JPS58117439A - Polarized fluorescence measuring apparatus - Google Patents

Abstract

PURPOSE:To automatically progress the measurement of a polarized fluorescence only through the operation for starting the measurement thereby to reduce the required labor, by arranging such that the operation for driving an excitation light polarizer and a fluorescence polarizer as well as for calculating a correction value and the operation for obtaining a degree of depolarization of fluorescence are sequentially conducted. CONSTITUTION:The excitation light is passed through an excitation light polarizer 2 from the right as shown by an arrow and made incident on a sample cell 1. The fluorescence is observed through a fluorescence polarizer 3 from the direction perpendicular to the direction of the excitation light. The polarizers 2, 3 are mounted on respective frames each having the outer periphery formed into a gear so that the polarizing directions can be changed by pulse motors 4, 5 through gears meshing with the respective frames. A controller 6 changes over the directions of the two polarizers 2, 3 according to a program, measures the fluorescence, carries out a predetermined operation on the measured fluorescence, and displays the operation result on a display 7 or records by means of a recorder 8.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an automated fluorescence polarimeter. To perform fluorescence polarization measurement, a polarizer is placed on the excitation light incident side and the fluorescence measurement side of the sample, and between - measurements, the polarizer on the fluorescence measurement side is moved vertically and horizontally. Switch. Furthermore, prior to measurement, it is necessary to measure the polarization characteristics of the fluorescence spectrometer and obtain a correction value.

The polarization characteristics of a spectrometer vary depending on the wavelength, so when measuring while changing the fluorescence wavelength, it is necessary to measure the polarization characteristics of the fluorescence spectrometer at that measurement wavelength for each measurement. The operation of light polarization measurement becomes even more complicated. Conventionally, this type of fluorescence polarization measurement was performed manually, so
Fluorescence polarization measurements are time consuming, and in particular, tracking changes over time in a sample is only possible if the changes are slow, and there is generally a high possibility of operational errors.

The present invention attempts to automate fluorescence polarization measurement in order to solve the above-mentioned problems in fluorescence polarization measurement. The present invention will be explained below with reference to Examples.

The figure shows an embodiment of the invention. 1 is a sample cell, 2 is an excitation polarizer, and 3 is a fluorescence polarizer. Although the excitation light source and fluorescence spectrometer are not shown in the figure, the excitation light enters the sample cell l from the right side through the excitation polarizer 2 as shown by the arrow, and the fluorescence polarization is generated from the direction perpendicular to the excitation light. Observe the fluorescence through the lens 3. Each of the polarizers 2 and 3 is attached to a frame whose outer periphery is a gear, and the polarization direction of the polarizer can be changed by pulse motors 4 and 5 via gears meshing with the respective fins. . The control device 6 switches the orientation of the two polarizers 2 and 3 according to the program described below, performs photometry of the fluorescent light, and starts a predetermined measurement operation for the photometric value.The control device 6 then performs the following sequence control. Execute.

(1) Place the excitation polarizer 2 in the horizontal (900) position.

In this orientation, the electric field of the excitation light is perpendicular to the plane of the paper in the horizontal direction. This position is set by detection by the frame photocoupler 22 of the excitation polarizer 2, and the direction of the polarizer 2 is detected by the count value of the drive pulse of the pulse motor 4 from this position.

(2) Next, place the fluorescent polarizer 3 in the vertical (0°) position.

In this position detection, as in the case of the polarizer 2, one hole provided in the gear 31 meshing with the frame of the polarizer 3 is connected to the photocoupler 3.
This is done by detecting in step 2. The general position of the polarizer 3 is detected based on the count value of the drive pulses of the pulse motor 5 with this position as a reference.

(3) After completing the settings in (1) and (2) above, perform fluorescence measurement.

Let's take a look at the measured values at this time.

(4) Memorize the steps above.

(5) Set the polarizer 3 to the 90° position.

(6) Perform fluorescence measurement. Let the measured value at this time be 1 above.

(Force: Memorize step 1 above.

(8) Correction value Gl by the above memorized value J-4 and ll
Calculate and store upper l and ll upper.

The above operation is an operation for measuring the polarization characteristics of the fluorescence spectrometer at one wavelength and calculating a correction value.

Let us outline the meaning of this operation. All samples are excited with polarized light such that the electric field is perpendicular to the plane of the diagram.

