CN104614324A - Method for measuring optical constant of microalgae culture medium and spectral attenuation coefficient of microalgae in combined way - Google Patents

Abstract

The invention discloses a method for measuring an optical constant of a microalgae culture medium and a spectral attenuation coefficient of microalgae in a combined way, relates to a method for measuring the optical constant of the culture medium and the spectral attenuation coefficient in a combined way, and aims to solve the problems that during measurement by the conventional method, relatively large deviation is caused because the influence of multiple reflections caused by glass and an interface cannot be eliminated accurately and the influence of cuvette glass is ignored. The method comprises the following steps: 1, establishing a three-layer medium light transmission model; 2, selecting microalgae and culture medium cuvette containers which obtain effective transmission data; 3, calculating the optical constant of the culture medium; 4, acquiring the attenuation coefficient betap of the microalgae. The method is applied to combined measurement of the optical constant of the culture medium and the spectral attenuation coefficient.

Description

The union measuring method of a kind of micro-algae culture medium optical constant and micro-algae Spectrum attenuation coefficient

Technical field

The present invention relates to the union measuring method of nutrient culture media optical constant and Spectrum attenuation coefficient, particularly the union measuring method of a kind of micro-algae culture medium optical constant and micro-algae Spectrum attenuation coefficient.

Background technology

The radiation characteristic of micro-algae is the basic parameter of research microalgae photobiological reaction efficiency.Spectrum attenuation coefficient is one of fundamental radiation characteristic, generally can be obtained by the measurement to normal direction transmitance by means of spectrometer.During Berberoglu employing monochromatic light journey method Measuring Oceanic chlorella attenuation coefficient, insert after chlorella is centrifugal in PBS solution and (absorb less solution at visible light wave range), with the cuvette of inserting PBS solution for reference to the impact eliminating the multiple reflections that glass and interface produce, Beer-Lambert law is utilized finally to obtain the attenuation coefficient of pure chlorella.Chlorella is after centrifugal treating, the change of external environment has no way of finding out about it on the impact that frustule measurement result produces, and with built-in PBS solution cuvette for reference to the impact accurately can not eliminating the multiple reflections that glass and interface produce, therefore this method has certain limitation.Yun and Park adopts monochromatic light journey method (utilizing Beer-Lambert law) the Spectrum attenuation characteristic to chlorella vulgaris (Chlorella vulgaris) suspension visible light wave range to be studied, find that light-decay characteristic exists obvious spectral dependency, but owing to have ignored the impact of cuvette glass when chlorella suspension is measured, so can relatively large deviation be produced.

Summary of the invention

The impact that the object of the invention is to produce frustule measurement result the change of external environment to solve prior art has no way of finding out about it, accurately can not eliminate the impact of the multiple reflections of glass and interface generation and prior art exists obvious spectral dependency, have ignored the impact of cuvette glass, produce the problem of relatively large deviation and the union measuring method of a kind of micro-algae culture medium optical constant of proposing and micro-algae Spectrum attenuation coefficient.

Above-mentioned goal of the invention is achieved through the following technical solutions:

Step one, according to improving the requirement of measuring accuracy, consider that glass is on measuring the impact producing multiple reflections, establishes three layers of medium light transmission model by Image Synthesis by Ray Tracing; Wherein, in three layers of medium light transmission model, the thickness of incident glass medium is L ₁, the complex index of refraction of incident glass medium is n ₁+ i κ ₁; The thickness of liquid medium is L ₂; The complex index of refraction of liquid medium is n ₂+ i κ ₂; The thickness of outgoing glass medium is L ₃; The complex index of refraction of outgoing glass medium is n ₃+ i κ ₃, n is refractive index, and κ is absorption index; I is plural number;

Step 2, when measuring micro-algae suspending liquid transmittance, select to obtain micro-algae cuvette container of effective transmission data; When measuring nutrient culture media transmittance, select two different light paths according to the absorption characteristic of nutrient culture media and meet the nutrient culture media cuvette container of transmission data measuring accuracy;

Step 3, nutrient culture media is contained in two different light path nutrient culture media cuvette containers, obtains two different nutrient culture media transmittance T with spectrometer measurement _{λ, EXP}with T ' _{λ, EXP}, adopt three layers of dielectric model simultaneous, two transmittance equations, utilize optimized algorithm and setting optimization object function f _λthe optical constant of nutrient culture media is calculated by the genetic algorithm in Matlab Optimization Toolbox;

