JP2013072727A - Spectrofluorometer - Google Patents

Abstract

PROBLEM TO BE SOLVED: To appropriately make a selection according to a sample concentration from an analog method suitable for high concentration sample analysis and a photon counting method suitable for low concentration sample analysis.SOLUTION: Signals from a spectrometer part 109 of a sample pass through low concentration and high concentration electric system control parts 107 and 108 corresponding to a low concentration and a high concentration, and measurement processing of the photon counting method and the analog method is performed respectively in an operation/control part 106. First, from measurement data obtained using a standard sample of a known concentration, a conversion equation for conversion from the analog method to the photon counting method, a calibration curve connected from the data of both methods, and a measurement limit value of the photon counting method are obtained. Then, in the case of an unknown sample, an appropriate electric system control part is discriminated in the operation/control part 106 from the signals from the spectrometer part 109 and the measurement limit value, and the signals are inputted to the low concentration electric system control part 107 in the case of the low concentration and the high concentration electric system control part 108 in the case of the high concentration.

Description

  The present invention relates to a spectrofluorometer used in the field of measuring samples in a wide concentration range.

  When a substance in the ground energy state absorbs light, it transitions to an excited state that is a high energy state. A substance that has transitioned to an excited state loses a part of its energy as vibration or thermal energy, and then returns to the original ground state with light radiation. At this time, the light emission is fluorescent or phosphorescent depending on the difference in the lifetime of the light emission. Called. Fluorescence or phosphorescence generated during the energy transition process is measured by an analyzer called a spectrofluorometer.

  As a detector mounted on this analyzer, a photomultiplier tube, a silicon photodiode, or an avalanche photodiode having a self-amplification system is used. However, when selecting a detector to be used, sufficient detection sensitivity is required for measurement. Or having sensitivity in the required measurement wavelength range is determined as a main factor. However, in a general-purpose spectrofluorometer, a photomultiplier tube having high measurement sensitivity in the ultraviolet region or visible region is generally used.

  Fluorescence or phosphorescence incident on the photocathode of the photomultiplier tube is gradually and repeatedly multiplied by secondary electron emission by a plurality of dynodes laid out in the tube, and then output as charge pulses from the anode. The signal is processed and recorded as fluorescence intensity.

  There are two methods for handling charge pulses: an analog method in which individual pulses are closely superimposed as an analog current signal, and a photon counting method in which charge pulses are treated as discrete pulses.

  The analog method is a method in which the output from the photomultiplier tube is further converted into a digital signal after the current-voltage conversion, and the charge pulse from the anode is superimposed with a number of pulse signals of a certain size as shown in FIG. It handles analog current signals with fluctuations.

  On the other hand, in the photon counting method, the number of pulses output from the anode is waveform-shaped and then counted by a counter circuit, and a signal level is output based on the number of counts obtained at a specific time.

  The photon counting method is advantageous in that it can measure extremely weak light with high accuracy. On the other hand, when the light entering the detector becomes large, the discrete charge pulses are superimposed on each other, so that all the incident light is not converted into discrete pulses. Measurement is impossible.

  For example, FIG. 4 shows a state in which the light incident on the detector is weak. In this situation, since all the incident light is converted into discrete pulses, it is possible to accurately measure even very weak light. However, as shown in FIG. 5, in the situation where very large light is incident, the appearing pulse intervals gradually start to closely approach each other, and if they overlap, the individual charge pulses are not accurately counted and measurement is performed with high accuracy. It becomes impossible.

  Further, when the incident light increases, the signal is completely saturated, and the pulse signal is counted as zero (0).

  In the photon counting method, the charge pulse output from the detector is amplified by an amplifier, and then processed through a discriminating circuit for discriminating non-fluorescent or phosphorescent signals such as electrical noise components or cosmic rays. Therefore, it is possible to measure with good S / N by extracting only the signal component of fluorescence or phosphorescence. Therefore, the photon counting method is more effective in measuring weaker light than the analog method, and the analog method is effective in the case of strong fluorescence intensity in which individual pulses are superimposed.

