Fluorescence polarization measurement method and device

Abstract

To provide a measuring device capable of measuring fluorescence anisotropy or fluorescence polarization degree of a sample in a short time, robustly against noise, and with high sensitivity, and to provide a measuring method.SOLUTION: The measuring device includes a polarizer on the excitation side that extracts linearly polarized light from light from a light source, a sample excited by the linearly polarized light, a fluorescent-side analyzer that extracts a polarization component in a predetermined direction from fluorescence emitted from the sample, a photodetector for detecting light intensity of the polarization component, and a signal processing unit that calculates the fluorescence anisotropy or fluorescence polarization degree of the sample on the basis of the detection value. The fluorescence-side analyzer is provided to ensure free continuous rotation about an incident axis of the fluorescence. The signal processing unit uses a reference signal synchronized with a rotational frequency to perform lock-in detection with respect to the detection value whose period changes with the continuous rotation to acquire an amplitude of an AC component in the signal and acquire a DC component in the signal from the photodetector. From these values, the maximum and minimum values of the period change of the signal are read to calculate the fluorescence anisotropy or fluorescence polarization degree of the sample.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a fluorescence polarization measuring device for measuring the fluorescence anisotropy (or degree of fluorescence polarization) of a fluorescent substance, and in particular to an improvement in the mechanism for switching the polarization direction of the fluorescence. [Background technology]

[0002] Principle of Fluorescence Polarization Measurement Traditionally, fluorescence polarization measurement devices have been used to investigate the association between biopolymers and bioactive molecules, the fluidity of biological membranes, and other such phenomena. The principle of fluorescence polarization measurement is briefly explained below. When a fluorescent substance in a solution is excited by linearly polarized light, if the polarization direction of the linearly polarized light matches the direction of the transition moment of the fluorescent substance, the fluorescent substance is highly likely to be excited and emit fluorescence. On the other hand, if the direction of the transition moment of the fluorescent substance is perpendicular to the polarization direction of the linearly polarized light, the fluorescent substance is not excited and does not emit fluorescence. The fluorescence emitted from an excited fluorescent substance has a polarization that corresponds to the direction of the transition moment of the fluorescent substance.

[0003] When dealing with a sample with isotropic transition moments of fluorescent substances, many fluorescent substances are dispersed in the solution with their transition moments pointing in various directions. Therefore, when the sample is excited with linearly polarized light, the linear polarization of the fluorescence emitted from many fluorescent substances will be biased in a direction that matches the polarization direction of the excitation light. This is called fluorescence anisotropy.

[0004] Configuration of a conventional fluorescence polarization measurement device Patent Document 1 shows the configuration of a typical fluorescence polarization measurement device. A method for measuring fluorescence anisotropy using this measurement device is explained with reference to Figure 8. Light emitted from a light source 2 is linearly polarized by a polarizer 4 and irradiates a sample filled in a sample cell 6 as excitation light L1. A linearly polarized component in a predetermined direction is extracted from fluorescence L2 emitted from the sample by an analyzer 8. This linearly polarized component enters a fluorescence side spectrometer 10 installed in the direction of propagation, where it is split into light components in an appropriate wavelength range. The light split by the fluorescence side spectrometer 10 is further converted into an electrical signal containing fluorescence intensity information by a detector 12 installed in the direction of propagation.

[0005] Here, in order to obtain fluorescence anisotropy, the polarizer 4 and the analyzer 8 are provided with drive mechanisms 14 and 16 that mechanically rotate them by 90 degrees and stop them. The polarization direction of the polarizer 4 is fixed vertically, and the polarization direction of the analyzer 8 is alternately switched between vertical and horizontal by the drive mechanism 16, and the fluorescence intensity (I / / ) and the fluorescence intensity (I ⊥ ) and the fluorescence anisotropy is calculated from this.

[0006] Here, the fluorescence anisotropy r is generally defined by the following formula: r=(I / / -I ⊥ ) / (I / / +2I ⊥ ) …(1) However, I / / is the intensity of the polarization component of the fluorescence L2 polarized in the same direction as the polarization direction of the excitation light L1. ⊥ is the intensity of the polarization component of the fluorescence L2 polarized perpendicular to the polarization direction of the excitation light L1. In equation (1), the denominator is a value proportional to the total fluorescence intensity emitted, and the numerator is the difference between the vertically and horizontally polarized components of the fluorescence. In addition, instead of the fluorescence anisotropy r, the fluorescence polarization P defined by the following formula may also be measured. P=(I / / -I ⊥ ) / (I / / +I ⊥ ) …(2) In both cases, two types of fluorescence intensity (I / / , I ⊥ ) and therefore, here, instruments that measure fluorescence anisotropy, fluorescence polarization, etc. will also be referred to as fluorescence polarization measurement instruments. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] JP 2002-98638 A (Fig. 2) Summary of the Invention [Problem to be solved by the invention]

[0008] In conventional fluorescence polarization measurement devices, a mechanical drive mechanism is used to rotate and stop the polarization direction of the analyzer by 90 degrees, alternately creating a state where the polarization direction is horizontal or vertical. / / ,I ⊥ Since we focused on directly detecting the rotational phase, we used only the intensity signal detected when the analyzer was completely stationary for calculating the fluorescence anisotropy. If the intensity signal during rotation were included, accurate fluorescence intensity would not be obtained.

