WO2013190618A1

WO2013190618A1 - Spectrophotofluorometer

Info

Publication number: WO2013190618A1
Application number: PCT/JP2012/065524
Authority: WO
Inventors: 渡邉　康之
Original assignee: 株式会社島津製作所
Priority date: 2012-06-18
Filing date: 2012-06-18
Publication date: 2013-12-27

Abstract

A synchronized fluorescent light spectrum for a sample is acquired by setting the wavelength difference between an excitation light wavelength and a fluorescent light wavelength to a minimum initial value, and synchronously scanning both wavelengths at a constant wavelength difference (S1 to S3). If there is a peak in the spectrum, the peak wavelength is treated as a fluorescent light wavelength (λ_EMn), and while holding this constant, an excitation light spectrum is acquired by scanning the excitation light wavelength (S4 to S7). A peak wavelength in the excitation light spectrum is treated as the optimal excitation light wavelength (λ_EXn), and while holding this constant, a fluorescent light spectrum obtained by scanning the fluorescent light wavelength is acquired, and the peak wavelength of the fluorescent light spectrum is treated as the optimal fluorescent light wavelength λ_EXn' (S8 to S10). The excitation light wavelength is increased by 10 nm and the fluorescent light spectrum is acquired in the same manner; if there is no wavelength shift in the acquired fluorescent light peak, the detected peak is the true fluorescent light-derived peak (S13 to S14), so the fact that fluorescence has been detected is output to a display along with the optimal wavelength information (S15, S17).

Description

Spectrofluorometer

The present invention relates to a spectrofluorometer that irradiates a sample with excitation light and detects fluorescence emitted from the sample.

In a general spectrofluorometer, the sample to be measured is irradiated with excitation light of a specific wavelength extracted by the excitation side spectroscope, and the fluorescence emitted from the sample by being excited by the irradiation light is subjected to fluorescence side spectroscopy. The wavelength is dispersed through the detector and introduced into the detector, and the light intensity is detected. In order to accurately measure the fluorescence intensity, it is necessary to appropriately set the wavelength of excitation light for generating fluorescence and to extract fluorescence having an appropriate wavelength from the fluorescence emitted from the sample. However, it is sometimes known that when a sample is measured, the sample emits fluorescence, but the appropriate excitation light wavelength condition and the fluorescence wavelength range in which fluorescence can be observed are unknown. is there. In addition, since substances that emit fluorescence are limited, it may be unclear whether or not the sample to be measured actually emits fluorescence.

Therefore, in order to investigate whether fluorescence is obtained from a sample to be measured, or to obtain measurement conditions such as the excitation light wavelength when fluorescence is obtained, a conventional general spectrofluorometer has a spectrum. A search function is provided for determining measurement conditions using the measurement function.

FIG. 9 is an explanatory diagram of measurement and processing for searching for excitation light and fluorescence wavelength in a conventional spectrofluorometer. As shown in FIG. 9A, in the graph with the excitation light wavelength on the vertical axis and the fluorescence wavelength on the horizontal axis, an oblique straight line L having the same excitation light wavelength and fluorescence wavelength indicates Rayleigh scattered light. When fluorescence is emitted, a peak due to fluorescence usually exists in a region below (or to the right of) the straight line L.

If the presence or absence of fluorescence is unknown in advance, or if the wavelength of the excitation light that produces the maximum fluorescence intensity and the wavelength of the fluorescence emitted at that time is unknown, although it is known that fluorescence will occur 9A, the wavelengths of the excitation light and the fluorescence are scanned in the order as indicated by the one-dot chain line, and the presence / absence of fluorescence is determined and the wavelengths of the excitation light and the fluorescence are calculated based on the spectrum obtained by the scanning. Is done. That is, first, the excitation light wavelength is set to the initial wavelength on the shortest wavelength side in a predetermined excitation light wavelength range, and the fluorescence wavelength is scanned at a specified wavelength interval over the predetermined wavelength range (FIG. 9A). ) The fluorescence spectrum is acquired while moving in the right lateral direction. When the fluorescence spectrum as shown in FIG. 9B is obtained for one excitation light wavelength, the excitation light wavelength is increased by the specified wavelength interval Δλ _X (in FIG. 9A). The fluorescence spectrum is acquired again while scanning the fluorescence wavelength at a specified wavelength interval over a predetermined wavelength range.

In this way, the fluorescence spectrum is acquired while increasing the excitation light wavelength by the specified wavelength interval Δλ _{X. In} the process, if a peak other than the Rayleigh scattered light is observed, the fluorescence wavelength of the peak top is stored. Keep it. Then, finally obtains a fluorescence wavelength showing the maximum peak intensity, and stores it as the optimal emission wavelength lambda _EM. Next, for the best fluorescence wavelength lambda _EM stored wavelength taken out by fluorescence side spectroscope, while scanning Te than at wavelength intervals specified excitation light wavelength over a predetermined wavelength range 9 ( An excitation light spectrum as shown in c) is acquired. Then, peak detection is performed on the obtained excitation light spectrum, and the wavelength exhibiting the maximum peak intensity is stored as the optimum excitation light wavelength λ _EX . The excitation light λ _EX and the fluorescence wavelength λ _EM thus obtained are output as the optimum excitation light wavelength and fluorescence wavelength for the sample to be measured, and the operator determines measurement conditions such as the measurement wavelength and the measurement wavelength interval with reference to this, A detailed measurement is performed on the sample to be measured.