Since the electric field of the fluorescent light is almost parallel to the electric field of the excitation light, it is emitted within the plane of the drawing, and is hardly emitted in the direction in which the fluorescent light is observed. However, due to the movement of the sample molecules and the time lag between excitation and fluorescence emission, the fluorescence contains components whose electric field direction is slightly different from that of the excitation light. The electric field of the excitation light is perpendicular to the plane of the diagram,
When the direction of fluorescence observation coincides with the direction of this electric field, the fluorescence components whose electric field direction is different from the excitation light described above are included in the same amount both horizontally and vertically as viewed from the observation direction. That is, in this case, the fluorescent light seen from the observation direction is not polarized at all. Therefore, this fluorescent light is used to measure the polarization characteristics of a fluorescent spectrometer.

After completing the operation of calculating the correction value described above, proceed to the sequence of fluorescence polarization measurement of the sample.

(9) Set excitation polarizer 2 to 00.

α [Perform fluorescence measurement. In this case, the operation step (
5), the fluorescent light polarizer 3 is at the 900 position. The measured value at this time is assumed to be above ll.

aυ Memorize the above I/work.

Set α fluorescence polarizer 3 to 00.

031 Perform fluorescence measurements. Edit the measured value at this time//
shall be.

α荀 Memorize the above I/ノ.

(1 [Existence] Determine by fluorescence polarization using the previously determined and memorized correction value G and the above-mentioned fluorescence measurement values Tech//L and Tech///, and display on a display device, or Record by recorder 8.

This completes one measurement sequence. Hereafter mentioned above (1
It is possible to select either a mode in which the operations from ) to a5 are continuously repeated, or a mode in which the process returns to (9) and repeats the operations from (9) to a5. The former mode is used when the wavelength of a fluorescent daylight device is changed for each measurement. Alternatively, even when the wavelength of the fluorescent light is fixed, particular precision is required, and this method is used when the correction value is recalculated every time the measurement is performed. It is also possible to calculate the workpiece S as the calculation in step aQ. The time required for one sequence of the above control operations is 5 to 20 seconds.

In the embodiment described above, both the excitation and fluorescence polarizers are moved back and forth between 00 and 1150 degrees for each measurement.
A configuration may also be adopted in which both polarizers are synchronized and one is rotated in one direction at twice the speed of the other, and the fluorescence photometry value is sampled at an appropriate timing. In this case, if the fluorescence polarizer is rotated twice as fast as the excitation polarizer and both polarizers are set to 90 at the start, then during one rotation of the excitation polarizer, 1 over 1, E L/, There are two chances to measure the force and step (3) mentioned above in the order of work 1 and finish, and take this chance to sample and store the above fluorescence measurement values. After performing this while changing the wavelength of the spectrometer, calculate the correction value G for each wavelength.Next, if the relationship between both polarizers is set to Oo at the start, then during one rotation of the excitation polarizer, Niko////, Il
There is a chance to measure the above-mentioned steps α +, Qt) in the order of 1, 2, 2 and 2, and at this chance, the 0 or excitation polarizer for sampling and storing the fluorescence measurement value is set to 0 or 0. It is also possible to fix it at 9o and rotate only the fluorescent light polarizer. According to this method, fluorescence polarization can be measured at a higher speed than in the above-described embodiment.

The fluorescence polarization measuring device of the present invention has the above-described configuration, and the measurement automatically proceeds by simply starting the measurement, and the measurement can be performed manually by opening the lid of the sample chamber one by one and rotating the polarizer. Compared to the conventional method, the measurement equipment can be set up quickly, there are no errors, it is easy to track changes in the sample over time, and above all, it has a large labor-saving effect.

[Brief explanation of the drawing]

The drawing is a perspective view of essential parts of an apparatus according to an embodiment of the present invention. DESCRIPTION OF SYMBOLS 1... Sample cell, 2... Excitation polarizer, 3... Fluorescence polarizer, 4.5... Pulse motor, 6... Control device, F... Display device, 8...・Meeting pointer. Agent Patent Attorney Kosuke Karu

Claims (1)

[Claims] means for rotating the excitation polarizer and the fluorescence polarizer; an operation of sampling, sampling and storing a photophotometric value; and an operation of sampling a fluorescence photometric value when the excitation polarizer is in a vertical position and the fluorescence polarizer is in a horizontal position and a vertical position; An operation of calculating a correction value based on the data stored by the above-mentioned storing operation, and an operation of introducing the above-mentioned correction value into the fluorescence photometry value sampled when the excitation polarizer is in the vertical position to calculate the degree of fluorescence depolarization, etc. It has an operation program that calculates the above, and drives both the polarizers and performs the correction
A fluorescence polarization measurement device characterized by comprising a control device that sequentially performs an operation of calculating a value and an operation of determining a degree of fluorescence depolarization.