Step 4, utilize the micro-algae suspending liquid transmittance be contained in micro-algae cuvette container of a spectrometer measurement light path, according to the optical constant of the nutrient culture media that micro-algae suspending liquid transmittance and step 3 obtain, try to achieve the attenuation coefficient β of micro-algae suspending liquid by monochromatic light journey method; The attenuation coefficient β of micro-algae suspending liquid deducts the attenuation coefficient α of nutrient culture media _mobtain the attenuation coefficient β of micro-algae _p; Namely the union measuring method of a kind of micro-algae culture medium optical constant and micro-algae Spectrum attenuation coefficient is completed.

Invention effect

The present invention abandons traditional short-cut method utilizing Beer-Lambert law, adopt the mode of decoupled method, the present invention takes into full account the multiple reflections impact between multilayered medium, compensate in Existing methods the calculating defect measuring micro-algae attenuation coefficient, improves measuring accuracy as Fig. 2.Fig. 2 is that traditional double light path method (utilizing Beer-Lambert law) contrasts with method of the present invention, and in figure, two groups of same transmittance experimental datas of result obtain, but the former ignores the impact of glass, and two groups of result differences are larger at this concentration.

Because the present invention has taken into full account that glass is on measuring the impact produced, set up three layers of medium ray tracing model, adopt the mode of decoupled method, namely first measure the optical constant of nutrient culture media by two light path method, and then try to achieve the Spectrum attenuation coefficient of micro-algae by monochromatic light journey method.In invention, two light path method measures with two that material is identical, light path is different and hold the cuvette transmittance of same liquid, adopt three layers of dielectric model and simultaneous two transmittance equations, utilize optimized algorithm inversion iterates to solve the optical constant of nutrient culture media to be measured.This invention takes into full account the multiple reflections between multilayered medium, effectively eliminates glass to measuring the impact produced.Basic ideas are the transmission signals recording micro-algae suspending liquid and nutrient culture media by experiment, then solve micro-algae culture medium optical constant and micro-algae Spectrum attenuation coefficient in conjunction with computation model.Compensate for traditional Beer-Lambert law that utilizes and measure the calculating defect of micro-algae attenuation coefficient short-cut method, take into full account the multiple reflections between multilayered medium, eliminating glass to measuring the impact produced, obtaining accurate result.The present invention provides one method accurately, for the research of microorganism and nanoparticle subgroup attenuation characteristic provides certain reference for the attenuation characteristic studying the population such as micro-algae.

Accompanying drawing explanation

Fig. 1 is three layers of medium ray tracing schematic diagram that embodiment one proposes;

Fig. 2 is classic method and the integrated processes measurement result comparison diagram of embodiment one proposition;

Fig. 3 is that the integrated processes that embodiment proposes measures distilled water optical constant and literature value comparison diagram;

Fig. 4 is the optical constant figure of the integrated processes measurement nutrient culture media that embodiment proposes;

Fig. 5 is the attenuation coefficient figure of three kinds of variable concentrations chlorella suspensions that embodiment proposes;

Fig. 6 is the attenuation coefficient figure of three kinds of variable concentrations chlorellas that embodiment proposes;

Fig. 7 is a kind of micro-algae culture medium optical constant of embodiment one proposition and the union measuring method process flow diagram of micro-algae Spectrum attenuation coefficient.

Embodiment

Embodiment one: a kind of micro-algae culture medium optical constant of present embodiment and the union measuring method of micro-algae Spectrum attenuation coefficient, specifically prepare according to following steps:

Step one, according to improving the requirement of measuring accuracy, consider that glass is on measuring the impact producing multiple reflections, establishes three layers of medium light transmission model by Image Synthesis by Ray Tracing; Wherein, being three layers of medium ray tracing schematic diagram according to Fig. 1 in three layers of medium light transmission model, supposing that light that light source sends is successively by arriving detector, I in Fig. 1 after incident air-incident glass-liquid-outgoing glass-outgoing air ₀the light intensity of transmission direction in incident air; I ₁it is transmission direction light intensity in a liquid; I ₂the light intensity of transmission direction in outgoing air; J ₀the light intensity of reflection direction in light source incidence air; J ₁the light intensity of reflection direction at liquid; The thickness (unit m) of incident glass medium is L ₁, the complex index of refraction of incident glass medium is n ₁+ i κ ₁; The thickness (unit m) of liquid medium is L ₂; The complex index of refraction of liquid medium is n ₂+ i κ ₂; The thickness (unit m) of outgoing glass medium is L ₃; The complex index of refraction of outgoing glass medium is n ₃+ i κ ₃, air is n ₀=1, n is refractive index, and κ is absorption index; I is plural number;

