JP4450750B2 - Fluorescence analyzer - Google Patents

Description

  The present invention relates to a fluorescence analyzer that irradiates a sample with excitation light and measures and analyzes fluorescence from the sample.

  As described in Patent Document 1, the fluorescence analyzer in the prior art detects a part of the excitation light with a detector and takes it as an excitation light signal. The amplification degree of the excitation light signal is determined by the slit width. The pumping light signal is set to a certain level or more by varying in accordance with one of the change and the wavelength change.

  Since the amount of fluorescence emitted from the sample fluctuates in proportion to the amount of excitation light, when performing quantitative measurement of the sample, a quantitative value that corrects fluctuations in the amount of excitation light by calculating (fluorescence amount) / (excitation light amount) Ask for. In this way, the calculation accuracy is improved and the measurement accuracy is improved.

Japanese Patent Laid-Open No. 53-50885

  By the way, in the fluorescence analyzer, the fluorescence emitted from the sample is measured by irradiating the sample with excitation light. Not only quantitative analysis in which the amount of the substance contained in the sample is obtained from the amount of fluorescence emitted by the substance, There is a demand for qualitative analysis in which the fluorescence spectrum is measured while scanning the wavelength to confirm the presence of the type of substance contained in the sample.

  However, when the qualitative analysis is performed by measuring the fluorescence spectrum while scanning the wavelength of the excitation light, the above-described conventional technique has the following problems (1) and (2).

(1) When a fluorescence spectrum is measured while scanning the wavelength of excitation light, a correct fluorescence spectrum cannot be obtained. The reason for this is that when the excitation wavelength is changed, if the amplification level of the excitation light signal is changed, the signal level of the excitation light becomes constant, but the fluctuation in the actual amount of excitation light irradiated to the sample remains unchanged. This is because the ratio between the excitation light amount and the excitation light signal is different before and after the change. The conventional technique cannot be used in an analysis method that measures the fluorescent substance concentration while changing the excitation wavelength, such as fluorescence three-dimensional spectrum measurement.
(2) Since the difference in the amount of fluorescence emitted by the sample is not taken into consideration, measurement conditions are generated in which the magnitudes of the fluorescence signal and the excitation light signal are greatly different. In this case, the resolution of both signals is different, and the correction accuracy of the excitation light fluctuation is deteriorated.

  Here, since the amount of fluorescent light emitted from the sample changes in proportion to the amount of excitation light, in order to obtain a signal (quantitative signal) that changes in proportion to only the amount of substance contained in the sample, the amount of fluorescent light is excited. Divide by the amount of light. That is, the quantitative signal can be obtained by calculating (fluorescence light amount signal) / (excitation light amount signal).

  In addition, the above calculation has an effect of removing noise components in which the amount of fluorescent light varies due to fluctuations in excitation light.

  In many cases, the excitation light is obtained by spectrally dividing light having a wavelength of 190 nm to 900 nm emitted from a xenon lamp. The amount of fluorescent light at each excitation light wavelength is measured while changing this wavelength, but the light amount difference of each wavelength generated by the light source, the fluorescent light amount difference due to the sample, the wavelength characteristics of the spectrometer, and the wavelength characteristics of the detector are affected. The excitation light amount changes 1000 times or more depending on the wavelength, and the fluorescence light amount changes 1,000,000 times or more depending on the wavelength and the sample.

  For this reason, the condition that the magnitude of the excitation light quantity signal becomes 1/100 or less of the magnitude of the fluorescence light quantity signal also occurs.

  The fluorescence spectrum shown in FIG. 7 is measured under measurement conditions where (excitation light amount signal) / (fluorescence light amount signal) irradiated onto the sample is 1/50. However, the excitation light signal is not amplified.

  FIG. 8 shows the result of measuring the time change of the quantitative value signal ((fluorescence light quantity signal: DEF) / (excitation light quantity signal: DEX)) at the peak wavelength (450 nm) of the fluorescence spectrum shown in FIG. It can be seen that is included.