[0009] However, precisely switching the polarization direction of such an analyzer requires a time of one to several seconds. This means that when attempting to capture changes in the fluorescence anisotropy of a substance over a reaction time of several seconds, the signal available for calculating the fluorescence anisotropy becomes intermittent due to the analyzer switching process, making it difficult to obtain a sufficient signal-to-noise ratio (SN ratio). In fact, there is a need to observe the time change in the fluorescence anisotropy of proteins in reactions in which a complex of several proteins is formed from a single protein. When the molecular weight increases before and after the reaction, the rotational relaxation time of the fluorescence anisotropy due to molecular motion becomes longer, so measuring the time change in the fluorescence anisotropy can accurately capture the reaction process. If the analyzer switching time is long, most of the time is spent rotating and stopping the analyzer. As a result, only a small portion of the intensity signal is actually used, and most of the intensity signal is wasted. This effectively shortens the acquisition time of the necessary fluorescence intensity signal, making it difficult to capture the reaction process with sufficient time resolution. The same problem occurs when measuring fluorescence anisotropy or the like while the polarization direction of the polarizer is alternately switched between vertical and horizontal directions and the polarization direction of the analyzer is fixed.

[0010] The present invention has been made in consideration of the above circumstances, and its purpose is to provide a measuring device and a measuring method that can measure the fluorescence anisotropy or fluorescence polarization of a sample in a shorter time than conventional methods, robust against noise, and with high sensitivity. [Means for solving the problem]

[0011] That is, the fluorescence polarization measurement device according to the present invention comprises a light source, an excitation-side polarizer, a sample cell, and a fluorescence-side analyzer. a fluorescence spectrometer; The optical sensor includes a photodetector and a signal processor. The excitation side polarizer extracts linearly polarized light from the light source. The sample cell is positioned so that the linearly polarized light extracted by the excitation-side polarizer excites the sample inside. The fluorescence analyzer extracts a polarized component in a predetermined direction from the fluorescence emitted from the sample. The fluorescence spectrometer is disposed between the fluorescence analyzer and the photodetector and is used to scan the wavelength of the fluorescence. The photodetector detects the light intensity of the polarized component extracted by the fluorescence-side analyzer and converts it into an electrical signal containing light intensity information. The signal processing unit is configured to calculate the fluorescence anisotropy or the degree of fluorescence polarization based on the electrical signal from the photodetector. Here, the excitation-side polarizer or the fluorescence-side analyzer is provided so as to be continuously rotatable about the axis of incidence. The wavelength scanning speed of the fluorescence spectrometer is set according to the speed of continuous rotation so that the spectrometer rotates continuously at the same wavelength a predetermined number of times or more. The signal processing unit performs lock-in detection using a reference signal synchronized with the rotation frequency for the electrical signal that periodically changes with continuous rotation. So, The maximum and minimum values ​​of the periodic change in the electrical signal are read to determine the fluorescence anisotropy or fluorescence polarization of the sample. During the wavelength scan The present invention is characterized in that it is configured to calculate Furthermore, the direction of travel of light from the light source is defined as the X axis, the excitation-side polarizer is provided so as to be able to change the polarization direction in the direction of the Y axis or the Z axis, The fluorescence analyzer is provided to capture fluorescence emitted from the sample in the Y-axis direction. The polarization direction of the excitation side polarizer is set to the Y axis to excite the sample, and an electrical signal from the photodetector that periodically changes with the continuous rotation of the fluorescence side analyzer is acquired, and a polarization dependency value of the fluorescence detection side is calculated, It is preferable that the polarization direction of the excitation side polarizer is set to the Z axis to excite the sample, and lock-in detection is performed on the electrical signal from the photodetector, which changes periodically with the continuous rotation of the fluorescence side analyzer, using a reference signal synchronized with the rotation frequency, to read the maximum and minimum values ​​of the periodic change in the electrical signal, correct them using the polarization dependency value of the fluorescence detection side, and calculate the fluorescence anisotropy or degree of fluorescence polarization of the sample. Furthermore, when calculating the polarization dependency value (αg, βg) on ​​the fluorescence detection side, the signal processing unit is configured to obtain the average value of one period of the electrical signal from the photodetector as αg, and to obtain the value obtained by multiplying this average value by a sine wave sin(θ+δ) and doubling the result as βg, where θ is twice the reference angle of the polarization direction of the fluorescence side analyzer, and δ is the phase difference. Furthermore, it is preferable that when calculating the fluorescence anisotropy or fluorescence polarization degree of the sample, the signal processing unit is configured to divide the maximum and minimum values ​​read by lock-in detection by a correction value (αg+βgsin(θ+δ)). Furthermore, it is preferable that the signal processing unit is configured to excite the sample by setting the polarization direction of the excitation side polarizer to the Z axis, acquire electrical signals from the photodetector that periodically change with the continuous rotation of the fluorescence side analyzer, acquire an average value of the electrical signals acquired at a certain phase difference δ, multiply the average value by a sine wave sin(θ+δ) and double the result to acquire a value Y, gradually change the phase difference δ to acquire the value Y for each phase difference δ, and acquire the variable δ that takes a maximum when the value Y is treated as a quadratic function of the variable δ as the phase difference δ.