Step 2, absorption characteristic according to micro-algae suspending liquid and nutrient culture media, because it is larger in different-waveband absorption difference, therefore be applicable to according to different measurement band selections the cuvette measuring micro-algae suspending liquid and nutrient culture media light path, namely when measuring micro-algae suspending liquid transmittance, select a suitable detect thickness according to the absorption characteristic of micro-algae suspending liquid, namely select the micro-algae cuvette container obtaining effective transmission data; When measuring nutrient culture media transmittance, select two different measuring thickness i.e. two different light paths according to the absorption characteristic of nutrient culture media and meet the nutrient culture media cuvette container of transmission data measuring accuracy;

Step 3, nutrient culture media is contained in two different light path nutrient culture media cuvette containers, obtains two different nutrient culture media transmittance T with spectrometer measurement _{λ, EXP}with T ' _{λ, EXP}, adopt three layers of dielectric model simultaneous, two transmittance equations, utilize optimized algorithm and setting optimization object function f _λthe optical constant of nutrient culture media is calculated by the genetic algorithm in Matlab Optimization Toolbox;

Step 4, utilize the micro-algae suspending liquid transmittance be contained in micro-algae cuvette container of a spectrometer measurement light path, according to the optical constant of the nutrient culture media that micro-algae suspending liquid transmittance and step 3 obtain, try to achieve the attenuation coefficient β of micro-algae suspending liquid by monochromatic light journey method; The attenuation coefficient β of micro-algae suspending liquid deducts the attenuation coefficient α of nutrient culture media _mobtain the attenuation coefficient β of micro-algae _p; Wherein, the attenuation coefficient of nutrient culture media is that two light path method obtains, and monochromatic light journey is that acquisition methods is different with two light path, and the order of magnitude is identical, is suitable for plus-minus relation as Fig. 7; Namely the union measuring method of a kind of micro-algae culture medium optical constant and micro-algae Spectrum attenuation coefficient is completed.

Present embodiment effect:

Present embodiment abandons traditional short-cut method utilizing Beer-Lambert law, adopt the mode of decoupled method, present embodiment takes into full account the multiple reflections impact between multilayered medium, compensate in Existing methods the calculating defect measuring micro-algae attenuation coefficient, improves measuring accuracy as Fig. 2.Fig. 2 is that traditional double light path method (utilizing Beer-Lambert law) contrasts with the method for present embodiment, and in figure, two groups of same transmittance experimental datas of result obtain, but the former ignores the impact of glass, and two groups of result differences are larger at this concentration.

Because present embodiment has taken into full account that glass is on measuring the impact produced, set up three layers of medium ray tracing model, adopt the mode of decoupled method, namely first measure the optical constant of nutrient culture media by two light path method, and then try to achieve the Spectrum attenuation coefficient of micro-algae by monochromatic light journey method.In invention, two light path method measures with two that material is identical, light path is different and hold the cuvette transmittance of same liquid, adopt three layers of dielectric model and simultaneous two transmittance equations, utilize optimized algorithm inversion iterates to solve the optical constant of nutrient culture media to be measured.This invention takes into full account the multiple reflections between multilayered medium, effectively eliminates glass to measuring the impact produced.Basic ideas are the transmission signals recording micro-algae suspending liquid and nutrient culture media by experiment, then solve micro-algae culture medium optical constant and micro-algae Spectrum attenuation coefficient in conjunction with computation model.Compensate for traditional Beer-Lambert law that utilizes and measure the calculating defect of micro-algae attenuation coefficient short-cut method, take into full account the multiple reflections between multilayered medium, eliminating glass to measuring the impact produced, obtaining accurate result.Present embodiment provides one method accurately, for the research of microorganism and nanoparticle subgroup attenuation characteristic provides certain reference for the attenuation characteristic studying the population such as micro-algae.