  There are two main causes of this noise, and one is because the fluctuation correction accuracy of the light source light quantity is deteriorated. This is because the resolution of the excitation light amount signal is only 1/50 of the fluorescence light amount signal in the calculation of the quantitative value signal = (fluorescence light amount signal) / (excitation light amount signal).

  For example, if the AD conversion value of the fluorescence light amount signal is 5000, the AD conversion value of the 1/50 excitation light amount signal is 100, and a resolution of 1/100 or less cannot be obtained from the AD conversion value of the excitation light amount signal. This is because it becomes impossible to correct fluctuations in the light source quantity of 1/100 or less.

  The second cause is due to the influence of analog signal circuit system noise. This is because the analog signal circuit noise is added to the measurement signal at a substantially constant level, so that the influence on the excitation light amount signal of 1/50 is almost 50 times that of the fluorescence light amount signal.

  In order to solve the above two causes of noise generation, it is conceivable to propose a technique for increasing the amplification degree when the excitation light amount signal is small.

The above proposed technology will be considered below.
FIG. 9 shows the results obtained by amplifying the excitation light amount signal by 50 times under the same conditions as the fluorescence spectrum measurement shown in FIG. Since the quantitative signal = (fluorescence light amount signal) / (excitation light amount signal) × 50, the quantitative value signal peak wavelength value is 1/50 of the peak wavelength shown in FIG.

  This indicates that if the amplification degree of the excitation light amount signal is changed depending on the measurement conditions, the quantitative value signal changes, and thus there remains a big problem that the correlation with the quantitative value signal cannot be obtained.

  In particular, in fluorescence spectrum measurement, it is necessary to obtain a quantitative value that continuously changes while continuously changing the spectral wavelength of excitation light and fluorescence. However, there is a problem that it is impossible to measure a spectrum that continuously changes.

  An object of the present invention is to realize a fluorescence analysis apparatus and method capable of measuring a fluorescence spectrum with high accuracy while continuously changing the spectral wavelength of excitation light and fluorescence while reducing fluctuations in light source light and disturbance noise. It is.

  In the present invention, every time the fluorescence light amount signal and the excitation light amount signal are AD converted and taken into the arithmetic processing unit, the AD conversion data of the fluorescence light amount signal and the excitation light amount signal becomes the data with the same accuracy (G ) Is calculated, and the excitation light signal is amplified by the amplification degree (G) and AD converted.

  Thereby, excitation light data and fluorescence data with the same accuracy can be obtained.

  Data obtained by dividing the fluorescence data thus obtained by the excitation light data is multiplied by an amplification factor (G) obtained by amplifying the excitation light signal in order to obtain this data.

  This processing makes it possible to obtain spectral data with low noise.

  However, the amount of fluorescence becomes zero depending on the wavelength of the excitation light and the fluorescence wavelength. In this case, the obtained amplification degree (G) may be 1 or less and may be 0. In this way, when the amount of fluorescence becomes a certain value or less, the amplification degree of the excitation light signal is set to a predetermined constant value.

  Further, under the condition that the excitation light signal is close to 0, the obtained amplification degree (G) becomes a large amplification degree that cannot be set, so the amplification degree of the excitation light signal is set to a predetermined constant value.

  According to the present invention, it is possible to realize a fluorescence analysis apparatus and method capable of measuring a fluorescence spectrum with high accuracy while continuously reducing the wavelength of excitation light and fluorescence while reducing fluctuations in light source light and disturbance noise. Can do.

  It enables fluorescence spectrum measurement with greatly reduced influences of light source fluctuations and disturbance noise, and enables analysis of trace samples.

Embodiments of the present invention will be described below with reference to the accompanying drawings.
FIG. 1 is a schematic configuration diagram of a fluorescence analyzer according to an embodiment of the present invention. In FIG. 1, the light from the light source 1 is split by the spectroscope 2. The spectroscope 2 is rotated by a motor 15 to change the spectral wavelength. The motor 15 is driven by a motor drive circuit 17. The motor drive circuit 17 specifies a rotation speed and a rotation angle by the data processing unit 14, and drives the motor 15 so that the specified rotation speed and rotation angle are obtained.