  In the field of fluorescence measurement using a spectrofluorometer, there are a wide variety of samples to be measured or the purpose of measurement, and as a result, an apparatus having a wide dynamic range for measuring samples having a wide range of concentrations is desired. For example, the patent introduced in Patent Document 1 relates to a technique for realizing a high speed, high sensitivity, and a wide dynamic range in a fluorescent image or a DNA inspection method.

JP 2002-55050 A

  For example, in the case of measuring fluorescence of a solution sample having very weak fluorescence, a spectrofluorophotometer using a photon counting method can be measured with high accuracy. However, when the measurement for obtaining the quantum efficiency of the sample is considered as a function evaluation for a film-like or thin solid sample or a powder-like sample, as shown in FIG. It is necessary to measure the fluorescence intensity of phosphor or phosphorescence while changing the wavelength.

  However, in the case of a solid or powder sample, the scattering property of excitation light on the sample surface tends to increase. In this case, in the measurement by the photon counting method, it is impossible to measure by making all the light amounts of the excitation light into discrete pulses, and the target measurement may not be performed accurately.

  Therefore, it is difficult to supplement all measurement purposes in the field of fluorescence measurement only with the photon counting method. Alternatively, when the measurement is performed by the photon counting method, there is a possibility that the measurement may be performed without the operator noticing the situation where the discrete pulse cannot be formed. Therefore, in order to solve these problems, it is necessary to select and process an analog method or a photon counting method in accordance with the magnitude of light to be detected.

  In order to solve the above problems, the present invention is based on a spectrofluorometer composed of a combination of a photon counting method suitable for a low concentration and an analog method suitable for a high concentration, and data obtained by both methods. The calibration curve created by the first to sixth steps shown below is used.

  That is, it has two types of electric system control units, an analog method suitable for high concentration and a photon counting method suitable for low concentration, and the two types of signals according to the output signal from the spectrometer. A spectrofluorometer having a wide dynamic range is realized by using a calibration curve provided with a signal switching mechanism for sending a signal to one of the electric system control units. Hereinafter, a method for creating a calibration curve according to the present invention will be described in the first to sixth steps.

  In the first step, a standard curve is used to obtain a calibration curve by both the photon counting method and the analog method. The second step is that the operator can extract two sets of data in the same concentration range from the measurement data of both methods, and the calibration curves of both methods satisfy the linearity with a contribution ratio of 0.999 or more. Check and judge. That is, the continuation is interrupted when the two conditions are satisfied, and is suspended when the two conditions are not satisfied.

  The third step receives the determination of continuation from the operator in the second step, the measured value and concentration value of the photon counting method exceeding the common concentration range selected by the operator, the measurement limit value below, Remember. In the fourth step, a linear simultaneous equation is solved from the two sets of data selected in the second step, and a coefficient for converting the measured value obtained by the analog method into the value of the photon counting method is obtained.

  The fifth step converts the measurement value obtained by the analog method equal to or higher than the measurement limit value of the photon counting method obtained in the third step, using the coefficient obtained in the fourth step. To do. In the sixth step, for the concentration below the measurement limit value obtained in the third step, the measurement value by the photon counting method is used, and for the concentration exceeding the measurement limit value, the value converted in the fifth step. Create a calibration curve using.

  At the time of sample measurement, it was judged that the measurement limit value obtained in the third step was exceeded, and if not exceeded, the signal from the spectroscope was sent to the electric system controller by the photon counting method and exceeded In this case, the switching mechanism sends the signal to an electric system controller using an analog method. The data measured by the electric system control unit is processed by the calibration curve created in the second to sixth steps, which will be described in detail below, so that a dynamic concentration that cannot be measured only by the photon counting method can be measured. A spectrofluorometer with a wide range can be realized.

