JPS624659B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an apparatus capable of measuring the luminescence lifetime of a sample, that is, the temporal change in its luminescence intensity.

In general, knowing the luminescence lifetime of a sample is an important element in knowing the physical or chemical properties of this sample.

By the way, when the light emission is strong, the output of the photoreceiver (mainly a photomultiplier tube) can be directly observed with a wide-band monitor, but the photoelectron output from the photomultiplier tube due to light emission is In the case of measuring relatively weak luminescence where only one or more photoelectrons are generated, the generation of photoelectrons at that time is discontinuous and does not directly correspond to the true luminescence intensity. The probability of photoelectron generation only corresponds to the intensity of light.

In such a case, light emission is repeated many times △t ₁ , △t ₂
When the number of photoelectrons generated in the time range is integrated for each time range, the obtained histogram shows the temporal change in luminescence intensity, that is, the luminescence lifetime.

Also, when the time range of △t is relatively long (0.1~
(1 microsecond or more), it can be directly integrated by a circuit using a digital IC (for example, a multi-channel scaler (MCS)).

Furthermore, by using an ultra-high-speed operation type IC, it is possible to shorten Δt to about 2 nanoseconds.

However, in this method, the resolution of the shortest time Δt is usually limited to 100 nanoseconds or more, and even in special cases, the resolution is limited to 2 nanoseconds due to limitations in the operating speed of the digital IC.

Time to voltage conversion method (TAC: time to amp)
A litude converter) converts the time from the reference time of light emission to the generation of photoelectrons into an analog voltage, which is then digitally converted to time, and can be used up to the picosecond range, and can be used to convert light emission in the nanosecond to microsecond range. It is one of the effective means of measuring life.

The measurement principle is as follows. In other words, the capacitor is charged with a constant current by the start pulse of the standard time of light emission, and the charging is stopped by the stop pulse of the photoelectron pulse. Furthermore, if this voltage is used as an input signal to a multichannel pulse height analyzer, a histogram of photoelectron generation frequency with voltage on the horizontal axis can be obtained. At this time, since voltage=time, this histogram represents the temporal change in luminescence intensity, that is, the luminescence lifetime.

Therefore, it has been proposed to use photoelectron counting means using the above-mentioned time-voltage conversion method as a means of measuring the luminescence lifetime (on the order of nanoseconds to microseconds) of fluorescence, phosphorescence, etc. of a sample. There is.

However, in this type of conventional means, only one set of time-voltage conversion circuits is provided.
As a result, the photoelectrons used for measurement are only the first ones generated from the reference time of light emission, and the generation of the second and subsequent photoelectrons gives an error in the measurement.To avoid this, photoelectrons are Considering that the occurrence of is a stochastic process, for example, 10
The light incident on the photomultiplier tube must be made small enough to generate on average one photoelectron per light emission.
As a result, the measurement time is over 10 hours when a pulsed laser with a repetition rate of 10 to 100 Hz is used as the light source.

In other words, in such conventional means, the number of photoelectron pulses that can be used in one light emission is limited to a maximum of one, so the efficiency of using photoelectron pulses is poor. There is a problem in that it takes a very long time to measure the luminescence lifetime.

The present invention aims to solve these problems, and by expanding the number of photoelectron pulses that can be used in one light emission to a plurality of photoelectron pulses and increasing the utilization efficiency of photoelectron pulses, it is possible to measure the luminescence lifetime of a sample. An object of the present invention is to provide an apparatus for measuring the luminescence lifetime of a sample that can shorten the time.

Therefore, the sample luminescence lifetime measurement device of the present invention includes a pulsed light emission source and a first photoelectron intensifier that generates a plurality of (n) photoelectron pulses upon receiving light generated from the sample that has received the pulsed light. It is equipped with a multiplier tube and a start switch that is closed when the pulsed light is emitted through a second photomultiplier tube that receives the pulsed light, and when the start switch is closed, it is charged by receiving current from the power source. First to nth condensers are arranged in parallel to start the photoelectron pulses, and sequentially receive the plurality of (n) photoelectron pulses from the first photomultiplier tube to start the photoelectron pulses from the first photomultiplier tube.
first to n stop switches are provided to sequentially open charging circuits for the n capacitors, and
The first to n photomultiplier tubes sequentially receive the plurality of (n) photoelectron pulses from the first photomultiplier tube and sequentially close the control circuits from the first photomultiplier tube to the second to n stop switches. It is characterized in that (n-1) preset switches are connected in series.