However, in the conventional excitation light / fluorescence wavelength searching method as described above, in general, the number of fluorescence spectra to be acquired in order to find one fluorescence is large, and the measurement takes time. In particular, for a sample in which fluorescence is obtained under an excitation light wavelength condition that is an extremely long wavelength (that is, a sample in which the fluorescence peak exists in the upper right part in FIG. 9A), the fluorescence peak is found. Thus, it is necessary to repeat the fluorescence wavelength scanning a number of times, and it takes a very long time to output the result. Of course, it is possible to initialize the excitation light wavelength to the long wavelength side instead of the short wavelength side, but this time, in this case, a sample that can obtain fluorescence under the excitation light wavelength condition that is an extremely short wavelength (that is, It takes a very long time to output the result for a sample having a fluorescence peak in the lower left part in FIG. 9A.

As described above, since many substances except for special substances have a fluorescence wavelength longer than the excitation light wavelength, only the Stokes region below the straight line L indicating the Rayleigh scattered light is subject to wavelength scanning. If the area is selected, a substantially sufficient wavelength search can be performed. As a result, the scanning range of the fluorescence wavelength for acquiring the fluorescence spectrum is limited, and the measurement time can be shortened to some extent. However, since the number of times of repeatedly acquiring the fluorescence spectrum itself while changing the excitation light wavelength is not reduced, the time required to output the result is not significantly reduced.

On the other hand, there is an apparatus described in Patent Document 1 as a spectrofluorophotometer for the purpose of quickly obtaining optimum excitation light wavelength and fluorescence wavelength. In the spectrofluorometer described in this document, one of the excitation light wavelength and the fluorescence wavelength is not fixed, and the wavelength scan is synchronously performed between the excitation side spectrometer and the fluorescence side spectrometer while keeping the wavelength difference constant. The fluorescence spectrum (the fluorescence spectrum obtained by synchronously scanning the excitation light wavelength and the fluorescence wavelength in this manner is referred to as “synchronous fluorescence spectrum” in this specification) is obtained. According to the description of this document, the slit width of the excitation-side spectrometer is set to 20 nm, the slit width of the fluorescence-side spectrometer is set to 40 nm, and the wavelength difference between the excitation light wavelength and the fluorescence wavelength is as wide as 100 nm and 150 nm. Thus, it is said that it is possible to cover almost all expected fluorescent regions by only acquiring two kinds of synchronous fluorescence spectra.

Although there is an advantage that a lot of information about the sample can be obtained at once by performing synchronous wavelength scanning under the above conditions to obtain a synchronous fluorescence spectrum, the excitation light wavelength and the fluorescence wavelength If the difference is small, effective information cannot be obtained. In addition, if the sample contains a plurality of components that emit fluorescence, the peaks derived from the components overlap on the synchronized fluorescence spectrum, resulting in a flat and large loose peak shape. There is a problem that there is a high possibility that wavelength information cannot be obtained. There is also a problem that it is difficult to selectively detect the fluorescence peak when the fluorescence peak and the peak due to Rayleigh scattered light are close to each other or when the peak due to Raman scattered light exists.

JP-A-60-239652

The present invention has been made to solve the above-mentioned problems, and the object of the present invention is to detect the presence or absence of fluorescence and search for the optimum excitation light wavelength and fluorescence wavelength in a short time when fluorescence is detected. Even if the fluorescence peak is close to the peak derived from the Rayleigh scattered light or the peak due to the Raman scattered light is generated, the fluorescence is surely excluded except for the influence thereof. An object of the present invention is to provide a spectrofluorometer capable of detecting a peak and obtaining an accurate excitation light wavelength and fluorescence wavelength.

In order to solve the above problems, the present invention includes an excitation light spectroscopic means for extracting light of a specific wavelength from light emitted from a light source and irradiating the sample as excitation light, and receiving the excitation light from the sample. In a spectrofluorometer comprising a fluorescence spectroscopic means for spectrally separating emitted fluorescence, and a detection means for detecting the spectroscopic fluorescence,
a) The excitation light spectroscopic means and the fluorescence spectroscopic means so as to scan the excitation light wavelength and the fluorescence wavelength synchronously while maintaining a constant wavelength difference between the excitation light wavelength and the fluorescence wavelength longer than the wavelength. The synchronous fluorescence spectrum acquisition means for creating a synchronous fluorescence spectrum based on the data obtained by the detection means for the synchronous wavelength scanning,
b) performing peak detection on the synchronous fluorescence spectrum, and when a peak is detected, peak detection means for obtaining an approximate value of at least one of the excitation light wavelength and the fluorescence wavelength corresponding to the peak;
c) fixing one of the excitation light wavelength and the fluorescence wavelength whose approximate value is obtained by the peak detection means, and controlling the excitation light spectroscopy means and the fluorescence spectroscopy means so as to scan the other of the excitation light wavelength and the fluorescence wavelength. While obtaining the detailed value of the excitation light wavelength or the fluorescence wavelength based on the data obtained by the detection means, fixing one of the excitation light wavelength or the fluorescence wavelength for which the detailed value was obtained, and fixing the other of the excitation light wavelength or the fluorescence wavelength. A wavelength detail value acquisition means for obtaining the other detail value of the excitation light wavelength or the fluorescence wavelength based on the data obtained by the detection means while controlling the excitation light spectroscopy means and the fluorescence spectroscopy means so as to scan;
d) While controlling the excitation light spectroscopic means and the fluorescence spectroscopic means so that the excitation light wavelength is fixed to a wavelength separated from the excitation light wavelength detailed value obtained by the wavelength detailed value acquisition means, and the fluorescence wavelength is scanned. The fluorescence wavelength is obtained based on the data obtained by the detection means, and the peak detected by the peak detection means based on the fluorescence wavelength and the fluorescence wavelength detail value obtained by the wavelength detail value acquisition means is derived from fluorescence. Fluorescence authenticity determination means for determining whether or not
It is characterized by having.