  When applied to the measurement for obtaining the quantum efficiency of a solid or powder sample with the above configuration, as shown in FIG. 7, the fluorescence or phosphorescence region was measured by the photon counting method, and the excitation wavelength region was measured by the analog method. Since the data is converted into a count value scale by the photon counting method, it is possible to handle the area calculation of the excitation light portion and the fluorescence or phosphorescence portion on the same scale.

  A spectrofluorophotometer having a wide measurement dynamic range can be realized, a wide concentration range can be accurately measured, and a quantum efficiency can also be accurately measured.

It is a schematic block diagram of the spectrofluorometer by the Example of this invention. 2 is an outline of an analysis work flow of a spectrofluorometer according to an embodiment of the present invention. It is an output state figure with large incident light of the conventional spectrofluorimeter. It is an output pulse figure by the weak incident light of the conventional spectrofluorimeter. It is an output pulse figure by the big incident light of the conventional spectrofluorimeter. It is a measurement image figure of the quantum efficiency of the conventional spectrofluorimeter. It is a quantum efficiency measurement image figure of this invention. It is an example of the measurement result by the standard sample of the conventional spectrofluorimeter. It is a calibration curve of the photon counting method of FIG. It is a calibration curve of the analog method of FIG. FIG. 9 is a calibration curve obtained by connecting the photon counting method and the analog method in FIG. 8. FIG.

  An embodiment of the present invention will be described with reference to FIGS. FIG. 1 is a schematic configuration diagram of a spectrofluorophotometer according to an embodiment of the present invention. FIG. 2 is a diagram showing an outline of analysis work according to the embodiment of the present invention.

  As shown in FIG. 1, a spectrofluorometer according to the present invention includes a sample chamber 103, a light source unit 101 for irradiating a sample with light, an excitation spectrometer unit 102, and a fluorescence spectrometer unit for converting the excited light into an electrical signal. In accordance with a signal from the operation / control unit 106, an electric signal from the spectroscope unit 109 composed of 104, the low concentration electrical system control unit 107, the high concentration electrical system control unit 108, and the fluorescence spectrometer unit 104 The switch 105 is configured to send an electrical signal to the low concentration electrical system control unit 107 or the high concentration electrical system control unit 108.

Fig. 8 shows a standard sample containing a sodium fluorescein solution having a concentration of ^10-6 to ^10-11 mol / L enclosed in a 10 mm cuvette cell. The excitation wavelength is 470 nm and the fluorescence wavelength is 510 nm. The measured values collected by analog method and photon counting method measured with slits are shown.

  Here, the value measured by the analog method is Ianalog, and the value measured by the photon counting method is Iphoton.

  If the photon counting method and the analog method are plotted, and both calibration curves show good linearity (correlation coefficient square (contribution rate) of 0.999 or more), the conversion coefficients are K1 and K2, and Iphoton = K1 x Ianalog + K2 is established. If the linearity of both or one of the calibration curves by both methods is not good (the square of the correlation coefficient (contribution rate) is less than 0.999), it is determined that the above equation does not hold and the present invention is not applicable.

  FIGS. 9 and 10 are graphs and contribution rates obtained by applying a straight line as an approximate expression of the photon counting method and the analog method created based on the data of FIG. 8. The respective contribution rates are 0.9998 and 1, respectively. The linearity condition of the calibration curve is satisfied. Therefore, if K1 and K2 are obtained using the measurement data shown in FIG. 8 in the above equation, the fluorescence intensity obtained by the analog method can be converted to a count value scale by the photon counting method. As is clear from the above, it is possible to extend the measurement range by combining the calibration curves obtained by both methods.