Hereinafter, an apparatus for measuring the luminescence lifetime of a sample as an embodiment of the present invention will be explained with reference to the drawings.
The figure is the overall configuration diagram, Figure 2 is the principle diagram, and Figure 3 is the diagram.
Figures a to j are time charts showing the waveforms of various parts in Figure 2, and Figures 4 and 5 are graphs showing the measurement results.

As shown in FIGS. 1 and 2, a pulse laser 1 is provided as a light source capable of emitting pulsed light P (see FIG. 3a).

Further, first and second photomultiplier tubes 2 and 3 are provided, and the first photomultiplier tube 2 receives the light L generated from the sample 4 that receives one pulse of light P [Fig. b], for example, 8 photoelectron pulses.
The second photomultiplier tube 3 receives the pulsed light P from the pulsed laser 1 and produces one corresponding pulse PP'.

In addition, in reality, as shown in Fig. 1, sample 4
The light generated from the is supplied to the first photomultiplier tube 2 via the spectrometer 5, and
The signals from the photomultiplier tubes 2 and 3 are shaped into waveforms by pulse shaping circuits 6 and 7, respectively, and then output.

Furthermore, a multi-channel time-voltage converter 8 receiving photoelectron pulses PP, PP' is provided,
This multi-channel time-voltage converter 8 has a second
As shown in the figure, power supply 9, start switch 1
0. 1st to 8th capacitors as charging means
C ₁ , C ₂ , C ₃ , ..., C ₈ , first to eighth stop switches S ₁ , S ₂ , S ₃ , ..., S ₈ and first to seventh preset switches PS ₁ , PS ₂ , It is composed of PS ₃ ,...etc.

The start switch 10 is normally open, and closes when it receives the photoelectron pulse PP' output from the second photomultiplier tube 3 when the pulsed light P is emitted from the pulsed laser 1, and is reset thereafter. It is configured to maintain the closed state unless it is applied.

In addition, the first to eighth capacitors C ₁ , C ₂ , C ₃ ,
..., _C8 are each connected in parallel to the power supply 9 via the start switch 10, so that when the start switch 10 is closed, the resistor paired with each capacitor receives the current from the power supply 9.
Charging is started with characteristics based on a time constant determined by the relationship with R ₁ , R ₂ , R ₃ , ..., R ₈ .

Furthermore, the first to eighth stop switches S ₁ , S ₂ ,
S ₃ , ..., S ₈ are each start switch 10
These stop switches are connected in series with each other and in parallel with each other, and specifically, these stop switches are interposed in the circuit parts (charging circuits of each capacitor) that branch from the start switch 10 to each capacitor. It is normally closed, opens when it receives a signal, and remains open unless reset.

That is, each stop switch S ₁ to S ₈ is closed when its excitation coil S _1EX to S _8EX is demagnetized, and opens when the excitation coil S _1EX to _{S 8EX} is excited, and then each excitation coil S _1EX to _{S 8EX} is demagnetized. The excitation coils S _1EX to S _8EX are connected in parallel to the first photomultiplier tube 2 via control circuit parts CC ₁ to CC ₈ . There is.

The second to eighth control circuit portions _CC2 to _CC8 are provided with first to seventh preset switches, respectively.
PS ₁ , PS ₂ , PS ₃ , . . . are interposed, so that the preset switches are connected in series.

Also, each of these preset switches PS ₁ ,
PS ₂ , PS ₃ , ... are the 1st to 7th stop switches
It is provided so as to be interlocked with _S1 to _S7 .

That is, the first preset switch _PS1 is
The stop switch S ₁ closes at the same time as it opens, and the second
The preset switches No. 7 to 7 are also configured to close simultaneously when the stop switches No. 2 to No. 7 open, respectively.

In addition, in FIG. 2, the symbols RS ₁ , RS ₂ , RS ₃ ,...
..., RS ₈ indicate reset switches for discharging the electrical energy charged in the capacitors C ₁ to _{C 8} , respectively.

Here, the process from the time the pulsed light P is emitted until the output is output from the multi-channel time-voltage conversion device 8 will be briefly described.

First, a target sample 4 that emits fluorescent light is irradiated with a pulsed laser 1 that emits a flash of light for a short time. At this time, the lifetime of the luminescence of the sample 4 after the pulsed light P is quenched is useful for determining the properties of the material, but the fluorescence is received by the first photomultiplier tube 2 and the luminescence intensity is relatively low. As an output, a photoelectron current appears in the form of discontinuous pulses, as shown in FIG. 3c.

On the other hand, the direct light from the pulsed laser 1 is received by the second photomultiplier tube 3, and its output PP' (see FIG. 3d) is used as a start pulse. When the start switch 10 is closed by this start pulse PP', the capacitors C ₁ , C ₂ , C ₃ , . . . , C ₈ start charging, and the voltage at each terminal increases.

