JPH0797080B2 - Spectrofluorometer - Google Patents

Description

TECHNICAL FIELD The present invention relates to a spectrofluorometer used in fields such as bioscience and medicine.

(Prior Art) The time course of the reaction is followed using a spectrofluorometer. For example, in order to quantitatively measure intracellular calcium ion, the chelating agent FURA II is added, but in this case, it is necessary to change the excitation wavelength as the reaction between the calcium ion and the chelating agent progresses. is there. Therefore, a method of alternately exciting the sample with two excitation wavelengths is adopted.

FIG. 8 shows a spectrofluorimeter in which the excitation wavelength is changed.

Reference numeral 2 is a light source, and the excitation light of the light source 2 is dispersed into a sample 4
Two excitation spectroscopes 6-1 and 6-2 are provided for irradiating the laser beam. The excitation spectroscope 6-1 is set so that the wavelength of the pumping light extracted is EX1.
2 is set so that the wavelength of the pumping light extracted is EX2.

Reference numeral 8 denotes a chopper, which has a wavelength EX1 from the excitation spectroscope 6-1.
And the excitation light of wavelength EX2 from the excitation spectroscope 6-2 are switched to irradiate the sample 4 alternately.

Reference numeral 10 is a fluorescence spectroscope, which disperses the fluorescence generated by the excitation of the sample 4 and makes it enter the detector 12. The output wavelength of the fluorescence spectroscope 10 is set to EM. As the detector 12, for example, a photomultiplier tube is used.

(Problems to be Solved by the Invention) In the spectrofluorometer shown in FIG.
Two excitation spectroscopes are used to extract one excitation light. Therefore, there is a problem that the cost becomes high.

The present invention uses a conventional spectrofluorometer equipped with one excitation spectroscope and one fluorescence spectroscope, and at least one of the excitation wavelength and the fluorescence wavelength is switched over at a plurality of wavelengths at fixed time intervals for reaction. An object of the present invention is to provide a spectrofluorometer capable of tracking changes over time and reducing costs.

(Means for Solving Problems) FIG. 1 shows the configuration of the present invention.

2 is an excitation light source, 6 is an excitation spectroscope that disperses the light from the excitation light source 2 and irradiates the sample 4, and 10 is a fluorescence spectroscope that disperses the fluorescence emitted from the excited sample 4 and sends it to the detector 12. is there. The above optical system is an optical system generally provided in the conventional spectrofluorimeter.

Reference numeral 14 denotes a spectroscope control unit, which includes an excitation spectroscope 6 and a fluorescence spectroscope 10.
In correspondence with each other or the excitation spectroscope 6 or the fluorescence spectroscope 10
Either one of the above is repeatedly switched between a plurality of wavelengths at regular time intervals to switch to each wavelength. A data processing unit 16 takes in the detection output of the detector 12 under each switched wavelength condition, calculates the ratio of the detection output under different wavelength conditions, and outputs the data in real time.

(Embodiment) FIG. 2 shows a system configuration diagram of an embodiment.

20 is an amplifier for amplifying the signal from the photomultiplier tube 12 for photometry, 22 is an output signal of the amplifier 20 is converted to a digital signal
It is an A / D converter. The operation of the A / D converter 22 is stopped by a signal from the CPU 38 during the wavelength shift of the excitation spectroscope 6 and the fluorescence spectroscope 10.

24 is A / depending on the output wavelength of the excitation spectroscope 6 and the fluorescence spectroscope 10.
It is a switch for switching the output of the D converter 22 to the latch 26a or the latch 26b for input. The control of the switching operation of the changeover switch 24 is also performed by the signal of the CPU 38.

The states of the latches 26a and 26b are sent to the response circuits 28a and 28b, which are digital filters, respectively.
Time constants are multiplied by a and 28b, and output ports 30a and 30b
Is output to. Ratters 26a, 26b and response circuits 28a, 2
The operation of 8b is controlled by the signal of the CPU 38.

Reference numerals 32a and 32b are memories for inputting signals from the output ports 30a and 30b, respectively, and storing them as time change data (file A, file B). Reference numeral 34 is a division circuit for performing a division operation between data stored in the memories 32 and 32b, and 36 is a CRT for displaying the division result. CRT36 also has memory 32
It is also possible to display the stored data of a and 32b.

42 is a wavelength motor of the excitation spectroscope 6, and 44 is a wavelength motor of the fluorescence spectroscope 10. The wavelength motors 42 and 44 are also controlled by the CPU 38.

All control by the CPU 38 is performed through the I / O port 40.

The spectroscope control unit 14 in FIG. 1 is realized by the CPU 38, and the data processing unit 16 is realized by the CPU 38, the changeover switch 24, the latches 26a and 26b, the response circuits 28a and 28b, the memories 32a and 32b, and the division circuit 34. It

FIG. 3 shows an example of wavelength conditions relating to the operation of this embodiment.

The excitation spectroscope 6 is switched between the excitation wavelengths EX1 and EX2, and scanning is performed so that the set wavelength of the fluorescence spectroscope 10 is switched between EM1 and EM2 in association with the respective excitation wavelengths EX1 and EX2. That is, the excitation wavelength and the fluorescence wavelength are made different, and the excitation spectroscope 6 changes from the A state to the B state and from the B state to the A state.
And the fluorescence spectroscope 10 are simultaneously scanned and switched alternately.