Embodiment two: present embodiment and embodiment one unlike: in step 2, micro-algae cuvette container and nutrient culture media cuvette container transmission data scope are 0.05 ~ 0.85.Other step and parameter identical with embodiment one.

Embodiment three: present embodiment and embodiment one or two unlike: adopt three layers of dielectric model simultaneous, two transmittance equations to be specially in step 3:

(1) utilize two light path method, carry out simultaneous according to the transmittance equation of three layers of dielectric model to two different-thickness:

T _λ＝f(n ₁,n ₂,n ₃；κ ₁,κ ₂,κ ₃；L ₁,L ₂,L ₃) (3)

T′ _λ＝f(n ₁,n ₂,n ₃；κ ₁,κ ₂,κ ₃；L ₁,L′ ₂,L ₃) (4)

Wherein, T _λwith T ' _λbe two different-thickness transmittances of three layers of dielectric model setting, in formula, only have n ₂and κ ₂unknown number, the optical constant n of glass ₁+ i κ ₁=n ₃+ i κ ₃, thickness L ₁=L ₃and two testing liquid sample thickness L ₂with L ' ₂be known, calculate total transmittance, boundary reflection rate, interface transmissivity and absorption coefficient according to optical constant;

(2) total transmittance is expressed as after deriving:

$\begin{array}{c}{T}_{\mathrm{\λ}}=\frac{I_2}{I_0}\\ =\left(\frac{t_{01}{t}_{12}{e}^{{-\mathrm{\α}}_1{L}_1}}{1-r_{10}{r}_{12}{e}^{-{2\mathrm{\α}}_1{L}_1}}\right)\left(\frac{t_{23}{t}_{34}{e}^{-{\mathrm{\α}}_3{L}_3}}{1-r_{32}{r}_{34}{e}^{-2{\mathrm{\α}}_3{L}_3}}\right)e^{-{\mathrm{\α}}_2{L}_2}{[1-(r_{23}+\frac{t_{23}{t}_{32}{r}_{34}{e}^{-{2\mathrm{\α}}_3{L}_3}}{1-r_{32}{r}_{34}{e}^{-2{\mathrm{\α}}_3{L}_3}})(r_{21}+\frac{t_{21}{t}_{12}{r}_{10}{e}^{-{2\mathrm{\α}}_1{L}_1}}{1-r_{12}{r}_{10}{e}^{-2{\mathrm{\α}}_1{L}_1}})e^{{-2\mathrm{\α}}_2{L}_2}]}^{-1}\end{array}---\left(5\right)$

(3) formula (5) median surface reflectivity r _ijbe expressed as:

$r_{\mathrm{ij}}=\frac{{(n_j-n_i)}^2+{({\mathrm{\κ}}_j-{\mathrm{\κ}}_i)}^2}{{(n_j+n_i)}^2+{({\mathrm{\κ}}_j+{\mathrm{\κ}}_i)}^2}---\left(6\right)$

(4) the interface transmissivity t of adjacent two layer medium i and j in formula (5) _ijbe expressed as:

t _ij＝1-r _ij(7)

(5) absorption coefficient in formula (5):

${\mathrm{\α}}_i=\frac{{4\mathrm{\π\κ}}_i}{\mathrm{\λ}}---\left(8\right)$

In formula: i, j represent adjacent two layer medium respectively, the sequence number span of medium from left to right: i=0 in Fig. 1,1,2,3 and 4, j=0,1,2,3 and 4; Wherein, 0 represents incident air medium, and 1 is incident glass medium, and 2 is liquid medium, and 3 is outgoing glass, and 4 is outgoing air; α ₁for incident glass medium absorption coefficient; α ₂for liquid medium absorption coefficient; α ₃for outgoing glass absorber coefficient.Other step and parameter identical with embodiment one or two.