In the spectrofluorometer according to the present invention, the synchronous fluorescence spectrum acquisition means is such that the difference between the wavelength of the excitation light extracted by the excitation light spectroscopy means and the wavelength of the fluorescence extracted by the fluorescence spectroscopy means is maintained constant. Both spectroscopic means are controlled to synchronously scan the excitation light wavelength and the fluorescence wavelength. Then, based on the data obtained by the detection means during the period in which the synchronous wavelength scanning is performed, a synchronous fluorescence spectrum in which the horizontal axis is the wavelength and the vertical axis is the intensity is created. Synchronous wavelength scanning with a constant wavelength difference is performed along a straight line parallel to a straight line (a straight line L in FIG. 9) indicated by a peak due to Rayleigh scattered light on a map having two axes of an excitation light wavelength and a fluorescence wavelength. This corresponds to scanning.

The peak detection means performs peak detection on the synchronous fluorescence spectrum, and if a peak is detected, the peak is extracted as a candidate for the fluorescence peak, and an outline of at least one of the excitation light wavelength and the fluorescence wavelength corresponding to the peak is extracted. Find the value. Since the peak obtained on the synchronous fluorescence spectrum is not necessarily the peak top of the original fluorescence peak, what is obtained by the peak detection means is only an approximate value of the wavelength. The wavelength detailed value acquisition means obtains the excitation light spectrum and the fluorescence spectrum by fixing one of the excitation light wavelength and the fluorescence wavelength and scanning only the other, not the synchronous wavelength scanning as described above. By obtaining the peak wavelength above, the detailed values of the excitation light wavelength and the fluorescence wavelength at a position closer to the peak top of the original fluorescence peak are obtained.

However, the peak detected by the peak detection means may actually be a peak derived from Raman scattered light rather than a peak derived from fluorescence. The fluorescence peak is an isolated peak in which the fluorescence intensity becomes maximum at a certain excitation light wavelength and fluorescence wavelength, and the intensity decreases as the wavelength shifts from the position. On the other hand, Raman scattered light is emitted from the sample as light having a wavelength that depends on the wavelength of incident light in a wide wavelength range, so the peak due to Raman scattered light is a mountain-shaped peak that covers a wide wavelength range in a band shape. Become. Therefore, when the wavelength of the excitation light (incident light) irradiated to the sample is slightly changed to the longer wavelength side, the fluorescence wavelength hardly shifts if it is a peak derived from fluorescence, but fluorescence ( The wavelength of the emitted light is clearly shifted to the long wavelength side. Therefore, the fluorescence authenticity determination means confirms whether or not there is a shift in the wavelength of the fluorescence peak on the fluorescence spectrum when the excitation light wavelength is changed by a predetermined amount (strictly speaking, the peak estimated to be fluorescence). Accordingly, it is determined whether or not the peak is derived from fluorescence. This eliminates false fluorescence peaks, that is, peaks due to Raman scattered light.

In the spectrofluorometer according to the present invention, the excitation light wavelength calculated by the wavelength detailed value acquisition unit and the detailed value of the fluorescence wavelength are displayed and output for the peak determined to be fluorescence-derived by the fluorescence authenticity determination unit. The display processing means may be further provided. As a result, when it is determined that fluorescence can be obtained with high accuracy, it is possible to inform the analyst of the optimum excitation light wavelength and fluorescence wavelength for obtaining the fluorescence as optimum measurement conditions.

In the spectrofluorometer according to the present invention, preferably, every time a synchronous fluorescence spectrum with respect to one wavelength difference is obtained, the peak detecting means performs peak detection, and the wavelength detailed value acquiring means is detected. Obtain the detailed values of the excitation light wavelength and the fluorescence wavelength for the peak, and the fluorescence authenticity determination means determines whether or not the detected peak is derived from fluorescence, and if it is determined that the detected peak is not derived from fluorescence For example, the synchronous fluorescence spectrum acquisition unit may acquire the next synchronous fluorescence spectrum by increasing the wavelength difference.

In a general fluorescent substance, the wavelength difference between the wavelength of excitation light causing fluorescence and the fluorescence wavelength caused by the excitation is not so large. Therefore, the initial value of the wavelength difference of the synchronous wavelength scanning is set as small as possible, and when the fluorescence-derived peak is not detected based on the obtained synchronous fluorescence spectrum, the wavelength difference is sequentially increased and newly set. If a simple synchronous fluorescence spectrum is acquired, it is possible to detect a fluorescence-derived peak in a short time.

When it is desired to know whether the sample is fluorescent or not, if at least one fluorescence-derived peak is detected, there is no need to continue acquisition of synchronous fluorescence spectrum or peak detection thereafter. . That is, the processing can be terminated when at least one fluorescence-derived peak is detected. On the other hand, when it is desired to detect all of a plurality of fluorescence-derived peaks having different excitation light wavelengths and fluorescence wavelengths, acquisition of a synchronized fluorescence spectrum, peak detection, etc. may be continued even if one fluorescence-derived peak is detected.

In any case, since the difference between the excitation light wavelength and the fluorescence wavelength is limited to some extent in a general fluorescent substance, a fluorescence-derived peak is not detected until the wavelength difference reaches a predetermined maximum wavelength difference. In this case, the display processing means may perform display output indicating that no fluorescence is detected. Thereby, it is possible to inform the analyst whether or not the sample generates fluorescence within a limited time.