  The light split by the spectroscope 2 is reflected by the mirror 3, passes through the quartz plate 4 and the slit 21, and is irradiated on the sample 6 as excitation light. A part of the excitation light reflected by the mirror 3 is irradiated to the detector 5 by the surface reflection of the quartz plate 4.

  The fluorescence emitted from the sample 6 is irradiated to the spectroscope 7 through the slit 22 and is split by the spectroscope 7. The spectroscope 7 is rotated by a motor 16 to change the spectral wavelength. The motor 16 is driven by a motor drive circuit 18. The motor driving circuit 18 designates the rotation speed and rotation angle of the motor 16 by the data processing unit 14 and drives the motor 16 so as to be the specified rotation speed and rotation angle. The light separated by the spectroscope 7 is irradiated to the detector 8.

  The detection signal from the detector 5 is amplified by the AMP 9. The amplification degree of the AMP 9 can be varied by the amplification variable circuit 10. The signal from the AMP 9 is AD converted by the ADC 12.

  The detection signal from the detector 8 is amplified by the AMP 11 and AD converted by the ADC 13. The ADC 12 and the ADC 13 perform AD conversion at a constant time period based on a signal from the timer 19. The timer cycle of the timer 19 is specified by the data processing unit 14.

  Data from the ADC 12 is read by the data processing unit 14 as excitation light data (DEX) for monitoring the amount of excitation light. The data processing unit 14 reads data from the ADC 13 as fluorescence data (DEF) for monitoring the amount of fluorescence from the sample. A data processing unit 14 that is a CPU processes data read from the ADC 12 and the ADC 13 and causes the display device 20 to display the data.

  The data processing unit 14 according to the embodiment of the present invention has the data processing function shown in FIG. 2 and performs an operation according to the time chart shown in FIG.

  In one embodiment of the present invention, every time AD conversion for measurement is performed, processing for equalizing the magnitudes of the excitation light amount signal and the fluorescence light amount signal and the change in the measurement signal generated by this processing are corrected. Process.

First, a process for equalizing the excitation light amount signal and the fluorescence light amount signal will be described.
The data processing unit 14 controls the motor drive circuits 17 and 18 in order to set the initial spectral wavelength shown in (1) of FIG. Next, in order to set the amplification degree of the AMP 9 shown in (2) of FIG. 3 to 1, the AMP amplification degree variable circuit 10 is controlled. Next, the timer 19 is controlled in order to set the AD conversion cycle time shown in (3) of FIG. Thereby, the timer 19 outputs the high-speed AD conversion start signals of the ADC 12 and the ADC 13 ((4) in FIG. 3). In this example, the high-speed AD conversion is, for example, 16 bits and 10 μs to 50 μs.

  In response to the high-speed AD conversion start signal, the ADC 12 and the ADC 13 perform high-speed AD conversion between the excitation light amount signal and the fluorescence light amount signal, and notify the data processing unit 14 that the AD conversion has been completed. When the high-speed AD conversion end signal is received from the ADC 12 and the ADC 13, the data processing unit 14 reads the converted data as shown in (5) of FIG. 3.

  From the read data, an amplification degree (G) for making the magnitude of the excitation light quantity signal equal to that of the fluorescence quantity signal is obtained by the dividing unit 54 of FIG. The amplification degree (G) is set in the amplification degree variable circuit 10 by the amplification degree setting processing unit 52. The amplification degree (G) is stored in the memory 55.

  With the above operation, the magnitudes of the excitation light amount signal and the fluorescence light amount signal input to the ADC 12 and ADC 13 become equal.

  Here, the reason why the ADC 12 and the ADC 13 perform high-speed AD conversion is that the measurement time for obtaining a signal proportional to the sample concentration, which is the main purpose, is reduced and the signal accuracy is not lowered.