Embodiment four: one of present embodiment and embodiment one to three unlike: adopt three layers of dielectric model simultaneous two transmittance equations in step 3, utilize optimized algorithm and setting optimization object function f _λthe optical constant detailed process being calculated nutrient culture media by the genetic algorithm in Matlab Optimization Toolbox is:

(1) inverting adopts optimized algorithm to realize; Due to the optical constant n of liquid ₂, κ ₂do not draw by formula direct solution, therefore adopt optimized algorithm to carry out inverting to it and solve; Genetic algorithm is adopted to set up optimization object function:

f _λ＝(T _λ-T _λ,EXP) ²+(T′ _λ-T′ _λ,EXP) ²(9)

In formula, wavelength at λ, liquid medium thickness L ₂time, the transmittance T of three layers of dielectric model setting _λ, T _λthe nutrient culture media transmittance T that corresponding spectrometer measurement obtains _{λ, EXP};

Wavelength at λ, when liquid medium thickness L ' 2, the transmittance T ' of three layers of dielectric model setting _λ, T ' _λthe nutrient culture media transmittance T ' that corresponding spectrometer measurement obtains _{λ, EXP};

(2) adopt three layers of dielectric model simultaneous, two transmittance equations (3) and (4), utilize optimized algorithm and setting optimization object function f _λthe optical constant n of nutrient culture media is calculated by the genetic algorithm in Matlab Optimization Toolbox ₂and κ ₂.Other step and parameter identical with one of embodiment one to three.

Embodiment five: one of present embodiment and embodiment one to four are unlike the attenuation coefficient β that obtains micro-algae in step 4 _pdetailed process is:

(1) the monochromatic light journey method computation model solving the attenuation coefficient of micro-algae is as follows: when light is vertically through micro-algae suspending liquid, the interface of glass and micro-algae suspending liquid is equal to the interface of glass and culture medium solution, now micro-algae suspending liquid transmittance is T _λbe expressed as:

$\begin{array}{c}{T}_{\mathrm{\λ}}=\left(\frac{t_{01}{t}_{12}{e}^{{-\mathrm{\α}}_1{L}_1}}{1-r_{10}{r}_{12}{e}^{-{2\mathrm{\α}}_1{L}_1}}\right)\left(\frac{t_{23}{t}_{34}{e}^{-{\mathrm{\α}}_3{L}_3}}{1-r_{32}{r}_{34}{e}^{-2{\mathrm{\α}}_3{L}_3}}\right)e^{-\mathrm{\β}{L}_2}{[1-(r_{23}+\frac{t_{23}{t}_{32}{r}_{34}{e}^{-{2\mathrm{\α}}_3{L}_3}}{1-r_{32}{r}_{34}{e}^{-2{\mathrm{\α}}_3{L}_3}})(r_{21}+\frac{t_{21}{t}_{12}{r}_{10}{e}^{-{2\mathrm{\α}}_1{L}_1}}{1-r_{12}{r}_{10}{e}^{-2{\mathrm{\α}}_1{L}_1}})e^{-2\mathrm{\β}{L}_2}]}^{-1}\end{array}---\left(10\right)$

After substituting into the optical constant of nutrient culture media, only have β to be unknown number in above formula, bring micro-algae suspending liquid transmittance of experiment measuring into obtain micro-algae suspending liquid attenuation coefficient β, wherein, the interface transmissivity t of adjacent two layer medium i and j _ij, the sequence number span of medium from left to right: i=0 in Fig. 1,1,2,3 and 4, j=0,1,2,3 and 4; 0 represents incident air medium, and 1 is incident glass medium, and 2 is liquid medium, and 3 is outgoing glass, and 4 is outgoing air;

(2) the attenuation coefficient β of micro-algae suspending liquid is represented by micro-algae and nutrient culture media attenuation coefficient sum:

β＝β _p+α _m(1)

In formula, β is total attenuation coefficient, cm ^-1; β _pfor micro-algae attenuation coefficient, cm ^-1; α _mfor nutrient culture media attenuation coefficient, cm ^-1; Because being difficult to the attenuation coefficient directly obtaining micro-algae, this mode of attenuation coefficient that the attenuation coefficient by micro-algae suspending liquid deducts nutrient culture media obtains indirectly;

(3) nutrient culture media attenuation coefficient α _mbe expressed as:

${\mathrm{\α}}_m=\frac{4\mathrm{\π}{\mathrm{\κ}}_m}{\mathrm{\λ}}---\left(2\right)$

In formula: κ _mfor the absorption index of nutrient culture media, m represents nutrient culture media; λ is incident light source wavelength, unit m;

(4) β deducts attenuation coefficient (absorption coefficient) α of nutrient culture media _mobtain the attenuation coefficient β of micro-algae _p.Other step and parameter identical with one of embodiment one to four.