[0012] Furthermore, the fluorescence polarization measurement method according to the present invention includes exciting fluorescent molecules with linearly polarized light by an excitation-side polarizer, extracting a polarized component in a predetermined direction from the fluorescence emitted from the molecules using a fluorescence-side analyzer, and measuring the light of the extracted polarized component. The wavelength of the polarized light component is scanned by a fluorescence spectrometer. The intensity is detected by a photodetector and converted into an electrical signal containing light intensity information. In the signal processing section A measurement method for calculating the fluorescence anisotropy or fluorescence polarization of the molecule based on the electrical signal, extracting a polarized component in a predetermined direction from the fluorescence while continuously rotating the excitation-side polarizer or the fluorescence-side analyzer around the incident axis; a wavelength scanning speed of the fluorescence spectrometer is set in accordance with a speed of the continuous rotation so that the fluorescence spectrometer rotates continuously a predetermined number of times or more in a state of the same wavelength; A lock-in detection is performed using a reference signal synchronized with the rotation frequency of the electrical signal that periodically changes with the continuous rotation of the excitation side polarizer or the fluorescence side analyzer. So, reading the maximum and minimum values ​​of the periodic change of the electrical signal; For each wavelength scan by the fluorescence spectrometer, Fluorescence anisotropy or fluorescence polarization of fluorescent molecules that change over a time range of 0.25 seconds to 1 minute Calculate, It is characterized by: The fluorescence analyzer is provided so as to capture the fluorescence emitted from the sample in the Y-axis direction, with the traveling direction of the light from the light source being the X-axis, The polarization direction of the excitation side polarizer is set to the Y axis to excite the sample, and an electrical signal from the photodetector that periodically changes with the continuous rotation of the fluorescence side analyzer is acquired, and a polarization dependency value of the fluorescence detection side is calculated. It is preferable to set the polarization direction of the excitation side polarizer to the Z axis to excite the sample, perform lock-in detection on the electrical signal from the photodetector, which changes periodically with the continuous rotation of the fluorescence side analyzer, using a reference signal synchronized with the rotation frequency, read the maximum and minimum values ​​of the periodic change in the electrical signal, correct them using the polarization dependency value of the fluorescence detection side, and calculate the fluorescence anisotropy or degree of fluorescence polarization of the sample. Furthermore, when calculating the polarization dependency value (αg, βg) on ​​the fluorescence detection side, the average value of the electrical signal for one period from the photodetector is obtained as αg, and the average value is multiplied by a sine wave sin(θ+δ) and doubled to obtain a value as βg, where θ is twice the reference angle of the polarization direction of the fluorescence side analyzer, and δ is a phase difference. When calculating the fluorescence anisotropy or fluorescence polarization of a sample, it is preferable to divide the maximum and minimum values ​​read by lock-in detection by a correction value (αg+βgsin(θ+δ)). Furthermore, it is preferable to excite the sample by setting the polarization direction of the excitation side polarizer to the Z axis, obtain electrical signals from the photodetector that periodically change with the continuous rotation of the fluorescence side analyzer, obtain the average value of the electrical signals obtained at a certain phase difference δ, multiply the average value by a sine wave sin(θ+δ) and double the result to obtain a value Y, gradually change the phase difference δ to obtain a value Y for each phase difference δ, and obtain the variable δ that takes a maximum when the value Y is a quadratic function of the variable δ as the phase difference δ. [Effects of the Invention]

[0013] In the present invention, the excitation-side polarizer or fluorescence-side analyzer is continuously rotated around the incident light axis, so that the light intensity of the polarized component of the fluorescence extracted by the fluorescence-side analyzer alternates between maximum and minimum values ​​with each quarter rotation. Therefore, with the fluorescence polarization measurement device of the present invention, lock-in detection is performed on the electrical signal from the photodetector to read the maximum and minimum values ​​of the electrical signal, and the fluorescence anisotropy or degree of fluorescence polarization is calculated based on these values. This eliminates the need to rotate the excitation-side polarizer (or fluorescence-side analyzer) back and forth by 90 degrees and then stop it. This allows the fluorescence anisotropy or degree of fluorescence polarization of a sample to be calculated in an extremely short time that corresponds to the rotation speed of the fluorescence-side analyzer, etc., without having to worry about the switching time of the fluorescence-side analyzer, etc. Furthermore, because lock-in detection is performed on the electrical signal from the photodetector, all of the detected electrical signal is used to calculate the fluorescence anisotropy or fluorescence polarization, which prevents the electrical signal used to calculate the fluorescence properties from being intermittent, thereby increasing the signal-to-noise ratio. As described above, the present invention enables measurement of the fluorescence anisotropy or fluorescence polarization of a sample in a short time, robust against noise, and with high sensitivity. In particular, it enables measurement of changes in the fluorescence anisotropy or fluorescence polarization of molecules that occur within a few seconds, robust against noise, and with high sensitivity. [Brief explanation of the drawings]

[0014] [Figure 1] 1 is a diagram showing a schematic configuration of a fluorescence polarization measurement device according to a first embodiment. [Figure 2] 10A and 10B are diagrams illustrating the operation of a sensor provided on the fluorescence-side analyzer. [Figure 3] FIG. 10 is a diagram showing a schematic configuration of a fluorescence polarization measurement device according to a second embodiment. [Figure 4] 4 is a graph showing a phase signal according to an embodiment of the present invention. [Figure 5] 6 is a graph showing the intensity of an AC component signal AC according to the example. [Figure 6] 6 is a graph showing a signal waveform after correction according to the example. [Figure 7] 7 is a graph in which the signal waveform of FIG. 6 is superimposed on a sine wave. [Figure 8] FIG. 1 is a diagram showing a schematic configuration of a conventional fluorescence polarization measurement device. DETAILED DESCRIPTION OF THE INVENTION

