CN104048922A - Method for measuring polarization degree and polarization angle of fluorescence spectrum - Google Patents

Abstract

The invention discloses a method for measuring a polarization degree and a polarization angle of a fluorescence spectrum. The method is used for solving the problems of different measurement polarization angles and errors changing over the fluorescence polarization angles during conventional fluorescence anisotropy measurement. The method is characterized by comprising the steps of regulating a polarization direction of a polarization detection polarizer to be parallel with a polarization direction of an emergent ray (completely polarized light) of a polarizer by adopting a common L measurement manner in fluorescence measurement through rotating the polarization detection polarizer, then clockwise rotating the polarization detection polarizer for 0 DEG, 45 DEG, 90 DEG and 135 DEG, respectively measuring by using a spectrograph and recording the fluorescence spectrum intensity, and calculating the polarization degree and the polarization angle by using the obtained fluorescence spectrum intensity. Compared with the prior art, the method has the beneficial effects that the polarization degree and the polarization angle of the fluorescence spectrum can be simultaneously obtained, and the polarization degree is more accurate.

Description

The measuring method of a kind of fluorescence spectrum degree of polarization and polarization angle

Technical field

The present invention relates to relate to a kind of polarization spectrum analytical technology, particularly the measuring method of a kind of fluorescence spectrum degree of polarization and polarization angle, is applicable to spectrum biological material analysis, belongs to field of spectral analysis technology.

Background technology

Tradition polarized spectrum technology is to carry out biological respinse analysis by surveying degree of polarization or the anisotropy of fluorescence spectrum.Do not measure with the polarization angle that Fluorescence Mechanism and the electronic structure of biology or material are associated, in degree of polarization is measured, acquiescence fluorescence polarization direction is consistent with the polarization direction of incident light, that is: polarization angle is 0 degree.And actual conditions are polarization angle are to change with incident light wavelength, its value is uncertain, and therefore traditional degree of polarization measuring method is inaccurate.Particularly, when the actual polarization angle of fluorescence is 45 while spending, the value of using traditional measurement method to measure degree of polarization is always 0, and its error rate reaches 100%.

In existing document, also do not find the measuring method of the polarization angle of fluorescence, there is the shortcomings such as inaccurate in the measurement of degree of polarization.

Summary of the invention

The object of the invention is, in order to overcome the defect of above-mentioned prior art, to propose the measuring method of a kind of fluorescence spectrum degree of polarization and polarization angle.

The inventive method is achieved through the following technical solutions.

The measuring method of a kind of fluorescence spectrum degree of polarization of the present invention and polarization angle, its concrete steps are as follows:

Step 1: LASER Light Source (1), an inclined to one side polarizer (2), cuvette (3), analyzing polarizer (4) and spectrometer (5) are positioned in same level (using symbol P).In cuvette (3), load testing sample.

Step 2: the continuous laser that LASER Light Source (1) is sent has been incident to inclined to one side polarizer (2), its emergent light is complete polarized light, polarization direction is perpendicular to surface level P;

Step 3: complete polarized light described in step 2 is incident on the testing sample in cuvette (3), inspires fluorescence.The incident direction of described complete polarized light is perpendicular to the side surface (Q1 represents with symbol) of cuvette (3).

Step 4: analyzing polarizer (4) is parallel to the surface (with symbol Q2 represent) adjacent with the surperficial Q1 of cuvette (3).Fluorescence described in step 3 is incident to analyzing polarizer (4), and its emergent ray enters spectrometer (5).

Step 5: the polarization direction of analyzing polarizer (4) is adjusted to parallel with the polarization direction of complete polarized light described in step 2.

Step 6: use spectrometer (5) to measure and record the spectral intensity of fluorescence (representing with symbol I (0)).

Step 7: analyzing polarizer (4) is rotated in a clockwise direction to 45 degree, and uses spectrometer (5) to measure and record photoluminescence spectrum intensity (representing with symbol I (45)) now.

Step 8: analyzing polarizer (4) is rotated to 45 degree along clockwise direction again, and use spectrometer (5) to measure and record photoluminescence spectrum intensity (representing with symbol I (90)) now.

Step 9: analyzing polarizer (4) is rotated to 45 degree along clockwise direction again, and use spectrometer (5) to measure and record photoluminescence spectrum intensity (representing with symbol I (135)) now.

Step 10: use formula (1) to calculate fluorescence polarization degree (DOP represents with symbol), use formula (2) to calculate fluorescence polarization angle (AOP represents with symbol).

$\mathrm{DOP}=\frac{\sqrt{{S_1}^2+{S_2}^2}}{S_0}---\left(1\right)$

Wherein, S ₀=I (0)+(90); S ₁=I (0)-(90); S ₂=2 * I (45)-S ₀.

$\mathrm{AOP}=\frac{1}{2}\arctan \left(\frac{S_2}{S_1}\right)---\left(2\right)$

Step 11: the graph of errors that calculates fluorescence polarization degree and polarization angle.Its process is:

Step 11.1: the check degree of polarization (DOP ' represents with symbol) that calculates fluorescence with formula (3).