The method for connecting the calibration curves of the analog method and the photon counting method will be described below. First, the operator selects two sets of data including measurement data of both the analog method and the photon counting method. In the case of the data in FIG. 8, it is 10 ⁻⁸ mol / L and 10 ⁻⁹ mol / L. When the above conversion coefficients K1 and K2 are obtained from these two sets of measurement results, the following equations K1 = 11675.66 and K2 = −103.951 are obtained.

Using these K1 and K2, the measured values of the analog method of 10 ⁻⁷ mol / L and 10 ⁻⁶ mol / L can be converted into the data values of the photon counting method. That is, the value after conversion at 10 ⁻⁷ mol / L is 99.084 × 11675.66-103.951 = 1156767, and the value after conversion at 10 ⁻⁶ mol / L is 965.987 × 11675.66-103.951 = 11278432.

  FIG. 11 shows a graph connecting the converted data and a linear approximation graph. A good linear calibration curve with a contribution ratio of 1 is obtained.

In this example, the conversion data from the analog method is used for concentrations of 10 ^-8 mol / L or higher, so the measurement limit value of the photon counting method is 10 ^-8 mol / L, 119303 cps. is there.

Generally, the sample concentration applicable by the analog method is 10 ⁻¹² to 10 ⁻⁵ mol / L, and there is a measurement dynamic range of about 7 digits. The photon counting method can handle sample concentrations up to 10 ^-13 mol / L. That is, a calibration curve with good linearity can be obtained by both methods. Assuming that there are at least two sets of measurement data at concentrations common to both methods, it is possible to construct a spectrofluorometer having a dynamic range of about 8 digits.

  The graph display of the calibration curve and the calculation of the contribution ratios K1 and K2 are performed by the operation / control unit 106 in an interactive manner with the operator.

  At the time of sample measurement, the operation / control unit 106 monitors the measurement value, and if it is within the measurement limit value, sets the switch 105 so that the signal of the spectroscope unit 109 is sent to the low-concentration electric system control unit 107, and the measurement is performed. If the limit value is exceeded, the switch 105 is set so that the signal from the spectroscope unit 109 is sent to the high concentration electrical system control unit 108. If the signal from the spectroscope unit 109 is obtained through the high-concentration electrical system control unit 108, the data is converted using K1 and K2, and then processed using the calibration curve. I do.

Next, the operation of the present invention will be described with reference to FIGS.
(1) Step 201, the operator prepares a low concentration standard sample.
(2) Step 202, the operator operates the operation / control unit 106, and sets the switch 105 so that the electrical signal from the spectroscope unit 109 is sent to the low concentration electrical system control unit 107.
(3) Step 203, standard sample measurement by the photon counting method is performed, measurement data is sent to the operation / control unit 106, and a calibration curve graph is obtained by linear approximation.
(4) Step 204, the operator prepares a high concentration standard sample.
(5) Step 205, the operator operates the operation / control unit 106, and sets the switch 105 so that the electrical signal from the spectroscope unit 109 is transmitted to the high-concentration electrical system control unit 108.
(6) Step 206, standard sample measurement by analog method is performed, measurement data is sent to the operation / control unit 106, and a calibration curve graph is obtained by linear approximation.
(7) Operation / control that the calibration curves obtained in steps 207, (3) and (6) both have good linearity of a predetermined contribution ratio or more, and that two or more sets of data having the same concentration exist. The unit 106 is operated for confirmation.
If the calibration curves do not show good linearity, or if data of two or more sets of common concentrations do not exist, the process proceeds to step 208 and the analysis operation is interrupted.
If both calibration curves show good linearity and there are two or more sets of data with a common concentration, the next step is executed.
(8) Step 209, a conversion formula is obtained from the data obtained by both methods.
(9) Step 210, obtaining the measurement limit of the photon counting method.
(10) Step 211, preparing a sample for measurement.
(11) Step 212, measuring the sample.
(12) Step 213, it is determined whether or not the measured value exceeds the measurement limit value of the photon counting method. If the measured value does not exceed the measurement limit value, (13) and (14) are executed to determine the measurement limit value. If the number exceeds, (15) to (17) are executed.
(13) Step 217: The switch 105 is switched to the low concentration electrical system controller 107.
(14) Step 218: Measurement is performed by the low concentration electrical system controller.
(15) Step 214, the switch 105 is switched to the high-concentration electric system controller 108.
(16) Step 215, measurement by the high concentration electrical system controller.
(17) Conversion is performed according to the conversion formulas obtained in step 216 and step 209.
(18) Step 219, outputting the measured value using the combined calibration curve.