[0015] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Preferred embodiments of the present invention will now be described with reference to the accompanying drawings. Fig. 1 shows a schematic configuration of a fluorescence polarization measurement device 20 according to a first embodiment of the present invention. The fluorescence polarization measurement device 20 shown in the figure includes an excitation light source 22, an excitation-side polarizer 24, a sample cell 26, a fluorescence-side analyzer 28, a monochromatic light means 30, a photodetector 32, and a signal processing means 34. In the figure, for convenience, the direction of propagation of the light beam from the light source 22 is represented as the X axis, and the direction of propagation of the detected fluorescence is represented as the Y axis. Fluorescence polarization measurement methods include a "continuous excitation method" in which the excitation light beam is continuously irradiated onto the sample, and a "pulse excitation method" in which the excitation light beam is irradiated in pulses; however, the present invention is based on the continuous excitation method.

[0016] The excitation-side polarizer 24 is placed in front of the direction of irradiation from the excitation light source 22. The excitation-side polarizer 24 is placed so that the polarizer itself can be rotated by 90 degrees, and the polarization direction can be changed in the vertical (Z-axis) or horizontal (Y-axis) direction. Here, we will explain the case where the polarization direction of the polarizer 24 is vertical. A light beam from the light source 22 is incident on the excitation-side polarizer 24, and linearly polarized light in a predetermined polarization direction is emitted from this polarizer. The linearly polarized light then irradiates the sample cell 26 placed in front of it as excitation light L1. A solution sample is placed in the sample cell 26, and the excitation light L1 emitted from the excitation-side polarizer 24 continuously excites the sample, causing the sample to emit fluorescent light L2.

[0017] The fluorescence-side analyzer 28 is placed on the optical axis (Y-axis) of the fluorescence light that is perpendicular to the optical axis (X-axis) of the excitation light L1 from the sample. The incident surface of the analyzer 28 is placed perpendicular to the optical axis of the fluorescence light, and the analyzer 28 is provided so that it can rotate continuously around the incident axis. A stepping motor, for example, is provided as a rotation drive mechanism 34 for the analyzer 28. During one rotation of the analyzer 28, there are two times when its polarization direction becomes parallel to the polarization direction of the excitation-side polarizer 24 (in the Z-axis direction) and two times when it becomes perpendicular to the polarization direction of the excitation-side polarizer 24 (in the X-axis direction). A portion of the fluorescence L2 from the sample enters the analyzer 28, and linearly polarized light with a polarization state that corresponds to the rotation position of the analyzer 28 is emitted from the analyzer 28.

[0018] As shown in Figure 1, the monochromatic light means 30 is composed of, for example, a fluorescence spectrometer, which disperses the linearly polarized light from the analyzer 28 and extracts light components in an appropriate wavelength range. Alternatively, a filter may be provided instead of the fluorescence spectrometer. The filter can remove scattered light from the excitation light L1. The light leaving the monochromatic light means 30 enters a photodetector 32, which may be, for example, a photomultiplier tube (PMT), and is converted by the photodetector 32 into an electrical signal containing fluorescence intensity information. As the fluorescence-side analyzer 28 continuously rotates, the photodetector 32 outputs a maximum electrical signal when the polarization direction becomes parallel to the polarization direction of the excitation-side polarizer 24, and outputs a minimum electrical signal when the polarization direction becomes perpendicular to the polarization direction of the excitation-side polarizer 24. Therefore, the photodetector 32 outputs an electrical signal that changes at a frequency twice the rotation frequency of the analyzer 28.

[0019] The following describes the signal processing means 34, which calculates the fluorescence anisotropy or the degree of fluorescence polarization of the sample by lock-in detection of the electrical signal from the photodetector 32. The main components of the signal processing means 34 are a means (DC amplifier 36) for extracting a direct current signal component DC from the electrical signal from the photodetector 32, a means (AC amplifier 38) for extracting an alternating current signal component AC, a means (lock-in amplifier 40) for acquiring an amplitude signal of the alternating current signal component AC, and a calculation means (CPU 42) for calculating the fluorescence anisotropy or the degree of fluorescence polarization based on the direct current signal and the amplitude signal.

[0020] The electrical signal obtained by the photodetector 32 is input to the preamplifier 44 where it is amplified. The signal output from the preamplifier 44 enters the DC amplifier 36, which amplifies and extracts only the DC signal component DC. This DC signal component DC has a value equivalent to the time average value of the electrical signal, and is input to the PMT voltage application circuit 46. The PMT voltage application circuit 46 can control the gain of the photodetector 32 by adjusting the voltage applied to the photodetector 32 so that the DC signal component DC is observed at a constant value. The electrical signal from the photodetector 32 also enters the AC amplifier 38 via the preamplifier 44, where the AC signal component AC is amplified. The AC signal component AC leaving the AC amplifier 38 enters the lock-in amplifier 40.

[0021] The lock-in amplifier 40 performs lock-in detection of the AC signal component AC using a reference signal from a reference signal generator 48. The reference signal generator 48 outputs a sine wave signal having a frequency (2f) that is twice the rotation frequency f of the analyzer 28 to the lock-in amplifier. In this embodiment, to match the phase of the AC signal component AC and the reference signal, the fluorescent-side analyzer 28 is provided with a sensor (photointerrupter) 50 that detects its rotational position.