In the spectrofluorometer according to the present invention, the initial value of the wavelength difference of the synchronous wavelength scanning is set to be small so that the fluorescence peak is not hidden behind the peak of the Rayleigh scattered light on the synchronous fluorescence spectrum. Is desirable. Specifically, the wavelength width of the detected fluorescence depends on the slit width included in the excitation light spectroscopic means and the slit width included in the fluorescent spectroscopic means, but when these slit widths are set to 3 nm, the wavelength difference of The initial value should be 5 nm. Thereby, it is possible to reliably detect a fluorescence peak having a very small wavelength difference without being affected by Rayleigh scattered light. Further, by acquiring the synchronous fluorescence spectrum while gradually increasing the wavelength difference from the initial value by about 10 nm, for example, the fluorescence peak can be found in a short time without missing the fluorescence peak.

According to the spectrofluorometer according to the present invention, a peak derived from fluorescence can be found reliably and in a short time without being affected by Rayleigh scattered light or Raman scattered light. Thereby, for example, when an analyst wants to know whether or not a sample emits fluorescence, it is possible to notify the presence or absence of fluorescence in a short time. In addition, when an analyst wants to know the optimum excitation light wavelength and fluorescence wavelength in a sample, these information can be provided in a short time. In addition, when the sample is a biological sample or the like, there is a risk of damaging the original characteristics due to photochemical changes when irradiated with light for a long time. By shortening, these problems can be avoided and good measurement can be performed.

BRIEF DESCRIPTION OF THE DRAWINGS The schematic block diagram of the spectrofluorometer which is one Example of this invention. The flowchart of the measurement for the excitation light and fluorescence wavelength search in the spectrofluorometer of a present Example, and a process. Explanatory drawing of the measurement and process for excitation light and fluorescence wavelength search in the spectrofluorometer of a present Example. Explanatory drawing of the method of identifying the fluorescence origin peak and Raman scattering light origin peak in the spectrofluorometer by a present Example. It is an actual measurement example by the spectrofluorometer by a present Example, (a) is a synchronous fluorescence spectrum, (b) is an excitation light spectrum, (c) is a figure which shows a fluorescence spectrum. The figure explaining the difference with the wavelength search method by the present Example and the present Example in case Rayleigh scattered light and fluorescence adjoin. The figure which shows an example of the display output of the wavelength search result in the spectrofluorometer by a present Example. The figure which shows the relationship between the fluorescence intensity mapping data about the sample which implemented the wavelength search function by a present Example, and the excitation light wavelength and fluorescence wavelength which were calculated based on this data. Explanatory drawing of the measurement and process for the excitation light and fluorescence wavelength search in the conventional spectrofluorometer.

Hereinafter, a spectrofluorometer which is an embodiment of the present invention will be described with reference to the accompanying drawings. FIG. 1 is a schematic configuration diagram of the spectrofluorophotometer of the present embodiment.

In FIG. 1, the light emitted from the light source unit 1 is introduced into an excitation-side spectroscope 2 having a diffraction grating and a slit (not shown), and monochromatic light having a specific wavelength set by the analysis control unit 8 is taken out as excitation light. The sample 3 is irradiated. The fluorescence emitted from the sample 3 by being excited by the excitation light is introduced into a fluorescence side spectroscope 4 having a diffraction grating and a slit (not shown), and the fluorescence having a specific wavelength set by the analysis control unit 8 (and other scattering). Light etc.) is taken out and introduced into the detector 5. The detector 5 outputs a detection signal corresponding to the intensity of the incident fluorescence. The detection signal is converted into a digital value by the A / D converter 6 and sent to the data processing unit 7.

The data processing unit 7 includes functional blocks such as a spectrum creation unit 71, a peak detection unit 72, a fluorescence / Raman light identification unit 73, and a wavelength information storage unit 74, and performs normal fluorescence spectrum creation processing and the like. Thus, the data processing for searching the measurement conditions (excitation light wavelength, fluorescence wavelength, etc.) for obtaining the fluorescence spectrum is performed. The analysis control unit 8 controls the operation of each unit in order to perform measurement, and particularly includes a spectrometer control unit 81 for setting or scanning the wavelength of excitation light or fluorescence. . In addition, an operation unit 10 and a display unit 11 are connected to the central control unit 9 that controls the entire apparatus in an integrated manner, and accepts input of measurement conditions by the operation unit 10 and displays measurement results on the display unit 11. Perform display control and so on.
All or part of the functions of the central control unit 9, the analysis control unit 8, and the data processing unit 7 should be achieved by executing dedicated control / processing software installed in the personal computer. Can do.

The spectrofluorometer of this embodiment has a search function for finding an appropriate measurement condition in order to obtain a highly accurate fluorescence spectrum. The measurement operation and data processing in this characteristic function will be described with reference to FIGS.
FIG. 2 is a flowchart of measurement and processing for searching for excitation light / fluorescence wavelength, FIG. 3 is an explanatory diagram of measurement and processing for searching for excitation light / fluorescence wavelength, and FIG. 4 is a graph showing fluorescence-derived peaks and Raman-scattered light-derived peaks. It is explanatory drawing of the method of identifying.