Following examples are adopted to verify beneficial effect of the present invention:

Embodiment one:

The union measuring method of the present embodiment a kind of micro-algae culture medium optical constant and micro-algae Spectrum attenuation coefficient, specifically prepare according to following steps:

Test and measure one by the V-VASE spectroscopic ellipsometer of J.A.Woollam company and fill with the transmittance of different light path cuvettes at ultraviolet, visible ray, near infrared (300nm-1800nm) continuous spectrum that chlorella suspending liquid monochromatic light journey cuvette and two fill with nutrient culture media.Experiment measuring temperature is 18 DEG C, and measurement pressure is standard atmospheric pressure.Experiment is limnetic chlorella with micro-algae, and nutrient culture media is BG11, and cultivation temperature is about 20 DEG C, cultivates about week age.The concentration of chlorella gets average after being counted under inverted fluorescence microscope by blood counting chamber to obtain.The detect thickness of cuvette is selected to be respectively L2=0.5cm and L ' according to the absorption characteristic of nutrient culture media in experiment ₂=1cm, selects the detect thickness of cuvette to be respectively L according to the absorption characteristic of micro-algae suspending liquid ₂=0.5cm.

Now the attenuation coefficient of micro-algae suspending liquid can be expressed as:

β＝β _p+α _m(1)

In formula: β is total attenuation coefficient, cm ^-1; β _pfor chlorella attenuation coefficient, cm ^-1; α _mfor nutrient culture media absorption coefficient, cm ^-1.

Nutrient culture media absorption coefficient is expressed as:

${\mathrm{\α}}_m=\frac{{4\mathrm{\π\κ}}_m}{\mathrm{\λ}}---\left(2\right)$

In formula: κ _mfor the absorption index of nutrient culture media; λ is wavelength, m.

Now be L thickness by two light path method ₂=0.5cm and L ' ₂=1cm two nutrient culture media transmittance equations simultaneousnesses:

T _λ＝f(n ₁,n ₂,n ₃；κ ₁,κ ₂,κ ₃；L ₁,L ₂,L ₃) (3)

T′ _λ＝f(n ₁,n ₂,n ₃；κ ₁,κ ₂,κ ₃；L ₁,L′ ₂,L ₃) (4)

N is only had in formula ₂, k ₂unknown number, the optical constant (n of glass ₁+ i κ ₁=n ₃+ i κ ₃) and thickness (L ₁=L ₃) and two testing liquid sample thickness (L ₂, L ' ₂) be known, the optical constant of nutrient culture media is obtained by inversion iterates.Total transmittance can be expressed as after deriving:

$\begin{array}{c}{T}_{\mathrm{\λ}}=\frac{I_2}{I_0}\\ =\left(\frac{t_{01}{t}_{12}{e}^{{-\mathrm{\α}}_1{L}_1}}{1-r_{10}{r}_{12}{e}^{-{2\mathrm{\α}}_1{L}_1}}\right)\left(\frac{t_{23}{t}_{34}{e}^{-{\mathrm{\α}}_3{L}_3}}{1-r_{32}{r}_{34}{e}^{-2{\mathrm{\α}}_3{L}_3}}\right)e^{-{\mathrm{\α}}_2{L}_2}{[1-(r_{23}+\frac{t_{23}{t}_{32}{r}_{34}{e}^{-{2\mathrm{\α}}_3{L}_3}}{1-r_{32}{r}_{34}{e}^{-2{\mathrm{\α}}_3{L}_3}})(r_{21}+\frac{t_{21}{t}_{12}{r}_{10}{e}^{-{2\mathrm{\α}}_1{L}_1}}{1-r_{12}{r}_{10}{e}^{-2{\mathrm{\α}}_1{L}_1}})e^{{-2\mathrm{\α}}_2{L}_2}]}^{-1}\end{array}---\left(5\right)$

Boundary reflection rate is expressed as:

$r_{\mathrm{ij}}=\frac{{(n_j-n_i)}^2+{({\mathrm{\κ}}_j-{\mathrm{\κ}}_i)}^2}{{(n_j+n_i)}^2+{({\mathrm{\κ}}_j+{\mathrm{\κ}}_i)}^2}---\left(6\right)$