${\mathrm{DOP}}^{\′}=\frac{\sqrt{{S_1}^2+{S_3}^2}}{S_0}---\left(3\right)$

Wherein, S ₃=I (45)-I (135).

Step 11.2: the graph of errors (representing with symbol δ DOP) that calculates fluorescence polarization degree with formula (4).

$\mathrm{\δDOP}=\left|\frac{{\mathrm{DOP}}^{\′}-\mathrm{DOP}}{\mathrm{DOP}}\right|---\left(4\right)$

Step 11.3: the check polarization angle (AOP ' represents with symbol) of calculating fluorescence with formula (5).

${\mathrm{AOP}}^{\′}=\frac{1}{2}\arctan \left(\frac{S_3}{S_1}\right)---\left(5\right)$

Step 11.4: the graph of errors (representing with symbol δ AOP) that calculates fluorescence polarization angle with formula (6).

$\mathrm{\δAOP}=\left|\frac{{\mathrm{AOP}}^{\′}-\mathrm{AOP}}{\mathrm{AOP}}\right|---\left(4\right)$

Beneficial effect

Compared with prior art, the invention has the beneficial effects as follows and can obtain fluorescence polarization degree and polarization angle simultaneously, and degree of polarization is more accurate.

Accompanying drawing explanation

Fig. 1 is the position view of arranging of each components and parts in the measuring method of fluorescence spectrum degree of polarization in the specific embodiment of the invention and polarization angle;

Wherein, 1-LASER Light Source, 2-play inclined to one side polarizer, 3-cuvette, 4-analyzing polarizer, 5-spectrometer;

Fig. 2 is that in the specific embodiment of the invention, to use wavelength be that the continuous laser of 473 nanometers triggers olive oil, under 473nm laser triggers, and degree of polarization and the polarization angle spectrogram of the polarization fluorescence spectra that analyzing polarizer obtains while spending at 0 degree, 45 degree, 90 degree and 135;

Fig. 3 is that in the specific embodiment of the invention, to use wavelength be that the continuous laser of 532 nanometers triggers olive oil, under 473nm laser triggers, and degree of polarization and the polarization angle spectrogram of the polarization fluorescence spectra that analyzing polarizer obtains while spending at 0 degree, 45 degree, 90 degree and 135;

Fig. 4 is that in the specific embodiment of the invention, to use wavelength be that the continuous laser of 600 nanometers triggers olive oil, under 473nm laser triggers, and degree of polarization and the polarization angle spectrogram of the polarization fluorescence spectra that analyzing polarizer obtains while spending at 0 degree, 45 degree, 90 degree and 135.

Embodiment

According to technique scheme, below in conjunction with accompanying drawing and embodiment, the present invention is described in detail.

In the present embodiment, utilize fluorescence spectrum degree of polarization that the present invention proposes and the measuring method of polarization angle to measure the degree of polarization of olive oil and polarization angle, its concrete steps are as follows:

Step 1: LASER Light Source 1, an inclined to one side polarizer 2, cuvette 3, analyzing polarizer 4 and spectrometer 5 are positioned over to same level P upper, as shown in Figure 1.In cuvette 3, load olive oil.

Step 2: the wavelength that LASER Light Source 1 is sent is that the continuous laser of 473 nanometers has been incident to inclined to one side polarizer 2, and its emergent light is complete polarized light, and polarization direction is perpendicular to surface level P;

Step 3: complete polarized light described in step 2 is incident on the testing sample in cuvette 3, inspires fluorescence.The incident direction of described complete polarized light is perpendicular to the side surface Q1 of cuvette 3.

Step 4: analyzing polarizer 4 is parallel to the surperficial Q2 adjacent with the surperficial Q1 of cuvette 3.Fluorescence described in step 3 is incident to analyzing polarizer 4, and its emergent ray enters spectrometer 5.

Step 5: the polarization direction of analyzing polarizer 4 is adjusted to parallel with the polarization direction of complete polarized light described in step 2.

Step 6: use spectrometer 5 to measure and record the spectral intensity I (0) of fluorescence.

Step 7: analyzing polarizer 4 is rotated in a clockwise direction to 45 degree, and uses spectrometer 5 to measure and record photoluminescence spectrum intensity I (45) now.

Step 8: analyzing polarizer 4 is rotated to 45 degree along clockwise direction again, and use spectrometer 5 to measure and record photoluminescence spectrum intensity I (90) now.

Step 9: analyzing polarizer 4 is rotated to 45 degree along clockwise direction again, and use spectrometer 5 to measure and record photoluminescence spectrum intensity I (135) now.

Step 10: use formula (1) to calculate fluorescence polarization degree DOP, use formula (2) to calculate fluorescence polarization angle AOP.

