CN102680438B - Quantitative measurement method for fluorescence lifetime and fluorescence dynamic anisotropic parameters - Google Patents

Abstract

The present invention relates to the fluorescent characteristic measurement methods of fluorescent samples, and in particular to a kind of method for quantitative measuring of fluorescence lifetime and fluorescence dynamic anisotropic parameters, comprising: pulse or sinusoidal polarized excitation light excitation sample generate fluorescence; Detect the polarized fluorescence light intensity for being parallel to pulse or sinusoidal polarized excitation light respectively by photodetector

With the polarized fluorescence light intensity perpendicular to pulse or sinusoidal polarized excitation light ; Calculate the average life span of sample

, initial time fluorescence anisotropy

, infinite moment of time fluorescence anisotropy

With the fluorescent molecule spin correlation time of sample

. Effectively quantitative measurment it can go out the fluorescence lifetime of all samples by means of the present invention, the invention proposes all parameter quantitative measurement methods of the fluorescence anisotropy of its fluorescent samples simultaneously, therefore the present invention has very big application value to the interaction in quantitative study active somatic cell between large biological molecule such as protein.

Description

The method for quantitative measuring of a kind of fluorescence lifetime and the dynamic anisotropic parameters of fluorescence

Technical field

The present invention relates to the fluorescent characteristic measuring method of fluorescent samples, be specifically related to the method for quantitative measuring of a kind of fluorescence lifetime and the dynamic anisotropic parameters of fluorescence.

Background technology

Due to the appearance of fluorescence protein molecule (FP), utilize the biophysical chemistry problem of fluorescence labeling and fluorescence measurement techniques research active somatic cell and biological tissue, become one of focus of life science.Along with the development of contemporary optics and computer technology, it is found that fluorescence anisotropic and fluorescence lifetime measurement technology can break through the restriction of Abbe principle, characterize between the inner biomacromolecule of active somatic cell and interact.As F.S.Wouters, " The physics and biology of fluorescence microscopy in the life science ", in Contemporary Physics (2006), Vol.47, described in pp.239-255, because fluorescence anisotropic and fluorescence lifetime measurement technology have the advantages such as good, highly sensitive, the anti-ground unrest interference performance of signal-selectivity is strong, therefore at biomedical sector, be widely used.

Fluorescence life τ refers to the averaging time of fluorescence molecule in excited state, and the fluorescence of fluorescence molecule transmitting is by certain time after exciting light stops, and its intensity time presents exponential damping.

As Q.S.Hanley, V.Subramaniam, D.J.Arndt-Jovin, and T.M.Jovin, " Fluorescence lifetime imaging:Multi-point calibration; minimum resolvable differences; and artifact suppression ", in Cytometry (2001), Vol.43, described in pp.248-260, by measuring the fluorescence lifetime of biological sample, we can be to the many biophysical chemistry parameters in target molecule microenvironment of living in, as pH value and ion concentration (Ca ⁺, Cl ^-deng) distribute and carry out quantitative measurment.

Fluorescence anisotropy principle refers to that fluorescence molecule in solution is when being subject to polarized light and exciting, and the fluorescence inspiring has partial polarization.

As D.S.Lidke, P.Nagy, B.G.Barisas, R.Heintzmann, J.N.Post, K.A.Lidke, A.H.A.Clayton, D.J.Arndt-Jovin, and T.M.Jovin, " Imaging molecular interactions in cells by dynamic and static fluorescence anisotropy (rFLIM and emFRET) ", in Meeting on Intermolecular Associations in2D and3D (Portland press, Nottingham, England, 2003), Vol.31, described in pp.1020-1027, by measuring the fluorescence anisotropy of sample, we can quantitative examination active somatic cell in biomacromolecule as the interaction between protein.