[0022] FIG. 2 is an explanatory diagram of the operation of the sensor 50 as the analyzer 28 rotates. As shown in FIG. 2A, the analyzer 28 is held by a rotating disk 52 and continuously rotates integrally with it. The detection surface of the sensor 50 is positioned facing the rotating disk 52, and a trigger signal is output when a notch 54 formed in one location on the rotating disk 52 is detected. FIG. 2B illustrates the relationship between the output of the trigger signal and the temporal change in the polarization direction of the analyzer 28. At the timing indicated by II in FIG. 2B, the polarization direction of the analyzer 28 becomes horizontal (H), and the sensor 50 detects the notch 54 and outputs a trigger signal. At the timing indicated by III, the polarization direction of the analyzer 28 becomes vertical (V). Thus, during one rotation of the analyzer until the output of the next trigger signal, the polarization direction changes from H → diagonal downward to V → diagonal upward to H → diagonal downward to V → diagonal upward to H. The signal processing means 34 processes the fluorescence intensity signal acquired during one rotation of the analyzer 28 as two cycles. FIG. 1A shows the positional relationship between the rotational position of the analyzer 28 and the sensor at the timings indicated by I to III in FIG. 1B.

[0023] 1, the reference signal generator 48 uses the trigger signal from the sensor 50 to output a reference signal with double the frequency and in phase with the AC signal component AC. Note that the sensor 50 is used to compensate for the synchronization between the reference signal and the AC signal component AC. If the reference signal generator 48 can separately generate a reference signal that is synchronized with the AC signal component, the sensor 50 is not necessary.

[0024] In the lock-in amplifier 40, the AC signal component AC is multiplied by a reference signal, and a DC voltage is obtained through a low-pass filter or the like. From this DC voltage, an amplitude signal of the AC signal component AC is obtained. The amplitude signal output from the lock-in amplifier 40 is input to an amplifier 56 where it is amplified. It is then converted into numerical data by an A / D converter 58. The amplitude signal converted into numerical data is input to the CPU 42 via the I / O.

[0025] On the other hand, the DC signal component DC from the DC amplifier 36 is also converted into numerical data by the A / D converter 60. Then, the DC signal converted into numerical data is input to the CPU 42 via the I / O. The CPU 42 calculates two types of fluorescence intensities (I / / ,I ⊥ ) is calculated, and the fluorescence anisotropy is calculated using formula (1), or the degree of fluorescence polarization is calculated using formula (2). If the angle of the polarization direction of the fluorescence side analyzer 28 with respect to the vertically polarized light on the excitation side is Φ, then the vertically polarized light on the fluorescence side is Φ=0. If the fluorescence intensity from the photodetector is I(Φ) using Φ, then the fluorescence intensity (I / / ,I ⊥ ) is related to the following equation:

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[0026] 1, the measuring device 20 of this embodiment is provided with drive control units 62 and 64 that respectively control the drive of the rotary drive mechanism 34 and the drive of the monochromatic light means 30. The drive control units 62 and 64 set the wavelength scanning speed of the monochromatic light means 30 to a speed corresponding to the rotation speed of the analyzer 28, so that the analyzer 28 rotates continuously a predetermined number of times or more in a state of substantially the same wavelength.

[0027] By configuring the fluorescence polarization measurement device 20 as in this embodiment, the fluorescence side analyzer 28 rotates continuously, so that the light intensity of the polarization component of the fluorescence extracted by the fluorescence side analyzer 28 alternates between maximum and minimum values ​​every quarter rotation. Furthermore, since the electrical signal from the photodetector 32 is detected by lock-in detection, the maximum and minimum values ​​of the electrical signal can be read. The maximum value is the fluorescence intensity (I) when the polarization directions of the excitation light and the fluorescence are the same. / / ), and the minimum value is the fluorescence intensity (I ⊥ ) The fluorescence anisotropy (r) or the degree of fluorescence polarization (P) can be calculated based on these values. As a result, it is no longer necessary to rotate and stop the fluorescence side analyzer by 90 degrees. Without having to worry about the switching time of the fluorescence side analyzer 28, the fluorescence anisotropy or the degree of fluorescence polarization of the sample can now be calculated in an extremely short time that corresponds to the rotation speed of the fluorescence side analyzer 28.

[0028] Furthermore, because lock-in detection is performed on the electrical signal from the photodetector 32, all of the detected electrical signal is used to calculate the fluorescence anisotropy or the degree of fluorescence polarization. This prevents the electrical signal used to calculate these fluorescence properties from being intermittent, and increases the signal-to-noise ratio of the measured values. As described above, the fluorescence anisotropy or the degree of fluorescence polarization of a sample can be measured in a short time, robustly against noise, and with high sensitivity.

[0029] FIG. 3 shows a schematic configuration of a fluorescence polarization measurement device 120 according to a second embodiment of the present invention. The optical elements that make up the optical system for excitation light and fluorescence are the same as those in the previously described embodiments. The difference between the two is the configuration of the signal processing unit that performs lock-in detection of the electrical signal from the photodetector 32. The previously described embodiment is configured for electrical (circuitry) lock-in detection as shown in FIG. 1. In contrast, in this embodiment, the hardware configuration is simplified, and the CPU 142 performs arithmetic processing for lock-in detection, as shown in FIG. 3.