Step 11: the graph of errors that calculates fluorescence polarization degree and polarization angle.Its process is:

Step 11.1: the check degree of polarization DOP ' that calculates fluorescence with formula (3).

Step 11.2: the graph of errors δ DOP that calculates fluorescence polarization degree with formula (4).

Step 11.3: the check polarization angle AOP ' that calculates fluorescence with formula (5).

Step 11.4: the graph of errors δ AOP that calculates fluorescence polarization angle with formula (6).

Through the operation of above-mentioned steps, the degree of polarization of polarization fluorescence spectra and polarization angular spectrum as shown in Figure 2.

And then the wavelength sending by LASER Light Source 1 is that the continuous laser of 532 nanometers and 660 nanometers repeats above-mentioned experiment, the fluorescence polarization degree obtaining and polarization angular spectrum are respectively as shown in Figure 3 and Figure 4.

Although described by reference to the accompanying drawings embodiments of the present invention, to those skilled in the art, under the premise without departing from the principles of the invention, can also make some improvement, these also should be considered as belonging to protection scope of the present invention.

Claims (2)

1. a measuring method for fluorescence spectrum degree of polarization and polarization angle, is characterized in that: its concrete steps are as follows:

Step 1: LASER Light Source (1), an inclined to one side polarizer (2), cuvette (3), analyzing polarizer (4) and spectrometer (5) are positioned over to same level P) on; In cuvette (3), load testing sample;

Step 2: the continuous laser that LASER Light Source (1) is sent has been incident to inclined to one side polarizer (2), its emergent light is complete polarized light, polarization direction is perpendicular to surface level P;

Step 3: complete polarized light described in step 2 is incident on the testing sample in cuvette (3), inspires fluorescence; The incident direction of described complete polarized light is perpendicular to the side surface Q1 of cuvette (3);

Step 4: analyzing polarizer (4) is parallel to the surperficial Q2 adjacent with the surperficial Q1 of cuvette (3); Fluorescence described in step 3 is incident to analyzing polarizer (4), and its emergent ray enters spectrometer (5);

Step 5: the polarization direction of analyzing polarizer (4) is adjusted to parallel with the polarization direction of complete polarized light described in step 2;

Step 6: use spectrometer (5) to measure and record the spectral intensity I (0) of fluorescence;

Step 7: analyzing polarizer (4) is rotated in a clockwise direction to 45 degree, and uses spectrometer (5) to measure and record photoluminescence spectrum intensity I (45) now;

Step 8: analyzing polarizer (4) is rotated to 45 degree along clockwise direction again, and use spectrometer (5) to measure and record photoluminescence spectrum intensity I (90) now;

Step 9: analyzing polarizer (4) is rotated to 45 degree along clockwise direction again, and use spectrometer (5) to measure and record photoluminescence spectrum intensity I (135) now;

Step 10: use formula (1) to calculate fluorescence polarization degree DOP, use formula (2) to calculate fluorescence polarization angle AOP;

$\mathrm{DOP}=\frac{\sqrt{{S_1}^2+{S_2}^2}}{S_0}---\left(1\right)$

Wherein, S ₀=I (0)+(90); S ₁=I (0)-(90); S ₂=2 * I (45)-S ₀;

$\mathrm{AOP}=\frac{1}{2}\arctan \left(\frac{S_2}{S_1}\right)---\left(2\right)$

Through the operation of above-mentioned steps, can obtain the value of fluorescence polarization degree DOP and the polarization angle AOP of testing sample.

2. the measuring method of a kind of fluorescence spectrum degree of polarization as claimed in claim 1 and polarization angle, is characterized in that: it also comprises step 11, calculates the graph of errors of fluorescence polarization degree and polarization angle; Its process is:

Step 11.1: the check degree of polarization DOP ' that calculates fluorescence with formula (3);

${\mathrm{DOP}}^{\′}=\frac{\sqrt{{S_1}^2+{S_3}^2}}{S_0}---\left(3\right)$

Wherein, S ₃=I (45)-I (135);

Step 11.2: the graph of errors δ DOP that calculates fluorescence polarization degree with formula (4);

$\mathrm{\δDOP}=\left|\frac{{\mathrm{DOP}}^{\′}-\mathrm{DOP}}{\mathrm{DOP}}\right|---\left(4\right)$

Step 11.3: the check polarization angle AOP ' that calculates fluorescence with formula (5);

${\mathrm{AOP}}^{\′}=\frac{1}{2}\arctan \left(\frac{S_3}{S_1}\right)---\left(5\right)$

Step 11.4: the graph of errors δ AOP that calculates fluorescence polarization angle with formula (6);

$\mathrm{\δAOP}=\left|\frac{{\mathrm{AOP}}^{\′}-\mathrm{AOP}}{\mathrm{AOP}}\right|---\left(4\right)$

Through the operation of above-mentioned steps, can obtain the graph of errors δ DOP of fluorescence polarization degree and the graph of errors δ AOP of polarization angle of testing sample.