Jameson.D.M, Gartton, E.and Hall.R, " The measurement and analysis of heterogeneous emissions by multifrequency phase and modulation fluorometry ", Appl.Spectrosc.Rev.20.55-106 (1984) and Joseph R.Lakowicz, Principles of Fluorescence Spectroscopy. (Springer, New York, 2006), 3rd ed has proposed the fluorescence lifetime method of quantitative measurment sample, but the fluorescence process for sample complexity, these measuring methods effectively quantitative measurment go out its fluorescence lifetime, and for the dynamic anisotropic parameters r of fluorescence ₀,

with

quantitative measurment, still do not provide at present concrete measuring method.

Summary of the invention

For the problems referred to above, the object of this invention is to provide the dynamic anisotropic parameters r of a kind of quantitative measurment fluorescent life-span and fluorescence ₀, with

method.

A method for quantitative measuring for fluorescence lifetime and the dynamic anisotropic parameters of time-resolved fluorescence, adopts measuring equipment to measure fluorescent samples, comprises following concrete steps:

Step 1, adopts pulse or sinusoidal polarized excitation light to irradiate fluorescent samples, and excited sample produces fluorescence;

Step 2, measures respectively the time resolution polarized fluorescence light intensity I that is parallel to pulse or sinusoidal polarized excitation light by photodetector _∏(t) with perpendicular to the time resolution polarized fluorescence light intensity I of pulse or sinusoidal polarized excitation light _⊥(t);

Step 3, total fluorescent intensity is I _∑(t)=I _∏(t)+2I _⊥(t);

Step 4, for angular frequency corresponding to sample, I _∑(t) position (ρ in polar coordinates phase space _∑, θ _∑) be,

Step 5, the fluorescence life τ of sample is,

Step 6, I _∏and I (t) _⊥(t) position (ρ in polar coordinates phase space _∏, θ _∏) and (ρ _⊥, θ _⊥) be respectively,

Step 7, the fluorescence molecule spin correlation time of sample for,

Wherein, according to the I in step 6 _∏(t) position (ρ in polar coordinates phase space _∏, θ _∏), ζ is,

$\mathrm{\ζ}=\frac{1+2{\mathrm{\ρ}}_{\mathrm{\Π}}\cos {\mathrm{\θ}}_{\mathrm{\Π}}-{2\mathrm{\ω\ρ}}_{\mathrm{\Π}}\sin {\mathrm{\θ}}_{\mathrm{\Π}}}{{2\mathrm{\ω\ρ}}_{\mathrm{\Π}}-({\sin \mathrm{\θ}}_{\mathrm{\Π}}-\mathrm{\ω\τ}-{\cos \mathrm{\θ}}_{\mathrm{\Π}})-{\mathrm{\ω}}^2\mathrm{\τ}};$

Step 8, initial time fluorescence anisotropy r ₀with infinity moment fluorescence anisotropy

be respectively:

Wherein,

According to the I in step 6 _∏and I (t) _⊥(t) position (ρ in polar coordinates phase space _∏, θ _∏) and (ρ _⊥, θ _⊥), parameter

and ε _{1 ⊥}for,

${\mathrm{\ϵ}}_{1\⊥}=\frac{{\tan \mathrm{\θ}}_{\⊥}-(\frac{1}{1+{\mathrm{\ω}}^2{\mathrm{\τ}}^2}-\frac{1}{2})-\frac{\mathrm{\ω\τ}}{1+{\mathrm{\ω}}^2{\mathrm{\τ}}^2}}{\frac{\mathrm{\ω\ζ}-{\tan \mathrm{\θ}}_{\⊥}}{1+{\mathrm{\ω}}^2{\mathrm{\ζ}}^2}+\frac{{\tan \mathrm{\θ}}_{\⊥}-\mathrm{\ω\τ}}{1+{\mathrm{\ω}}^2{\mathrm{\τ}}^2}}.$