[0030] That is, in the signal processing unit 134 of this embodiment, the electrical signal from the photodetector 32 is amplified by the preamplifier 44, converted into numerical data by the A / D converter 60, and then input to the CPU 142 via the I / O. The CPU 142 also exchanges signals with the drive control unit 62 for the analyzer 28 and the PMT application voltage circuit 46 for the photodetector 32. The CPU 142 generates a synchronization signal from its own reference signal or the like, and acquires the output signal of the photodetector 32 synchronized with the rotational drive of the analyzer 28 as numerical data. The CPU 142 then performs arithmetic processing to read the direct current signal component DC and the alternating current signal component AC of the fluorescence intensity I(Φ) from the acquired numerical data, thereby being able to calculate the fluorescence anisotropy or the degree of fluorescence polarization of the sample. The CPU can also perform calculations related to the correction of fluorescence polarization characteristics and the acquisition of phase synchronization data, which will be described later.

[0031] In the above embodiments, the excitation-side polarizer 24 is fixed, and the fluorescence-side analyzer 28 is rotated to detect the fluorescence anisotropy of the sample based on the electrical signal of the photodetector 32. However, the device and measurement method of the present invention can also be applied to a case in which the excitation-side polarizer 24 is rotated by the drive mechanism 14 in FIG. 1, the fluorescence-side analyzer 28 is fixed, the electrical signal of the photodetector 32 is detected, and the fluorescence anisotropy of the sample is obtained based on this signal. In FIG. 3, if the CPU 142 exchanges signals with the drive control unit 15 of the excitation-side polarizer 24 via the I / O, it becomes possible to rotate the excitation-side polarizer 24 and detect fluorescence while the fluorescence-side analyzer 28 is fixed.

[0032] (Example) An example is shown in which an FPGA, a type of programmable logic device, is used as the CPU 42 in FIG. 1 to perform rotational driving of the analyzer and acquisition of intensity signals. This FPGA acquires two types of fluorescence intensities (I / / ,I ⊥) can be calculated. Here, a method of lock-in detection that effectively utilizes the timing of the trigger signal of the sensor 50 will be described. That is, in this embodiment, data sampling of the photodetector 32 is performed at intervals of 5 msec, and 50 pieces of data are acquired per half rotation of the analyzer 28, which are then processed as data for one cycle. From these data, the fluorescence intensity (I / / ,I ⊥ ) is calculated.

[0033] The main specifications of the measuring device according to this embodiment are as follows: Stepping motor: 0.9 degrees / step (half-step drive) Motor drive clock: 3200Hz Analyzer: 2 rotations / sec (2Hz) Sampling interval: 5 msec AD converter conversion speed: 40000Hz

[0034] First, in the above equation (3), if θ=2Φ is defined, the following equation is obtained.

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[0035] However, the actual fluorescence intensity signal I(Φ) is the product of the signal due to the polarization dependency of the fluorescence detection system and the signal due to the fluorescence anisotropy of the sample, which is the original measurement target (see the following equation).

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[0036] Correction of polarization characteristics of the fluorescent light The excitation-side polarizer 24 can change its polarization direction to either vertical or horizontal by the driving mechanism 14. Therefore, the excitation light is polarized in two different directions, and the fluorescence intensity (I / / ,I ⊥ ) to correct for the polarization dependency of the fluorescence detection system. When the excitation-side polarizer 24 is set horizontally (Y-axis), the horizontally polarized intensity (X-axis) and vertically polarized intensity (Z-axis) of the fluorescence will, in principle, coincide. When the analyzer 28 is rotated in this state, the signal Ig(Φ) will contain only the polarization characteristics of the fluorescence detection side (monochromatic light means 30 and photodetector 32).

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[0037] In this example, the average value for one cycle (50 points) is αg, and this is multiplied by the sine wave sin(θ+δ) and doubled to obtain βg. For one cycle of data, if the fluorescence intensity at the time when sensor 50 changes from off to on is defined as I(0), and the fluorescence intensity of 50 points (j=0 to 49) for one cycle is represented as I(j), the following equation is obtained:

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[0038] Here, the G factor is defined as the vertically polarized fluorescence intensity (αg + βg,I HV ) versus horizontally polarized fluorescence intensity (αg-βg,I HH When using the G factor for correction, it is advisable to normalize αg to 1.0.

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[0039] In equation (9), the corrected signal I * The average value of one period of s (50 points) is αa, and multiplying this by the sine wave sin(θ+δ) and doubling it results in βa, which can be expressed by the following equation.

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[0040] In this way, αa and βa are calculated, and the fluorescence intensity (I / / ,I ⊥ ) can be calculated.

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[0041] Next, the acquisition of phase synchronization data will be described. When acquiring phase synchronization data (phase difference δ), it is necessary to acquire it with the maximum when the polarization of the excitation side is vertical and the minimum when it is horizontal. Failure to do so will cause a 180-degree phase shift. In this embodiment, the unit of the phase difference δ is radian, but the actual measurement is performed at mechanical intervals of 3.6 degrees, which is twice the angle of equation (6), that is, at 7.2 degrees. Therefore, the explanation will be given using the data interval unit Δ, which is the phase difference δ divided by 7.2 degrees.

