superposed and obtained. 10: Photomultiplier tube 12: Amplifier / Discriminator 14: Shift register 16: Clock pulse generator 18: Controller 22: Counter 24: RAM 30: Coaxial cable 32: Refresh amplifier 34: Coincidence circuit 36: Scaler 42, 42A , 58: Effective data extraction circuit 46: On-delay circuit 50: AND gate 52, 56: Monostable multivibrator

Claims (1)

(57) [Claims] 1. In response to an excitation light pulse applied to a sample,
The fluorescence photons emitted from the sample are converted into photoelectron pulses, and the converted photoelectron pulses are supplied to a shift register (14) that is shifted by a clock pulse, and the information has the information corresponding to the photon generation time. A multi-channel fluorescence decay waveform measuring apparatus for measuring a fluorescence decay waveform by creating a pattern of a photoelectron pulse train in the shift register (14) and reading the pattern to perform statistical integration processing, the shift register (14) A multi-channel characterized by being provided with an effective data extracting means (58) for extracting bit data at constant intervals among the continuous plural bit data corresponding to photoelectron pulses stored in Fluorescence decay waveform measuring device.