It takes time to switch from one state to the other because the excitation spectroscope 6 and the fluorescence spectroscope 10 are scanned. Both spectroscopes 6 and 10 are simultaneously scanned in order to reduce the transition time. Therefore, the transition time is determined on the side of the spectroscope having a wavelength separation.

In the case of time scanning recording, the time needs to be accurate, and data of a fixed time interval is collected. Therefore, it is necessary that both the transition time from the A state to the B state and the transition time from the B state to the A state are shorter than the data collection time.

FIG. 4 shows an example of wavelength switching between the A state and the B state and data collection. t is the wavelength switching time of the spectroscopes 6 and 10, Ta is the data acquisition time on the A state side, and Tb is the data acquisition time on the B state side.

Next, the operation of this embodiment will be described.

While the wavelengths of the spectroscopes 6 and 10 are moving, an A / D converter is generated by the signal from the CPU 38
22 operation is stopped. Excitation spectrometer 6 and fluorescence spectrometer
When the state of 10 becomes the state A, the signal from the photomultiplier tube 12 is amplified by the amplifier 20 and input to the A / D converter 22, converted into a digital signal, and the digital signal output is stored in the latch 26a. It The state of the latch 26a is sent to the response circuit 28a,
It is multiplied by a time constant, output from the output port 30a, and stored in the memory 32a.

After a certain period of time, the spectroscope shifts to the B state, and similarly, the signal from the photomultiplier tube 12 is stored in the latch 26b via the amplifier 20, the A / D converter 22, and the response circuit 28b and the output port 30b are set. After that, it is stored in the memory 32b.

Then, the time-varying data stored in the memories 32a and 32b is processed by the dividing circuit 34, and the result is displayed on the CRT 36.

The method of using this embodiment is not limited to the conditions shown in FIG. For example, as shown in FIG.
Alternatively, the fluorescence wavelength may be fixed by EM1 and EM2, or conversely, the fluorescence wavelength may be fixed by EM and the excitation wavelength may be changed by EX1 and EM2 as shown in FIG. .

In Fig. 7, the generated fluorescence spectra are T1 and T depending on time.
It shows the case where it changes like 2, T3. In the case of such a sample, if the wavelength c and the wavelength d are alternately monitored and the output is set to c / d, the data is collected at any fixed wavelength. Almost 2
Time change can be measured with double sensitivity.

In the embodiment, at least one of the excitation wavelength and the fluorescence wavelength is switched in two states to track the time change, but at least one of the excitation wavelength and the fluorescence wavelength is sequentially switched under three or more wavelength conditions, and each state is changed. You may make it measure the time change of the detection output of.

When the ratio of the photometric output is taken as in the embodiment, even if there is a change in the concentration of the measurement object (for example, particle breakage or sedimentation), it is not affected by them. For example, the output of I ₁ in the wavelength lambda ₁ is becomes a kI ₁ by the concentration change, the intensity I ₂ is also of course be kI ₂ at a wavelength lambda _2. However, when the specific output kI ₁ / kI ₂ is calculated, this value becomes I ₁ / I ₂ and is not affected by the change in concentration. For example, when measuring cells, the cells will settle, so the memory 32a,
The time-varying data stored in 32b changes even with the sedimentation of cells. However, the effect of cell sedimentation can be removed by dividing those time-varying data.

(Effect of the Invention) In the present invention, in an apparatus provided with one excitation spectroscope and one fluorescence spectroscope, at least one of the excitation spectroscope and the fluorescence spectroscope is switched at regular time intervals, and in a plurality of states. Since the photometric output is taken in, the excitation spectroscope is
The cost is reduced as compared with a device provided with one piece.

Then, by measuring under a plurality of wavelength conditions and tracking the change over time, it is possible to measure the change over time of the sample in which the spectrum shifts more efficiently than in the fixed wavelength method. Further, for example, since the fluorescence at two wavelengths can be monitored, the states before and after the chemical change can be correctly determined.

[Brief description of drawings]

FIG. 1 is a block diagram showing the present invention, FIG. 2 is a block diagram showing an embodiment, FIG. 3 is a diagram showing one use condition of the embodiment, and FIG. 4 is a timing showing an operation of the embodiment. Figure, fifth
FIG. 6 and FIG. 6 are diagrams showing other conditions of use, FIG. 7 is a diagram showing an example of time change of fluorescence, and FIG. 8 is a block diagram showing a conventional spectrofluorimeter. 2 ... Light source, 4 ... Sample, 6 ... Excitation spectrometer, 10 ... Fluorescence spectrometer, 12 ... Detector, 14 ... Spectrometer control unit, 16 ... Data processing unit.

Claims (1)

[Claims] 1. An excitation spectroscope for separating light from an excitation light source to irradiate a sample, and one fluorescence spectroscope for separating fluorescence emitted from the excited sample and sending it to a detection section. In a spectrofluorometer, a spectrometer that switches the excitation spectroscope and the fluorescence spectroscope in correspondence with each other, or repeats either the excitation spectroscope or the fluorescence spectroscope at a constant time interval among a plurality of wavelengths. The present invention is characterized by including a control unit and a data processing unit that takes in detection output under each switched wavelength condition, calculates a ratio of detection output under different wavelength conditions, and performs data processing for outputting in real time. Spectrofluorometer.