Interface transmissivity is expressed as:

t _ij＝1-r _ij(7)

Absorption coefficient:

${\mathrm{\α}}_i=\frac{{4\mathrm{\π\κ}}_i}{\mathrm{\λ}}---\left(8\right)$

In formula, i, j distinguish the sequence number of medium from left to right in representative graph 1, and both sides ambient symbology is 0.Due to the optical constant n of liquid ₂, κ ₂do not draw by formula direct solution, therefore adopt genetic algorithm to carry out inverting to it and solve.Genetic algorithm objective function is defined as follows:

f _λ＝(T _λ-T _λ，EXP) ²+(T′ _λ-T′ _λ，EXP) ²(9)

In formula, T _λwith T ' _λrepresent that wavelength is when λ, two different-thickness L ₂with L ' ₂calculate gained transmittance, T _{λ, EXP}with T ' _{λ, EXP}it is the nutrient culture media transmittance that the spectrometer measurement corresponding with it obtains.

First the accuracy measuring liquid medium optical property model in invention is verified, here with the spectroscopic optics constant of distilled water for benchmark is verified.In the experiments of measuring of distilled water, be respectively L at the detect thickness of 300-1000nm wave band cuvette ₂=10cm and L ' ₂=5cm, is respectively L at the detect thickness of 1000-1800nm wave band cuvette ₂=0.5cm and L ' ₂=0.3cm, water is less in visible absorption, is more greatly so select the reason of thickness gap near infrared absorption.Fig. 3 records distilled water to compare in the optical constant of 300-1800nm wave band and the data of Segelstein (1981) in the present invention.Can find out in Fig. 3 that distilled water optical constant and data in literature are coincide better, model computational accuracy is higher.Fig. 4 is the optical constant recording nutrient culture media in the present invention.

When light is vertically through thickness L ₂during micro-algae suspending liquid of three kinds of variable concentrations of=0.5cm, now transmittance is expressed as:

$\begin{array}{c}{T}_{\mathrm{\λ}}=\left(\frac{t_{01}{t}_{12}{e}^{{-\mathrm{\α}}_1{L}_1}}{1-r_{10}{r}_{12}{e}^{-{2\mathrm{\α}}_1{L}_1}}\right)\left(\frac{t_{23}{t}_{34}{e}^{-{\mathrm{\α}}_3{L}_3}}{1-r_{32}{r}_{34}{e}^{-2{\mathrm{\α}}_3{L}_3}}\right)e^{-\mathrm{\β}{L}_2}{[1-(r_{23}+\frac{t_{23}{t}_{32}{r}_{34}{e}^{-{2\mathrm{\α}}_3{L}_3}}{1-r_{32}{r}_{34}{e}^{-2{\mathrm{\α}}_3{L}_3}})(r_{21}+\frac{t_{21}{t}_{12}{r}_{10}{e}^{-{2\mathrm{\α}}_1{L}_1}}{1-r_{12}{r}_{10}{e}^{-2{\mathrm{\α}}_1{L}_1}})e^{-2\mathrm{\β}{L}_2}]}^{-1}\end{array}---\left(10\right)$

After substituting into the optical constant of nutrient culture media, only have β to be unknown number in above formula, the transmittance of experiment measuring is brought into the attenuation coefficient β that can obtain micro-algae suspending liquid.Fig. 5 gives three kinds of concentration and is respectively 5.87x10 ¹²individual/m ³, 2.95x10 ¹²individual/m ³, 1.57x10 ¹²individual/m ³chlorella suspension at the Spectrum attenuation coefficient of 300-1800nm wave band.The attenuation coefficient of micro-algae suspending liquid deducts the absorption coefficient of nutrient culture media _mjust the attenuation coefficient β of pure micro-algae can be obtained _p.Fig. 6 gives three kinds of concentration and is respectively 5.87x10 ¹²individual/m ³, 2.95x10 ¹²individual/m ³, 1.57x10 ¹²individual/m ³chlorella cells at the Spectrum attenuation coefficient of 300-1800nm wave band.

The result of above example shows, the method in the present invention not only accurately can obtain the optical constant of the neat liquids such as nutrient culture media, can also obtain the attenuation coefficient of the population suspending liquid such as micro-algae, its result accurate and effective more.