First, the analyst sets the sample 3 to be measured at a predetermined position of the apparatus (step S1), and the scanning start excitation light wavelength (or scanning start) is set as a wavelength scanning condition for acquiring the synchronous fluorescence spectrum from the operation unit 10. In addition to the fluorescence wavelength) and the scanning end excitation light wavelength (or scanning end fluorescence wavelength), an initial value of the wavelength difference (wavelength interval) Δλ between the excitation light wavelength and the fluorescence wavelength is input (step S2). For example, in an actual measurement example to be described later, the scanning start excitation wavelength is 220 nm, the scanning end fluorescence wavelength is 900 nm, and the initial value of the wavelength difference Δλ is 5 nm. These parameters may be default values. Although it differs depending on the substance, in general, the difference between the excitation light wavelength and the fluorescence wavelength is not so large. Therefore, the initial value of the wavelength difference Δλ is preferably small in order to prevent the fluorescence from being overlooked. However, under the condition that the excitation light wavelength and the fluorescence wavelength are the same, a large intensity of Rayleigh scattered light is observed instead of fluorescence. Therefore, if the wavelength difference Δλ is too small, it is difficult to distinguish it from the Rayleigh scattered light. Become. The peak width depends on the slit width in each of the

spectrometers

2 and 4, but when the slit width is set to 3 nm or less in order to obtain high wavelength resolution, the initial value (minimum value) of the wavelength difference Δλ is about 5 nm. Is appropriate.

When measurement is started in response to an instruction from the analyst, the spectroscope control unit 81 detects the wavelength of the excitation light extracted by the excitation side spectroscope 2 and the fluorescence extracted by the fluorescence side spectroscope 4 (strictly speaking, it is fluorescence. The excitation-side spectrometer 2 and the fluorescence-side spectroscopy are so scanned that the excitation light wavelength and the fluorescence wavelength are scanned synchronously while the difference from the wavelength of the light emitted from the sample 3 is not limited to Δλ. Each device 4 is controlled. That is, on the map having the excitation light wavelength and the fluorescence wavelength as two axes shown in FIG. 3A, the scanning is performed on the straight line P indicated by the alternate long and short dash line from the lower left scanning start point P1 to the upper right scanning end point P2. During this scanning, the light intensity signal obtained by the detector 5 is digitized by the A / D converter 6 at regular time intervals and input to the data processing unit 7. Therefore, fluorescence spectrum data (synchronous fluorescence spectrum data) accompanying synchronous scanning with a constant Δλ from the scanning start point P1 to the scanning end point P2 is obtained (step S3).

When a peak due to fluorescence or the like exists in the synchronous fluorescence spectrum obtained at this time, the peak is in a wavelength region where the excitation light spectrum and the fluorescence spectrum in the sample 3 overlap as shown in FIG. It is characterized by appearing sharply (see FIG. 3C). When fluorescence does not occur, the fluorescence spectrum in FIG. 3 (b) has no undulations and is in a state along the baseline, so that no peak appears in the synchronous fluorescence spectrum. For this reason, it can be immediately judged that there is no fluorescence by confirming that there is no peak in this synchronous fluorescence spectrum.

Therefore, in the data processing unit 7, the spectrum creating unit 71 creates a synchronized fluorescence spectrum as shown in FIG. 3 (c) based on the collected synchronized fluorescence spectrum data, and the peak detecting unit 72 is displayed on the synchronized fluorescence spectrum. Peak detection is executed (step S4). Then, it is determined whether or not at least one peak has been detected (step S5). If even one peak has been detected, the process proceeds from step S5 to S6, where the wavelength of the peak top (fluorescence) of the detected peak is detected. Wavelength) is temporarily stored in the wavelength information storage unit 74 as _λEMn . Here, n is a serial number assigned to the detected peak. When only one peak is detected in one synchronous wavelength scan, n is only 1, and a plurality of peaks are detected. In this case, n = 1, 2,...

Depending on the type of sample, the amount of Stokes shift between the excitation light wavelength causing fluorescence and the fluorescence wavelength may be large, and fluorescence may be observed in a wavelength region far away from the excitation light wavelength to the long wavelength side. In that case, when the wavelength difference Δλ between the excitation light wavelength and the fluorescence wavelength is small, no peak appears on the synchronous fluorescence spectrum. Therefore, if it is determined in step S5 that no peak is detected, it is first determined whether or not the wavelength difference Δλ at that time is 200 nm or less, which is the maximum wavelength difference (step S18). If the wavelength difference Δλ is 200 nm or less, the wavelength difference Δλ is increased by 10 nm (step S19), and the process returns to step S3. For example, if no peak is detected on the synchronous fluorescence spectrum at 5 nm, which is the initial value of the wavelength difference Δλ, then the wavelength difference Δλ is changed to 15 nm and the synchronous fluorescence spectrum is acquired again.

Then, when no peak is detected again on the newly acquired synchronous fluorescence spectrum, the acquisition of the synchronous fluorescence spectrum and the peak detection are repeated while further increasing the wavelength difference Δλ by 10 nm. In general, in many fluorescent substances, the Stokes shift amount between the excitation light wavelength and the fluorescence wavelength is smaller than 200 nm. Therefore, when the sample 3 has fluorescence, it is usually unnecessary to repeat acquisition of the synchronous fluorescence spectrum until the wavelength difference Δλ reaches 200 nm, and before that (that is, the wavelength difference Δλ reaches 200 nm). By the time, a synchronous fluorescence spectrum with at least one peak is obtained.

As described above, since the fluorescence wavelength is not usually longer than 200 nm longer than the excitation light wavelength, if it is determined in step S18 that the wavelength difference Δλ exceeds 200 nm, the process proceeds to step S22, and the sample 3 Is determined that no fluorescence is detected, a display output indicating that no fluorescence is detected is performed on the display unit 11 through the fluorescence central control unit 9 (step S17), and the process is terminated.