  As a process in the case where the excitation light amount signal is too small and exceeds the amplification level settable by the amplification variable circuit 10, in one embodiment of the present invention, the magnitude comparison unit 53 in FIG. And a fixed value (D), it is determined whether or not a settable amplification degree (D) is exceeded.

  As a result of the determination, if the amplification degree that can be set is exceeded, the magnitude comparison unit 53 sets the amplification degree to a fixed value (E) (100 times in one embodiment of the present invention).

  The effect of this process is to prevent AMP system noise from increasing and increasing the S / N ratio when the degree of amplification of AMP 9 is increased to a certain level or more.

  Further, whether or not the fluorescence light amount signal is small and the amplification degree of the excitation light amount signal is 1 or less is determined by comparing the fluorescence light amount signal with the fixed value (A) by the magnitude comparison unit 51 of FIG. To do. As a result, when it is determined that the fluorescence light amount signal is small, the amplification degree is set to a fixed value (B) (1 time in one embodiment of the present invention).

  The effect of this processing is to prevent the resolution of the excitation light quantity signal from being reduced and the S / N from being deteriorated if the amplification degree of the excitation light quantity signal is set to 1 or less.

  Next, a description will be given of processing for correcting a change in the measurement signal generated by the main purpose measurement and the processing for equalizing the magnitudes of the excitation light amount signal and the fluorescence light amount signal.

  After the amplification degree of the AMP 9 is set, a low-speed and high-precision AD conversion start signal is output from the timer 19 to the ADC 12 and the ADC 13 according to the control operation of the timer control unit 57 ((6) in FIG. 3). Here, in this example, the low-speed and high-precision AD conversion is, for example, 24 bits and 10 ms to 50 ms.

  When the timer 19 notifies the data processing unit 14 that the low-speed and high-precision AD conversion has been completed, the data processing unit 14 reads the AD conversion data from the ADC 12 and the ADC 13.

  Then, measurement and processing for obtaining a signal proportional to the sample concentration, which is the main purpose, are performed by the division processing by the division unit 54 of FIG.

  However, since the value obtained at this time includes an error caused by changing the amplification degree of the excitation light amount signal, the multiplication unit 56 multiplies the amplification degree (G) value stored in the memory 55. .

FIG. 4 is a flowchart of signal processing.
In FIG. 4, first, the amplification degree of the excitation light AMP9 is set to 1 (step (a)), and high-speed AD conversion is performed in the ADC 12 and ADC 13 (step (b)).

  When the data processing unit 14 receives the AD conversion end signals from the ADC 12 and the ADC 13, the signal (DEX1n) obtained by AD-converting the signal (AEX1n) obtained by amplifying the excitation light signal with the amplification degree 1 and the fluorescence signal ( AEFn) is read from the AD-converted signal (DEFn) (steps (c) and (d)).

  In step (ca), the data processing unit 14 compares the value of the AD conversion data (DEX1n) of the excitation light with a fixed value (D). As a result, when the AD conversion data (DEX1n) is equal to or less than the fixed value (D), the amplification degree (G) of the excitation light AMP is determined by the fixed value (E) stored in advance in the memory 55 of the data processing unit 14. (Step (cb)).

  Next, the data processing part 14 compares the fluorescence signal AD conversion data (DEFn) from the ADC 13 with a fixed value (A) stored in advance in the memory of the data processing unit 14 (step (e)).

  As a result, when the value of the AD conversion data (DEFn) of the fluorescence signal is larger than the fixed value (A), the amplification degree (G) of the excitation light AMP9 is obtained by calculating (DEFn) / (AEX1n). (Step (f)).

  When the value of the AD conversion data (DEFn) of the fluorescence signal is equal to or less than the fixed value (A), the fixed value (B) stored in advance in the memory of the data processing unit 14 is used as the amplification degree (G ) (Step (g)).