[0042] To acquire phase-locked data, the excitation wavelength and the fluorescence wavelength are specified in addition to the sample. (1) A dilute ethylene glycol solution of rhodamine B is set as a sample. (2) The excitation wavelength is set to 550 nm and the fluorescence wavelength is set to 620 nm. (3) The excitation side polarizer is set vertically. (4) 2000 points are measured at 5 msec intervals by triggering from sensor 50. (5) Using the phase difference δ in equation (7) as Δ=-22 (δ=7.2×Δ), calculate βg for 20 rotations, and take the average of 40 values ​​as Y(-22). (6) Similarly, let Δ=-21,-20,...,-4,-3,-2, and find Y(-21), Y(-20),..., Y(-4), Y(-3), Y(-2). (7) Assuming Y is a quadratic function of Δ, find the maximum ΔMAX.

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[0043] The signal Ig(θ) shown in equation (6) is a function of the angle θ of the analyzer 28. The phase difference of the signal is δ in equation (6). When δ is positive, the phase advances, and when δ is negative, the phase lags. See Figure 4. The actual signal is triggered when the analyzer sensor 50 turns from off to on, and 100 pieces of data are continuously acquired at 5 msec intervals. This corresponds to one rotation of the analyzer 28. If this fluorescence side signal is I(0), ..., I(99), equation (6) can be expressed as follows:

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[0044] Here, δ is the phase difference between the analyzer sensor 50 and the signal expressed in degrees, and Δ is the phase difference between the sensor 50 and the signal expressed in terms of the data sampling period. If the installation accuracy of the sensor 50 is ±2.5 degrees, the actual trigger is obtained when the analyzer 28 has a horizontal angle of ±2.5 degrees. δ should be -45±2.5 degrees, and Δ should be -12.5±0.7 periods. Since this phase difference is considered to be almost constant, the same value is used until this operation is performed again. In this example, ΔMAX=−11.4818 was obtained, as shown in FIG.

[0045] Using the phase-locked data measured in this way, the G factor (Gj) was calculated based on equation (10), and an example of a signal waveform corrected for the fluorescence side polarization characteristics is shown in Figure 6. The signal waveform in this figure was successfully fitted with a sine wave. Figure 7 shows a graph superimposing the corrected signal waveform and the sine wave. The αa and βa obtained from the signal waveform in Figure 6 were αa = 920.095 and βa = 366.863. From these values, the fluorescence intensity (I / / ,I ⊥ The P value (fluorescence polarization) was 0.398722903 and the r value (fluorescence anisotropy) was 0.306559346, which showed good agreement with the values ​​measured manually using the same sample.

[0046] (Change in molecular fluorescence anisotropy) In particular, the fluorescence polarization measurement device 20 of this embodiment can measure changes in the fluorescence anisotropy of molecules (e.g., molecular weights of about 3000) that occur over a period of several seconds, robustly against noise and with high sensitivity. When fluorescence polarization measurement is used for biopolymers, for example, the fluorescence from fluorescent molecules bound to proteins or their aggregates is often measured. For this reason, "fluorescence anisotropy," which is relatively insignificant when measuring fluorescent molecules alone, becomes extremely important when measuring polymers. This is because the larger the fluorescent molecule, the longer its rotational relaxation time, and hydrogen bonding of water molecules to the fluorescent molecule also tends to lengthen the rotational relaxation time.

[0047] For example, when measuring polymers such as proteins, (1) the speed of molecular rotation of the polymer in solution can be analyzed from its fluorescence anisotropy to determine the fluidity of the polymer, or (2) the change in rotational relaxation time of a biopolymer before and after a reaction such as an antigen-antibody reaction can be analyzed from its fluorescence anisotropy or fluorescence polarization to determine whether a product has been produced.

[0048] Changes in molecular fluidity and biological reactions often occur within a few seconds, making it difficult for conventional fluorescence polarization measurement devices to measure fluorescence anisotropy during such rapid changes and reactions. By using the measurement device 20 of this embodiment, it is possible to obtain a high signal-to-noise ratio (SN) for the measurement value and achieve good time resolution, even when fluorescence anisotropy or fluorescence polarization changes within a few seconds to several tens of seconds (for example, within a time range of 0.25 seconds to 1 minute). [Explanation of symbols]

[0049] 20, 120 Fluorescence polarization measurement device 22 Excitation light source 24 Excitation side polarizer 26 Sample Cell 28 Fluorescence side analyzer 32 Photodetector 34, 134 Signal processing means