If one or more fluorescence peak wavelengths are recorded as _λEMn in step S6, then the spectroscope control unit 81 selects _λEMn in ascending order of n and sets the fluorescence wavelength to _λEMn. The side spectroscope 4 is controlled, and the excitation side spectroscope 2 is controlled so as to scan the excitation light wavelength in a predetermined wavelength range while fixing the side spectroscope 4. Thereby, wavelength scanning is achieved so that the vertical dotted line in FIG. Then, based on the light intensity signal obtained by the detector 5 during this wavelength scanning, the spectrum creation unit 71 creates an excitation light spectrum (step S7). This excitation light spectrum shows a wavelength distribution of excitation light that generates fluorescence having a wavelength of _λEMn . Therefore, the peak detector 72 performs peak detection on the excitation light spectrum, and sets the peak top wavelength (excitation light wavelength) of the detected peak as the optimum excitation light wavelength λ _EXn for _obtaining the optimum fluorescence wavelength. Stored in the wavelength information storage unit 74 (step S8).

Next, the spectroscope control unit 81 controls the excitation side spectroscope 2 so as to set the excitation light wavelength to λ _EXn , and the fluorescence side spectroscope so as to scan the fluorescence wavelength in a predetermined wavelength range with this being fixed. 4 is controlled. Thereby, wavelength scanning is achieved so that the horizontal dotted line in FIG. Then, during this scanning, the spectrum creation unit 71 creates a fluorescence spectrum based on the light intensity signal obtained by the detector 5 (step S9). The peak detection unit 72 performs peak detection on the fluorescence spectrum, and stores the peak top wavelength (fluorescence wavelength) of the detected peak in the wavelength information storage unit 74 as the optimum fluorescence wavelength _λEMn ′ (step S10). .

With the above processing, the optimum fluorescence wavelength λ _EMn ′ and the optimum excitation light wavelength λ _{EXn for obtaining} it are obtained, but the peak detected in step S4 is only a fluorescence peak candidate and is derived from fluorescence. That is not guaranteed. This is because in addition to fluorescence, Raman scattered light may be observed in a region below (rightward) the straight line L indicating Rayleigh scattered light in FIG. Therefore, the peak derived from the fluorescence and the peak derived from the Raman scattered light are identified by the measurement and data processing in subsequent steps S11 to S14.

That is, the wavelength of the excitation light applied to the sample 3 is changed to a wavelength λ _EXn +10 nm _obtained by adding 10 nm to the excitation light wavelength λ _EXn (step S11), and the excitation light wavelength is changed to the wavelength λ _EXn +10 nm. The excitation-side spectroscope 2 and the fluorescence-side spectroscope 4 are each controlled so that the fluorescence wavelength is scanned in a predetermined wavelength range while being fixed to λ. And during this wavelength scanning, the spectrum preparation part 71 newly produces a fluorescence spectrum based on the light intensity signal obtained by the detector 5 (step S12). Then, the peak detector 72 performs peak detection on the fluorescence spectrum, and whether or not the peak top wavelength (fluorescence wavelength) of the detected peak is shifted from the previously stored optimum fluorescence wavelength λ _EMn ′. Is determined (step S13). Specifically, an allowable range in consideration of a measurement error or the like is set for the wavelength λ _EMn ′, and if the newly detected fluorescence wavelength is within the allowable range, it may be determined that there is no peak shift.

As shown in FIG. 4 (b), the peak derived from the Raman scattered light is different from the fluorescence peak and appears over a relatively wide excitation light wavelength range, and the relationship between the excitation light wavelength and the Raman scattered light wavelength is substantially band-shaped. Extend. Therefore, when the fluorescence spectrum is acquired by slightly changing the excitation light wavelength to the longer wavelength side as described above, the peak wavelength appearing in the fluorescence spectrum is shifted from the peak wavelength before the excitation light wavelength change. On the other hand, since the fluorescence peak is an isolated peak that maximizes the light intensity at a certain excitation light wavelength and fluorescence wavelength, the fluorescence spectrum is acquired by slightly changing the excitation light wavelength to the longer wavelength side. Even so, the peak wavelength appearing in the fluorescence spectrum is substantially the same as the peak wavelength before the excitation light wavelength change. Therefore, if it is determined in step S13 that the fluorescence wavelength has shifted, it is determined in step S6 that the peak stored as the fluorescence wavelength as _λEMn is not actually derived from fluorescence but from Raman scattered light. Proceed from S13 to S20.

In step S20, it is determined whether or not a peak derived from fluorescence that is not derived from Raman scattered light has already been detected, and if detected, the process proceeds to step S16 described later. On the other hand, if no fluorescence-derived peak has been detected, it is next determined whether or not the measurement and processing from steps S3 to S5 have been performed until the wavelength difference Δλ reaches 200 nm (step S21). If not, a fluorescence-derived peak may still be found, so the process proceeds to step S19 described above, and the wavelength difference Δλ is set again to measure the synchronous fluorescence spectrum. On the other hand, if it is determined Yes in step S21, there is almost no possibility of finding a fluorescence peak, and thus the process proceeds to step S22 described above. As a result, even if a false fluorescence peak derived from Raman scattered light is found prior to a peak derived from true fluorescence, it is identified that the false fluorescence peak is actually a peak derived from Raman scattered light. It is possible to return to the search for the fluorescence peak again. As a result, as long as even one fluorescence-derived peak exists in a range where the wavelength difference Δλ is 200 nm or less, the fluorescence-derived peak can be reliably found even if a false peak due to Raman scattered light exists. it can.

If it is determined in step S13 that the fluorescence peak wavelength is not shifted, it is determined that the peak is not derived from Raman scattered light but is actually derived from fluorescence (step S14), and excitation light is emitted in steps S7 and S8. The combination of the excitation light wavelength λ _EXn obtained from the spectrum and the fluorescence wavelength λ _EMn ′ obtained from the fluorescence spectrum in steps S9 and S10 is stored in the wavelength information storage unit 74 as output information (step S15).