  Next, the signal amplification degree from the excitation light AMP9 is set to the amplification degree (G) calculated in any one of steps (f), (g), and (cb) (step (h)).

  As a result, the pumping light signal (AEX1n) is amplified with the amplification degree (G), subjected to AD conversion at low speed and high accuracy (step (i)), and read by the data processing unit 14 as AD conversion data (DEXn) (step (j) )). At the same time, the fluorescence signal is read by the data processing unit 14 as AD conversion data (DEFn) (step (k)).

  The data processing unit 14 calculates (DEFn) × G / (DEXn) by multiplying the result after calculating (DEFn) / (DEXn) by the amplification degree (G) to obtain DATAn (step ( l)).

  By the above processing, a signal having the same measurement signal level as that when the excitation light signal is not amplified with the amplification degree (G) can be obtained with high accuracy.

  Then, the data processing unit 14 stores DATAn in the memory (step (m)), and displays DATAn on the display device 20 (step n). Subsequently, after changing the wavelength of the excitation light (step o), the process returns to step (a).

  FIG. 5 is a fluorescence spectrum obtained by scanning the sample from which the data shown in FIG. 7 was obtained in one embodiment of the present invention, and FIG. 6 is a fluorescence spectrum peak wavelength of the sample in one embodiment of the present invention. It is time change measurement data at, and is data obtained by measuring S / N of peak data.

  As can be understood by comparing the data obtained by the embodiment of the present invention (FIGS. 5 and 6) with the data obtained by the prior art (FIGS. 7 and 8), the embodiment of the present invention. According to the above, it is possible to obtain a result in which the S / N is 5 times or more that of the conventional technique and the peak value is the same as that of the conventional technique.

  In addition, according to the present invention, depending on the analysis conditions, it is possible to reduce the light source light amount fluctuation and the influence of electrical system noise to 1/6 or less of the prior art, and it has been difficult to measure by being buried in noise. It becomes possible to measure substances, and it is possible to expand R & D, high-precision quality control, and other fields of application.

  The widths of the slits 21 and 22 shown in FIG. 1 are adjusted to improve the wavelength resolution. However, the amplification degree (G) is calculated for each wavelength setting and the width of the slits 21 and 22. It is also possible to configure so as to be performed for each adjustment.

It is a schematic block diagram of the fluorescence analyzer which is one Embodiment of this invention. It is an internal function block diagram of the data processing part in the fluorescence analyzer shown in FIG. It is operation | movement explanatory drawing of the data processing part in one Embodiment of this invention. It is an operation | movement flowchart of the signal processing in one Embodiment of this invention. It is a figure which shows the fluorescence spectrum which carried out the scan measurement of the sample with the fluorescence analyzer in one Embodiment of this invention. It is a figure which shows the measurement data of the time change of the quantitative value in the fluorescence spectrum peak wavelength shown in FIG. It is a figure which shows the fluorescence spectrum measured by wavelength-scanning the sample in a prior art. It is a figure which shows the measurement data of the time change of the quantitative value in the fluorescence spectrum peak wavelength shown in FIG. 7 in a prior art. It is a figure which shows the fluorescence spectrum which amplified the excitation light signal 50 times in the prior art, and measured the sample.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Light source 2 Spectrometer 3 Mirror 4 Quartz plate 5 Detector 6 Sample 7 Spectrometer 8 Detector 9, 11 AMP
10 AMP amplification variable circuit 12, 13 ADC
Reference Signs List 14 Data processing unit 15, 16 Motor 17, 18 Motor drive circuit 19 Timer 20 Display device 21, 22 Slit 51, 53 Size comparison unit 52 Amplification setting processing unit 54 Division processing unit 55 Memory 56 Multiplication unit 57 Timer control unit

Claims (6)