  The configuration and operation of the present invention are as described above, and a spectrofluorometer that accurately measures over a wide concentration range can be realized.

  As a modified embodiment of the present invention, when a measurement limit point is exceeded during measurement by the photon counting method, a notification means for notifying that the measurement limit point has been exceeded during measurement or after completion of measurement, for example, a buzzer, a blinking lamp Means can be added. Alternatively, it is possible to provide a spectrofluorometer equipped with a recording mechanism capable of recording in the measurement data that the measurement limit point has been exceeded.

  The present invention can be used as a spectrofluorometer that requires a wide dynamic range such as the quantum efficiency.

DESCRIPTION OF SYMBOLS 101 Light source part 102 Excitation spectrometer part 103 Sample chamber part 104 Fluorescence spectrometer part 105 Switch 106 Operation / control part 107 Low concentration electric system control part 108 High concentration electric system control part 109 Spectrometer part 201 Low concentration standard sample Preparation 202 The switch 105 is set so that the electrical signal from the spectroscope unit 109 is sent to the low-concentration electrical system control unit 107 203 Standard sample measurement 204 by the photon counting method 204 Preparation of the high-concentration standard sample 205 Spectroscope unit 109 The switch 105 is set so that the electrical signal from the high-concentration electric system controller 108 is sent to the high-concentration electric system control unit 206. Standard sample measurement by the analog method 207. Check 208 Stop measurement 209 Create conversion formula 210 Create linked calibration curve and photon cow Setting of measurement method limit value 211 Preparation of measurement sample 212 Sample measurement 213 Has the measurement limit been exceeded?
214 Switch the switch 105 to the high-concentration electrical system control unit 215 Measurement 216 by the high-concentration electrical system control unit 217 Conversion of measured values 217 Switch the switch 105 to the low-concentration electrical system control unit 218 Low-concentration electrical Measurement by system controller 219 Outputs measurement value using connected calibration curve

Claims (2)

  Two types of electric system control units that process signals from the spectroscope, a signal switching mechanism that transmits an output signal from the spectroscope to one of the two types of electric system control units, and each electric system control A spectrofluorometer comprising an operation / control unit for processing data from the unit, and a mechanism for controlling the signal switching mechanism by a signal from the operation / control unit.   The operation / control unit uses a standard sample to obtain a calibration curve by a photon counting method and an analog method, and both calibration curves obtained by both methods satisfy linearity with a contribution ratio of 0.999 or more. The method of extracting two sets of data in the same concentration range from the measurement data of the method, the step of solving the linear simultaneous equations from the two sets of extracted data, and the measurement value obtained by the analog method A step of obtaining a coefficient to be converted into a value of the counting method, a step of storing a high concentration value and a corresponding measurement value among the two sets of concentration values, and a measurement value obtained by an analog method equal to or higher than the concentration value. The conversion using the coefficient, the measured value by the photon counting method for the density less than the stored density value, and the conversion for the density exceeding the stored density value. A calibration curve using values, a step of monitoring whether the sample measurement value from the spectrometer exceeds or does not exceed the stored concentration value, and if not, the signal from the spectrometer has a low concentration 2. The electrical system control unit according to claim 1, further comprising a step of transmitting a signal from the spectrometer to the high concentration electrical system control unit when exceeding, and a step of processing data using the calibration curve. The spectrofluorometer described in 1.

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