Thereafter, it is determined whether or not another peak wavelength λ _{EM_n} stored in step S6 exists (step S16). For example, if only n = 1 is completed, there is still a peak wavelength of n = 2. Then, the process returns to step S7, and the above-described processing is repeated for the peak wavelength.

If it is determined in step S16 that another peak wavelength _{λEM_n} does not exist, the combination of the excitation light wavelength and the fluorescence wavelength stored as output information in step S15 is displayed through the central control unit 9 to the display unit 11. Are displayed on the display screen in a predetermined format (step S17), and the process is terminated. FIG. 7 shows a display example when a peak derived from fluorescence is found.

The above processing will be described according to a specific measurement example. FIG. 5A is an actual measurement example of a synchronous fluorescence spectrum obtained by measuring a cell filled with tap water as a sample 3. Here, as described above, the scanning start excitation light wavelength was set to 220 nm, the scanning start fluorescence wavelength was set to 225 nm, and the wavelength difference Δλ was maintained at 5 nm until the fluorescence wavelength reached 900 nm. In addition, the slit width in the excitation side spectroscope 2 and the fluorescence side spectroscope 4 is set to 3 nm, and the wavelength width of the extracted light is about 3 nm.

As shown in FIG. 5A, peaks are observed at two wavelengths of 505 nm and 590 nm on the synchronous fluorescence spectrum. Accordingly, two peaks are detected in step S4, and the fluorescence wavelengths for obtaining the optimum excitation light wavelength in step S6 are stored as λ _EM1 = 505 nm and λ _EM2 = 590 nm. FIG. 5B is an actually measured excitation light spectrum obtained by scanning the wavelength of the excitation light with which the sample 3 is irradiated with the fluorescence wavelength fixed at λ _EM1 = 505 nm. On this excitation light spectrum, a peak is observed at a wavelength of 495 nm. Therefore, in step S8, this is stored as the excitation light wavelength λ _EX1 for _{obtaining the} optimum fluorescence wavelength. Next, a fluorescence spectrum is acquired by scanning the fluorescence wavelength to be detected while fixing the wavelength of the excitation light applied to the sample 3 to λ _EX1 = 495 nm. The solid line in FIG. 5C is a fluorescence spectrum obtained by setting the excitation light wavelength to λ _EX1 = 495 nm. In this fluorescence spectrum, since a peak is observed at a wavelength of 510 nm, this is stored as an optimum fluorescence wavelength λ _EM1 ′.

Subsequently, the excitation light wavelength is changed to 505 nm obtained by adding 10 nm to λ _EX1 = 495 nm obtained above, and this is fixed to obtain a fluorescence spectrum. The dotted line in FIG. 5C is this fluorescence spectrum. As can be seen by comparing the spectrum of the solid line and the dotted line, although the peak intensity itself decreases, the peak is detected at a wavelength of 510 nm with almost no peak shift, as before the change of the excitation light wavelength. This indicates that the peak having the maximum intensity at 510 nm is not due to Raman scattered light but originates from fluorescence. Therefore, the optimum excitation light wavelength and fluorescence wavelength for obtaining the fluorescence exhibiting the maximum fluorescence intensity are output as λ _EX11 = 495 nm and λ _EM11 = 510 nm, respectively.

Similarly, with respect to the other fluorescence wavelength λ _EM2 = 590 nm, the excitation light spectrum and the fluorescence spectrum are obtained in order to obtain the optimum excitation light wavelength and fluorescence wavelength, and the optimum excitation light wavelength although the fluorescence intensity itself is small. Wavelength information of λ _EX22 = 560 nm and fluorescence wavelength λ _EM22 = 590 nm is obtained. The wavelength information about the two detected fluorescence peaks is displayed in an easy-to-understand manner, for example, as shown in FIG.

FIG. 8 shows the fluorescence intensity mapping data of the excitation light wavelength range: 300 to 600 nm and the fluorescence wavelength range: 300 to 600 nm, and the excitation light wavelength and the excitation wavelength calculated based on the data for the sample subjected to the above-described wavelength search function. It is the figure which showed the relationship of the fluorescence wavelength. In addition to the wavelength pair of λ _EX11 and λ _EM11 showing the highest fluorescence intensity, the wavelength pair of λ _EX22 and λ _EM22 _having the lowest fluorescence intensity is correctly determined.

In the example of FIG. 8, Raman scattered light does not appear, but even when the wavelengths of the fluorescence-derived peak and the Raman scattered light-derived peak are close by the above-described method, they can be accurately identified. Further, even when the fluorescence-derived peak exists in the very vicinity of the Rayleigh scattered light, the fluorescence-derived peak can be accurately detected. This point will be described with reference to FIG.

FIG. 6A is a diagram showing a fluorescence spectrum obtained by fixing the excitation light wavelength as in the prior art when a fluorescence-derived peak occurs in the vicinity of Rayleigh scattered light. In this case, the wavelength scanning traverses the Rayleigh scattered light, and the spectrum peak derived from the fluorescence overlaps with the large peak derived from the Rayleigh scattered light in the fluorescence spectrum. Therefore, the fluorescence peak cannot be detected, or the fluorescence peak is completely overlapped with the base of the peak derived from Rayleigh scattered light, and the fluorescence peak itself may not be recognized. On the other hand, in the synchronous fluorescence spectrum obtained by keeping the above-mentioned wavelength difference Δλ constant, even if the wavelength difference Δλ is as small as about 5 nm, the influence of Rayleigh scattered light hardly appears and is derived from fluorescence. Only the peak to be detected can be detected. Thus, the distinguishability between the Rayleigh scattered light and the fluorescence peak is also good.