That is, the pulsed light P from the pulsed laser 1
By irradiating the sample 4 with 8 photoelectron pulses PP are generated from the first photomultiplier tube 2 in time series, and the pulsed light P is received by the second photomultiplier tube 3 to start. By closing the switch 10, the first to eighth capacitors C ₁ to
Charging by C ₈ is started at the same time as the pulsed light P is emitted.

Then, the first of the eight photoelectron pulses PP
When the th photoelectron pulse PP ₁ is generated, the excitation coil S _1EX is excited and the first stop switch S ₁
At the same time that PS1 opens, the first preset switch _PS1 closes.

This stops charging by the first capacitor _C1 . In this case, the waveform diagram of the current I ₁ flowing through the resistor R ₁ from the start to the stop of charging is as shown in Figure 3e, and the waveform diagram of the terminal voltage V ₁ of the first capacitor C ₁ is as shown in Figure 3f. become.

Next, when the second photoelectronic pulse PP ₂ occurs, since the first preset switch PS _{1 is already closed, the excitation coil S 2EX of the second stop switch S 2 is} energized and the second stop switch S ₂ is activated.
opens, and at the same time the second preset switch PS ₂ closes.

This stops charging by the second capacitor _C2 . In this case, the waveform diagram of the current I ₂ flowing through the resistor R ₂ from the start to the stop of charging is as shown in Figure 3g, and the terminal voltage V ₂ of the second capacitor C ₂
The waveform diagram of is shown in Fig. 3h.

Similarly, the third to eighth photoelectron pulses are
As PP ₃ ,... occurs, the 3rd to 8th stop switches are activated by the action of the preset switch.
Since S ₃ , . . . , S ₈ are closed one after another, charging by the third to eighth capacitors C ₃ , . . . , C ₈ is stopped one after another. In this case as well, the current flowing through the resistances R ₃ to _{R 8} from the start of charging to the stop is I ₃ to
Waveform diagram of I ₈ and 3rd to 8th capacitors C ₃ to _{C 8}
The waveform diagrams of the terminal voltages V ₃ to _{V 8} change depending on the length of the charging time. Resist an example
R ₃ and capacitor C ₃ are shown in Figure 3 i and j.

Here, each terminal voltage V ₁ to V ₈ is determined by the charging of each capacitor with a constant current, and each terminal voltage is determined by each charging time, in other words, the first to 8th voltage after the start switch 10 is closed. is proportional to the time it takes for the stop switches S ₁ to S ₈ to open.

As a result, the first to eighth photoelectron pulses PP ₁ , PP ₂ , PP ₃ , . . . after the laser pulse 1 is emitted are emitted.
each time until the capacitor
The terminal voltages of C ₁ to C ₈ are converted into terminal voltages V ₁ to _{V 8} and output.

Thereafter, these terminal voltages V ₁ to V ₈ are sequentially switched by a switching switch CS such as a multiplexer and used as input signals of a multi-channel pulse height analyzer.

As shown in Figure 1, this multi-channel pulse height analyzer uses an analog-to-digital converter (hereinafter referred to as "A-D").
called a converter. ) 11 and a microcomputer 12, this multi-channel wave height analyzer sequentially converts the eight sets of voltages into analog-
Digitally converted and microcomputer 12
Additive storage is performed at .

After measuring 8 sets of voltages in this way, operate the reset switches RS ₁ to _{RS 8} to reset each capacitor.
Discharge the charge stored in C ₁ to C ₈ to prepare for the next measurement.

At this time, the start switch 10, stop switches _S1 to _S8 , and preset switch _PS1 ,
PS ₂ , ... are also reset at the same time. That is, the start switch 10 is returned to the open state, each stop switch is returned to the closed state, and each preset switch is returned to the open state.

Note that the reference numeral 13 in FIG. 1 indicates an XY plotter.

Furthermore, in reality, all of the operations of the above-mentioned switches are performed by IC circuits due to the necessity of high-speed switching.

With the above configuration, in order to measure the luminescence lifetime of the sample 4, first the pulsed light P from the pulsed laser 1 is
is input to the second photomultiplier tube 3, and the output PP' is shaped by the pulse shaping circuit 7 and used as a start pulse.

On the other hand, fluorescence emitted from the sample 4 by receiving the remainder of the pulsed light P passes through the spectrometer 5 and enters the first photomultiplier tube 2 .