A light source, a spectroscopic means for splitting light emitted from the light source, and irradiating the sample by changing its wavelength as excitation light, a means for irradiating a part of the excitation light to the excitation light detector, and the excitation An excitation light signal amplifier that amplifies the signal from the light detector, an excitation light AD converter that AD converts the signal from the excitation light signal amplifier, a fluorescence detector that detects the fluorescence emitted by the sample, and the fluorescence detection A fluorescence signal amplifier that amplifies the fluorescence signal from the detector and a fluorescence AD converter that AD converts the signal from the fluorescence signal amplifier, and analyzes the sample based on the fluorescence signal from the fluorescence AD converter In the fluorescence analyzer,
The output data of the fluorescence AD converter is divided by the output data of the excitation light AD converter, and the input signal to the excitation light AD converter and the input signal to the fluorescence AD converter have the same magnitude. and division means for the amplification degree of the excitation light signal amplifier, it calculates for each change the wavelength of the excitation light,
Storage means for storing the calculated amplification degree;
Amplification changing means for changing the amplification of the pumping optical signal amplifier,
Excitation light data that has been changed to the amplification degree calculated by the division means by the amplification degree changing means, amplified with the changed amplification degree, and AD converted by the excitation light AD converter, and the fluorescence AD converter Multiplication means for multiplying the division data with the fluorescence data AD-converted by the multiplication factor stored in the storage means ,
A fluorescence analysis apparatus comprising: The fluorescence analyzer according to claim 1, wherein
First comparison means for comparing the output signal of the fluorescent AD converter with a predetermined value;
Amplification setting processing for determining whether the amplification factor of the pumping optical signal amplifier is the amplification factor stored in the storage unit or a predetermined fixed amplification value according to the comparison result of the first comparison unit Means,
A fluorescence analysis apparatus comprising: The fluorescence analyzer according to claim 2, wherein
A second comparison unit that compares the output signal of the pump light AD converter with a predetermined value; and the amplification level setting processing unit outputs the output signal of the pump light AD converter as a result of the comparison by the second comparison unit. When is smaller than a predetermined value, the fluorescence analyzer is characterized in that the amplification factor of the excitation light signal amplifier is set to a predetermined fixed amplification factor value. 2. The fluorescence analyzer according to claim 1, wherein an AD conversion speed of the excitation light AD converter and the fluorescence AD converter in a period in which the division means calculates an amplification degree is calculated by the amplification degree of the excitation light signal amplifier. A fluorescence analyzer characterized by being faster than the AD conversion speed of the excitation light AD converter and the fluorescence AD converter after being changed to the amplification degree. 2. The fluorescence analyzer according to claim 1, further comprising slit means for passing the excitation light from the spectroscopic means and irradiating the sample. The slit means can change the slit width, and the dividing means is the wavelength. A fluorescence analyzer, wherein the amplification degree is calculated for each change and each slit width change. A light source, means for spectrally dividing light emitted from the light source, changing the wavelength of the light as an excitation light, irradiating the sample, means for irradiating a part of the excitation light to the excitation light detector, and the excitation light An excitation light signal amplifier that amplifies the signal from the detector, an excitation light AD converter that AD converts the signal from the excitation light signal amplifier, a fluorescence detector that detects the fluorescence emitted by the sample, and the fluorescence detector Fluorescence for analyzing a sample based on the fluorescence signal from the fluorescence AD converter using a fluorescence signal amplifier for amplifying the fluorescence signal from the fluorescence signal and a fluorescence AD converter for AD converting the signal from the fluorescence signal amplifier In the analysis method,
The output data of the fluorescence AD converter is divided by the output data of the excitation light AD converter, and the input signal to the excitation light AD converter and the input signal to the fluorescence AD converter have the same magnitude. calculating sushi the amplification degree of the excitation light signal amplifier for each change the wavelength of the excitation light,
Memorize the calculated degree of amplification,
Amplified with the calculated amplification degree, and the division data of the excitation light data AD-converted by the excitation light AD converter and the fluorescence data AD-converted by the fluorescence AD converter is multiplied by the stored amplification degree. And analyzing the sample.

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