Claims

1. A light source and an excitation-side polarizer that extracts linearly polarized light from the light source; a sample cell arranged so that the linearly polarized light extracted by the excitation side polarizer excites a sample therein; a fluorescence analyzer for extracting a polarized component in a predetermined direction from the fluorescence emitted from the sample; a photodetector that detects the light intensity of the polarized component extracted by the fluorescence-side analyzer and converts it into an electrical signal having light intensity information; a fluorescence spectrometer disposed between the fluorescence analyzer and the photodetector for wavelength scanning the fluorescence; a signal processing unit configured to calculate the fluorescence anisotropy or the degree of fluorescence polarization of the sample based on the electrical signal from the photodetector, the propagation direction of light from the light source is defined as an X axis, the excitation-side polarizer is provided so as to be able to change the polarization direction in a Y-axis direction or a Z-axis direction, and the fluorescence-side analyzer is provided so as to capture fluorescence emitted from a sample in the Y-axis direction, the fluorescence-side analyzer is provided so as to be continuously rotatable around the incident axis; a wavelength scanning speed of the fluorescence spectrometer is set according to a speed of the continuous rotation so that the fluorescence-side analyzer is continuously rotated a predetermined number of times or more in a state of the same wavelength; The signal processing unit The polarization direction of the excitation side polarizer is set to the Y axis to excite a sample, and an electrical signal from the photodetector that periodically changes with the continuous rotation of the fluorescence side analyzer is acquired, and polarization-dependent values ​​(αg, βg) on ​​the fluorescence detection side are calculated; the polarization direction of the excitation side polarizer is set to the Z axis to excite the sample, and lock-in detection is performed on the electrical signal from the photodetector, which periodically changes with the continuous rotation of the fluorescence side analyzer, using a reference signal synchronized with the rotation frequency, to read the maximum and minimum values ​​of the periodic change in the electrical signal, and correct the values ​​using the polarization dependency values ​​(αg, βg) of the fluorescence detection side, thereby calculating the fluorescence anisotropy or the degree of fluorescence polarization of the sample during the wavelength scanning; When calculating the polarization dependency values ​​(αg, βg) on ​​the fluorescence detection side, an average value of the electrical signal for one period from the photodetector is obtained as αg, and the average value is multiplied by a sine wave sin(θ+δ) and doubled to obtain a value as βg, where θ is twice the reference angle of the polarization direction of the fluorescence-side analyzer, and δ is the phase difference. A fluorescence polarization measuring device characterized in that, when calculating the fluorescence anisotropy or fluorescence polarization of a sample, the maximum and minimum values ​​read by lock-in detection are divided by a correction value (αg + βg sin(θ + δ)).

2. 2. The fluorescence polarization measurement device according to claim 1, The signal processing unit The polarization direction of the excitation side polarizer is set to the Z axis to excite the sample, and an electrical signal from the photodetector that periodically changes with the continuous rotation of the fluorescence side analyzer is acquired. An average value of the electrical signal acquired at a certain phase difference δ is obtained, and the average value is multiplied by a sine wave sin(θ+δ) and doubled to obtain a value Y. The phase difference δ is gradually changed to obtain a value Y for each phase difference δ. A fluorescence polarization measuring device characterized in that it is configured to acquire, as the phase difference δ, the variable δ that takes a maximum when the value Y is a quadratic function of the variable δ.

3. A measurement method comprising: exciting a fluorescent molecule with linearly polarized light from an excitation-side polarizer; extracting a polarized component in a predetermined direction from the fluorescence emitted from the molecule using a fluorescence-side analyzer; wavelength-scanning the extracted polarized light component with a fluorescence spectrometer; detecting the light intensity of the wavelength-scanned polarized component with a photodetector and converting it into an electrical signal carrying light intensity information; and calculating the fluorescence anisotropy or degree of fluorescence polarization of the molecule based on the electrical signal in a signal processing unit, The fluorescence analyzer is provided so as to capture the fluorescence emitted from the molecules in the Y-axis direction, with the traveling direction of the light from the light source being the X-axis, extracting a polarized component in a predetermined direction from the fluorescence while the fluorescence-side analyzer is continuously rotated around the incident axis; a wavelength scanning speed of the fluorescence spectrometer is set in accordance with a speed of the continuous rotation so that the fluorescence-side analyzer is continuously rotated a predetermined number of times or more in a state of the same wavelength; The polarization direction of the excitation-side polarizer is set to the Y-axis to excite the molecule, and an electrical signal from the photodetector that periodically changes with the continuous rotation of the fluorescence-side analyzer is acquired, and the polarization-dependent values ​​(αg, βg) of the fluorescence detection side are calculated; the polarization direction of the excitation side polarizer is set to the Z axis to excite the molecule, and lock-in detection is performed on the electrical signal from the photodetector, which periodically changes with the continuous rotation of the fluorescence side analyzer, using a reference signal synchronized with the rotation frequency, to read the maximum and minimum values ​​of the periodic change in the electrical signal, and correct the values ​​using the polarization dependency (αg, βg) on ​​the fluorescence detection side to calculate the fluorescence anisotropy or degree of fluorescence polarization of the fluorescent molecule, which changes over a time range of 0.25 seconds to 1 minute for each wavelength scan by the fluorescence spectrometer; When calculating the polarization dependency values ​​(αg, βg) on ​​the fluorescence detection side, the average value of the electrical signal for one period from the photodetector is obtained as αg, and the average value is multiplied by a sine wave sin(θ+δ) and doubled to obtain a value as βg; where θ is twice the reference angle of the polarization direction of the fluorescence-side analyzer, and δ is the phase difference. A fluorescence polarization measurement method characterized in that, when calculating the fluorescence anisotropy or fluorescence polarization of the molecule, the maximum and minimum values ​​read by lock-in detection are divided by a correction value (αg + βg sin(θ + δ)).

4. 4. The fluorescence polarization measurement method according to claim 3, The polarization direction of the excitation-side polarizer is set to the Z-axis to excite the molecules, and an electrical signal from the photodetector is acquired that periodically changes with the continuous rotation of the fluorescence-side analyzer; An average value of the electrical signal acquired at a certain phase difference δ is obtained, and the average value is multiplied by a sine wave sin(θ+δ) and doubled to obtain a value Y. The phase difference δ is gradually changed to obtain a value Y for each phase difference δ. A fluorescence polarization measurement method, characterized in that the variable δ that takes a maximum when the value Y is a quadratic function of the variable δ is obtained as the phase difference δ.

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