Described measuring equipment comprises: exciting bank, for pulse or sinusoidal Polarized Excitation irradiation fluorescent samples; Photoelectric detector, measures respectively the time resolution polarized fluorescence light intensity I that is parallel to pulse or sinusoidal polarized excitation light _∏(t) with perpendicular to the time resolution polarized fluorescence light intensity I of pulse or sinusoidal polarized excitation light _⊥(t); Data processing equipment, for to the I being recorded by described photoelectric detector _∏and I (t) _⊥(t) carry out data analysis, this data analysis comprises calculates total fluorescent intensity I _∑(t) and by Fourier transform and polar coordinate transform determine I _∑(t), I _∏and I (t) _⊥(t) position on polar coordinates, and then calculate fluorescence lifetime or mean lifetime and the dynamic anisotropic parameters of fluorescence of sample.

Further, described photoelectric detector comprises: CCD camera, CMOS camera, ICCD camera, EMCCD camera and/or other photoelectronic imaging equipment.

Relative prior art, the present invention has following beneficial effect:

For the fluorescence process of sample complexity, the present invention effectively quantitative measurment goes out its fluorescence lifetime.The present invention proposes the method for its fluorescence anisotropy parameter of quantitative measurment simultaneously.Initial time fluorescence anisotropy r due to fluorescence molecule or molecular group ₀only relevant with the angle between transmitting transition distance with the absorption jump distance of fluorescence molecule, so r ₀can become the signature identification signal of fluorescence molecule, thus the present invention to biomacromolecule in quantitative examination active somatic cell as the interaction between protein has very large using value.

Accompanying drawing explanation

Fig. 1 is the measuring principle of fluorescence lifetime and fluorescence anisotropy;

Fig. 2 is the polar coordinates phase space of the glycerine water solution mixed liquor of 10 μ M rhodamine 6Gs and 91%; Point A represents I _∑(t) phase space position (ρ _∑, θ _∑); Point B represents I _∏(t) phase space position (ρ _∏, θ _∏); Point C represents I _⊥(t) phase space position (ρ _⊥, θ _⊥).

Embodiment

Below in conjunction with the drawings and specific embodiments, the present invention will be further described in detail.

Rhodamine 6G is the common used material of biological fluorescent labelling technology, is widely used in DNA and protein research.Existing sample solution is the glycerine water solution mixed liquor of 10 μ M rhodamine 6Gs and 91%, the measurement mechanism of its fluorescence lifetime and fluorescence anisotropy as shown in Figure 1, in this example, adopt from x direction to be parallel to the 100fs pulse polarized excitation light excited sample solution of z axle, with I _∏(t) represent to be parallel to the time resolution polarized fluorescence light intensity of pulse polarized excitation light, with I _⊥(t) represent the time resolution polarized fluorescence light intensity perpendicular to pulse polarized excitation light.Total fluorescent intensity I _∑(t) be,

I _∑(t)＝I _∏(t)+2I _⊥(t)＝I ₀-exp(-t/τ), (1)

In above formula, I ₀for initial light intensity, τ is the fluorescence lifetime of rhodamine 6G in sample solution.

The dynamic anisotropy r of time-resolved fluorescence (t) is

In above formula, r ₀for the fluorescence anisotropy of rhodamine 6G molecule initial time, for the infinitely great fluorescence anisotropy constantly of rhodamine 6G molecule,

for the spin correlation time of rhodamine 6G molecule in sample solution.

The present invention below provides the fluorescence life τ of rhodamine 6G molecule in sample solution, the fluorescence anisotropy r of rhodamine 6G molecule initial time ₀, infinitely great fluorescence anisotropy constantly

and the spin correlation time of rhodamine 6G molecule in sample solution

measuring method.

First determine I _∑(t), I _∏and I (t) _⊥(t) position (ρ in polar coordinates phase space _∑, θ _∑), (ρ _∏, θ _∏) and (ρ _⊥, θ _⊥),

In this example, get f=60MHz.As shown in the A point of Fig. 2, record I _∑(t) polar coordinates position is (0.5,108.28); As shown in the B point of Fig. 2, record I _∏(t) polar coordinates position is (0.4963,99.396); As shown in the C point of Fig. 2, record I _⊥(t) polar coordinates position is (0.5335,122.70).