The photoelectronic output generated by this light is pulsed by the pulse shaping circuit 6, and at this time, the preset switch of the multi-channel time-voltage converter 8 is activated.
Adjacent photoelectron pulses PP ₁ , PP ₂ , . . . are completely separated by the action of PR ₁ , PR ₂ , . . . , and each pulse PP ₁ , PP ₂ , . . . acts as a stop pulse.

These start pulses and stop pulses cause the multichannel time-voltage converter 8 to operate as described above.

Eight sets of voltages from the multi-channel time-voltage converter 8 are sequentially switched by the switch CS, digitized by the AD converter 11, and added and stored by the microcomputer 12.
At this time, as described above, the A-D converter 11 and the microcomputer 12 operate as a multichannel pulse height analyzer.

After that, the histogram, which is added and stored by repeating the emission many times, represents the temporal change in the emission intensity.
This is the basic data for luminescence life.

This data is then processed through the necessary calculations.
Plotted with XY plotter 13, etc.

FIG. 4 is a graph showing temporal changes in luminescence intensity obtained with this device, where the horizontal axis represents time and the vertical axis represents the number of photoelectrons generated in each time range.

In FIG. 5, the vertical axis is changed to a logarithm.

Note that hematoporphyllin was used as Sample 4, and approximately 40,000 photoelectrons were required to create this histogram.

At this time, the pulse laser 1 was operated at about 20 Hz, and the measurement time was about 40 minutes.

If this is done using the conventional method, it will take about 3 hours.

From these measurement results, it was found that the measurement time could be shortened, and it was also found that the measurement light amount could be easily adjusted.

In addition, as in the above-mentioned embodiment, in addition to providing 8 sets of capacitors and stop switches and 7 sets of preset switches, other arbitrary sets of capacitors and stop switches are provided, and the number of preset switches is one less than the above number. It is also possible to provide only a set of , and the circuit in this case can be easily constructed according to the above embodiment.

Also, instead of interlocking the stop switch and preset switch, it is possible to operate them separately, but even in this case, the first to
When the (n-1) stop switch is opened, the first to (n-1) preset switches are opened correspondingly.

As described in detail above, according to the sample luminescence lifetime measuring device of the present invention, the following effects and advantages can be obtained.

(1) Multiple photoelectron pulses generated from the first photomultiplier tube in response to the light emitted from the sample 4 can be used in one measurement, increasing the efficiency of photoelectron pulse utilization and significantly reducing measurement time. Can be shortened.

(2) Since the measurement time can be significantly shortened in this way, it is also possible to measure the luminescence lifetime of biological cell samples, which are difficult to maintain in a constant state for a long time.

(3) It is relatively easy to increase the number of photoelectron pulses generated from the first photomultiplier tube in response to the light emitted from the sample by one light emission, and as a result, the amount of measured light can be reduced. Adjustment becomes easier.

[Brief explanation of the drawing]

The figures show an apparatus for measuring the luminescence lifetime of a sample as an embodiment of the present invention. Figure 1 is its overall configuration diagram, Figure 2 is its principle diagram, and Figures 3 a to j are each part of Figure 2. 4 and 5 are graphs showing the measurement results. 1...Pulsed laser as a light source, 2,3
... photomultiplier tube, 4 ... sample, 5 ... spectrometer,
6, 7...Pulse shaping circuit, 8...Multi-channel time-voltage converter, 9...Power source, 10...Start switch, 11...A-D converter, 12...
_{Microcomputer , 13 ... _ _ _ _} CC ₁ ~ CC ₈
...Control circuit part to stop switch, CS...
...Changing switch, PS ₁ , PS ₂ , PS ₃ ... Preset switch, RS ₁ to _{RS 8} ... Reset switch.

Claims (1)

[Scope of Claims] 1. A light emitting source of pulsed light, a first photomultiplier tube that receives light generated from a sample receiving the pulsed light and generates a plurality (n) of photoelectron pulses, and the pulsed light and a start switch that is closed when the same pulsed light is emitted through a second photomultiplier tube that receives the pulsed light. are provided with first to nth condensers, which sequentially receive the plurality of (n) photoelectron pulses from the first photomultiplier tube to
first to n stop switches are provided to sequentially open charging circuits for the n capacitors, and
The first to n photomultiplier tubes sequentially receive the plurality of (n) photoelectron pulses from the first photomultiplier tube and sequentially close the control circuits from the first photomultiplier tube to the second to n stop switches. An apparatus for measuring the luminescence lifetime of a sample, characterized in that (n-1) preset switches are connected in series. 2. Luminescence lifetime measurement of a sample according to claim 1, wherein the first to (n-1) preset switches are provided in such a manner that the first to (n-1) preset switches are respectively linked to the first to (n-1) stop switches. Device.