Actually, in many fluorescent substances, the wavelength difference between the excitation light wavelength and the fluorescence wavelength is small, and the fluorescence peak often exists in the vicinity of the Rayleigh scattered light. In the above method, the fluorescence-derived peak can be reliably detected even in such a case. In addition, by acquiring a synchronized fluorescence spectrum while gradually increasing the wavelength difference from a state where the initial value of the wavelength difference is as small as 5 nm, for example, a peak derived from fluorescence can be found with a small number of synchronized fluorescence spectra. it can. For example, even when two fluorescent peaks exist as in the actual measurement example shown in FIG. 8, the two fluorescent peaks can be detected from the synchronous fluorescent spectrum obtained by one synchronous wavelength scanning. In the case of the conventional technique as shown in FIG. 9, it is necessary to acquire a large number of fluorescence spectra in order to detect two fluorescence peaks, and the time shortening effect by the wavelength search method according to the present invention is clear. .

It should be noted that the above-described embodiment is an example of the present invention, and it is a matter of course that modifications, corrections, and additions are appropriately made within the scope of the present invention.

DESCRIPTION OF SYMBOLS 1 ... Light source part 2 ... Excitation side spectroscope 3 ... Sample 4 ... Fluorescence side spectroscope 5 ... Detector 6 ... A / D converter 7 ... Data processing part 71 ... Spectrum preparation part 72 ... Peak detection part 73 ... Fluorescence / Raman Optical identification unit 74 ... wavelength information storage unit 8 ... analysis control unit 81 ... spectroscope control unit 9 ... central control unit 10 ... operation unit 11 ... display unit

Claims

An excitation light spectroscopic means for extracting light of a specific wavelength from light emitted from a light source and irradiating the sample as excitation light, a fluorescence spectroscopic means for receiving the excitation light and dispersing fluorescence emitted from the sample, and its spectrum A spectrofluorometer comprising: a detecting means for detecting the emitted fluorescence;
a) The excitation light spectroscopic means and the fluorescence spectroscopic means so as to scan the excitation light wavelength and the fluorescence wavelength synchronously while maintaining the wavelength difference between the excitation light wavelength and the fluorescence wavelength longer than the wavelength constant. The synchronous fluorescence spectrum acquisition means for creating a synchronous fluorescence spectrum based on the data obtained by the detection means for the synchronous wavelength scanning,
b) performing peak detection on the synchronous fluorescence spectrum, and when a peak is detected, peak detection means for obtaining an approximate value of at least one of the excitation light wavelength and the fluorescence wavelength corresponding to the peak;
c) fixing one of the excitation light wavelength and the fluorescence wavelength whose approximate value is obtained by the peak detection means, and controlling the excitation light spectroscopy means and the fluorescence spectroscopy means so as to scan the other of the excitation light wavelength and the fluorescence wavelength. While obtaining the detailed value of the excitation light wavelength or the fluorescence wavelength based on the data obtained by the detection means, fixing one of the excitation light wavelength or the fluorescence wavelength for which the detailed value was obtained, and fixing the other of the excitation light wavelength or the fluorescence wavelength. A wavelength detail value acquisition means for obtaining the other detail value of the excitation light wavelength or the fluorescence wavelength based on the data obtained by the detection means while controlling the excitation light spectroscopy means and the fluorescence spectroscopy means so as to scan;
d) While controlling the excitation light spectroscopic means and the fluorescence spectroscopic means so as to scan the fluorescence wavelength while fixing the excitation light wavelength to a predetermined wavelength separated from the excitation light wavelength detailed value obtained by the wavelength detailed value acquisition means The fluorescence wavelength is obtained based on the data obtained by the detection means, and the peak detected by the peak detection means based on the fluorescence wavelength and the fluorescence wavelength detail value obtained by the wavelength detail value acquisition means is derived from fluorescence. Fluorescence authenticity determination means for determining whether or not
A spectrofluorometer characterized by comprising:
The spectrofluorometer according to claim 1, wherein
A display processing means for displaying and outputting the excitation light wavelength and the detailed value of the fluorescence wavelength calculated by the wavelength detailed value acquisition means for the peak determined to be fluorescence-derived by the fluorescence authenticity determination means. Spectral fluorometer.
The spectrofluorometer according to claim 1 or 2,
Each time a synchronous fluorescence spectrum for one wavelength difference is obtained, the peak detection means performs peak detection, and the wavelength detailed value acquisition means obtains the detailed values of the excitation light wavelength and the fluorescence wavelength for the detected peak, The fluorescence authenticity determining means determines whether or not the detected peak is derived from fluorescence, and if it is determined that the detected peak is not derived from fluorescence, the synchronized fluorescence spectrum acquiring means increases the wavelength difference. A spectrofluorometer characterized by acquiring the next synchronized fluorescence spectrum.
The spectrofluorometer according to claim 3, wherein
A spectrofluorometer characterized in that, when a peak derived from fluorescence is not detected before the wavelength difference reaches a predetermined maximum wavelength difference, the display processing means performs display output indicating that fluorescence is not detected.
The spectrofluorometer according to any one of claims 1 to 4,
The initial value of the wavelength difference of the synchronous wavelength scanning in the synchronous fluorescence spectrum acquisition means is set to be small so that the peak due to fluorescence is not hidden behind the peak due to Rayleigh scattered light on the synchronous fluorescence spectrum. Fluorescence spectrophotometer.
The spectrofluorometer according to claim 5, wherein
An initial value of the wavelength difference is 5 nm, a fluorescence spectrophotometer.