According to I _∑(t) position (ρ in polar coordinates phase space _∑, θ _∑), the fluorescence lifetime of sample is,

$\mathrm{\τ}={\mathrm{\ω}}^{-1}-\sqrt{\frac{1-\cos {\mathrm{\θ}}_{\mathrm{\Σ}}}{1+\cos {\mathrm{\θ}}_{\mathrm{\Σ}}}},---\left(6\right)$

In this example, the fluorescence life τ of rhodamine 6G molecule in sample solution is 3.67ns.

Fluorescence molecule spin correlation time in sample

for,

Wherein,

According to I _∏(t) at the position of polar coordinates phase space (ρ _∏, θ _∏), ζ is,

$\mathrm{\ζ}=\frac{1+2{\mathrm{\ρ}}_{\mathrm{\Π}}\cos {\mathrm{\θ}}_{\mathrm{\Π}}-{2\mathrm{\ω\ρ}}_{\mathrm{\Π}}\sin {\mathrm{\θ}}_{\mathrm{\Π}}}{{2\mathrm{\ω\ρ}}_{\mathrm{\Π}}-({\sin \mathrm{\θ}}_{\mathrm{\Π}}-\mathrm{\ω\τ}-{\cos \mathrm{\θ}}_{\mathrm{\Π}})-{\mathrm{\ω}}^2\mathrm{\τ}},---\left(8\right)$

In this example, ζ=2.84ns, so the spin correlation time of rhodamine 6G molecule in sample solution for 12.51ns.

The fluorescence anisotropy r of initial time ₀with infinity fluorescence anisotropy constantly

for,

Parameter ε _{1 ⊥}with

can pass through I _⊥and I (t) _∏(t) at the coordinate (ρ of phase space _∏, θ _∏) and (ρ _⊥, θ _⊥) try to achieve,

${\mathrm{\ϵ}}_{1\⊥}=\frac{{\tan \mathrm{\θ}}_{\⊥}-(\frac{1}{1+{\mathrm{\ω}}^2{\mathrm{\τ}}^2}-\frac{1}{2})-\frac{\mathrm{\ω\τ}}{1+{\mathrm{\ω}}^2{\mathrm{\τ}}^2}}{\frac{\mathrm{\ω\ζ}-{\tan \mathrm{\θ}}_{\⊥}}{1+{\mathrm{\ω}}^2{\mathrm{\ζ}}^2}+\frac{{\tan \mathrm{\θ}}_{\⊥}-\mathrm{\ω\τ}}{1+{\mathrm{\ω}}^2{\mathrm{\τ}}^2}},$

In this example, sample solution parameter ε _{1 ∏}=0.3824 and ε _{1 ⊥}=2.07.Therefore, record the fluorescence anisotropy r of rhodamine 6G molecule initial time ₀=0.4 and infinitely great fluorescence anisotropy constantly this and Y.Zhou, J.M.Dickenson, and Q.S.Hanley, " Imaging lifetime and anisotropy spectra in the frequency domain, " in Journal of Microscopy-Oxford (2009), Vol.234, pp.80-88. and A.H.A.Clayton, Q.S.Hanley, D.J.Arndt-Jovin, V.Subramaniam, and T.M.Jovin, " Dynamic fluorescence anisotropy imaging microscopy in the frequency domain (rFLIM) ", in Biophys.J. (2002), Vol.83, the measurement result of pp.1631-1649 is consistent.

Claims (3)

1. a method for quantitative measuring for fluorescence lifetime and the dynamic anisotropic parameters of fluorescence, adopts measuring equipment to measure fluorescent samples, it is characterized in that, described method comprises following concrete steps:

Step 1, adopts pulse or sinusoidal polarized excitation light to irradiate fluorescent samples, and excited sample produces fluorescence;

Step 2, measures respectively the time resolution polarized fluorescence light intensity I that is parallel to pulse or sinusoidal polarized excitation light by photodetector _∏(t) with perpendicular to the time resolution polarized fluorescence light intensity I of pulse or sinusoidal polarized excitation light _⊥(t);

Step 3, total fluorescent intensity is I _∑(t)=I _∏(t)+2I _⊥(t);

Step 4, for angular frequency corresponding to sample, I _∑(t) position (ρ in polar coordinates phase space _∑, θ _∑) be,

Step 5, the fluorescence life τ of sample is,

Step 6, I _∏and I (t) _⊥(t) position (ρ in polar coordinates phase space _∏, θ _∏) and (ρ _⊥, θ _⊥) be respectively,

Step 7, the fluorescence molecule spin correlation time of sample

for,

Wherein, according to the I in step 6 _∏(t) position (ρ in polar coordinates phase space _∏, θ _∏), ζ is,

$\mathrm{\ζ}=\frac{1+2{\mathrm{\ρ}}_{\mathrm{\Π}}\cos {\mathrm{\θ}}_{\mathrm{\Π}}-{2\mathrm{\ω\ρ}}_{\mathrm{\Π}}\sin {\mathrm{\θ}}_{\mathrm{\Π}}}{{2\mathrm{\ω\ρ}}_{\mathrm{\Π}}-({\sin \mathrm{\θ}}_{\mathrm{\Π}}-\mathrm{\ω\τ}-{\cos \mathrm{\θ}}_{\mathrm{\Π}})-{\mathrm{\ω}}^2\mathrm{\τ}};$

Step 8, initial time fluorescence anisotropy r ₀with infinity moment fluorescence anisotropy

be respectively:

Wherein,

According to the I in step 6 _∏and I (t) _⊥(t) position (ρ in polar coordinates phase space _∏, θ _∏) and (ρ _⊥, θ _⊥), parameter

and ε _{1 ⊥}for,

${\mathrm{\ϵ}}_{1\⊥}=\frac{{\tan \mathrm{\θ}}_{\⊥}-(\frac{1}{1+{\mathrm{\ω}}^2{\mathrm{\τ}}^2}-\frac{1}{2})-\frac{\mathrm{\ω\τ}}{1+{\mathrm{\ω}}^2{\mathrm{\τ}}^2}}{\frac{\mathrm{\ω\ζ}-{\tan \mathrm{\θ}}_{\⊥}}{1+{\mathrm{\ω}}^2{\mathrm{\ζ}}^2}+\frac{{\tan \mathrm{\θ}}_{\⊥}-\mathrm{\ω\τ}}{1+{\mathrm{\ω}}^2{\mathrm{\τ}}^2}}.$

2. measuring method according to claim 1, is characterized in that: described measuring equipment comprises: exciting bank, for pulse or sinusoidal Polarized Excitation irradiation fluorescent samples;

Photoelectric detector, measures respectively the time resolution polarized fluorescence light intensity I that is parallel to pulse or sinusoidal polarized excitation light _∏(t) with perpendicular to the time resolution polarized fluorescence light intensity I of pulse or sinusoidal polarized excitation light _⊥(t); Data processing equipment, for to the I being recorded by described photoelectric detector _∏and I (t) _⊥(t) carry out data analysis, this data analysis comprises calculates total fluorescent intensity I _∑(t) and by Fourier transform and polar coordinate transform determine I _∑(t), I _∏and I (t) _⊥(t) position on polar coordinates, and then calculate fluorescence lifetime and the dynamic anisotropic parameters of fluorescence of sample.

3. measuring method according to claim 2, is characterized in that: described photoelectric detector comprises: CCD camera, CMOS camera, ICCD camera, EMCCD camera and/or other photoelectronic imaging equipment.

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