The present invention also can have other various embodiments; when not deviating from the present invention's spirit and essence thereof; those skilled in the art are when making various corresponding change and distortion according to the present invention, but these change accordingly and are out of shape the protection domain that all should belong to the claim appended by the present invention.

Claims (5)

1. a union measuring method for micro-algae culture medium optical constant and micro-algae Spectrum attenuation coefficient, is characterized in that: the union measuring method of a kind of micro-algae culture medium optical constant and micro-algae Spectrum attenuation coefficient specifically carries out according to following steps:

Step one, according to improving the requirement of measuring accuracy, consider that glass is on measuring the impact producing multiple reflections, establishes three layers of medium light transmission model by Image Synthesis by Ray Tracing; Wherein, in three layers of medium light transmission model, the thickness of incident glass medium is L ₁, the complex index of refraction of incident glass medium is n ₁+ i κ ₁; The thickness of liquid medium is L ₂; The complex index of refraction of liquid medium is n ₂+ i κ ₂; The thickness of outgoing glass medium is L ₃; The complex index of refraction of outgoing glass medium is n ₃+ i κ ₃, n is refractive index, and κ is absorption index; I is plural number;

Step 2, when measuring micro-algae suspending liquid transmittance, select to obtain micro-algae cuvette container of effective transmission data; When measuring nutrient culture media transmittance, select two different light paths according to the absorption characteristic of nutrient culture media and meet the nutrient culture media cuvette container of transmission data measuring accuracy;

Step 3, nutrient culture media is contained in two different light path nutrient culture media cuvette containers, obtains two different nutrient culture media transmittance T with spectrometer measurement _{λ, EXP}with T ' _{λ, EXP}, adopt three layers of dielectric model simultaneous, two transmittance equations, utilize optimized algorithm and setting optimization object function f _λthe optical constant of nutrient culture media is calculated by the genetic algorithm in Matlab Optimization Toolbox;

Step 4, utilize the micro-algae suspending liquid transmittance be contained in micro-algae cuvette container of a spectrometer measurement light path, according to the optical constant of the nutrient culture media that micro-algae suspending liquid transmittance and step 3 obtain, try to achieve the attenuation coefficient β of micro-algae suspending liquid by monochromatic light journey method; The attenuation coefficient β of micro-algae suspending liquid deducts the attenuation coefficient α of nutrient culture media _mobtain the attenuation coefficient β of micro-algae _p; Namely the union measuring method of a kind of micro-algae culture medium optical constant and micro-algae Spectrum attenuation coefficient is completed.

2. the union measuring method of a kind of micro-algae culture medium optical constant according to claim 1 and micro-algae Spectrum attenuation coefficient, is characterized in that: in step 2, micro-algae cuvette container and nutrient culture media cuvette container transmission data scope are 0.05 ~ 0.85.

3. the union measuring method of a kind of micro-algae culture medium optical constant according to claim 1 and micro-algae Spectrum attenuation coefficient, is characterized in that: adopt three layers of dielectric model simultaneous, two transmittance equations to be specially in step 3:

Utilize two light path method, carry out simultaneous according to the transmittance equation of three layers of dielectric model to two different-thickness:

T _λ＝f(n ₁,n ₂,n ₃；κ ₁,κ ₂,κ ₃；L ₁,L ₂,L ₃) (3)

T _λ′＝f(n ₁,n ₂,n ₃；κ ₁,κ ₂,κ ₃；L ₁,L′ ₂,L ₃) (4)

Wherein, T _λand T _λ' be two different-thickness transmittances that three layers of dielectric model set, only have n in formula ₂and κ ₂unknown number, the optical constant n of glass ₁+ i κ ₁=n ₃+ i κ ₃, thickness L ₁=L ₃and two testing liquid sample thickness L ₂with L ' ₂known.

4. the union measuring method of a kind of micro-algae culture medium optical constant according to claim 1 and micro-algae Spectrum attenuation coefficient, it is characterized in that: in step 3, adopt three layers of dielectric model simultaneous, two transmittance equations, utilize optimized algorithm and setting optimization object function f _λthe optical constant detailed process being calculated nutrient culture media by the genetic algorithm in Matlab Optimization Toolbox is:

(1) genetic algorithm is adopted to set up optimization object function:

f _λ＝(T _λ-T _λ,EXP) ²+(T′ _λ-T′ _λ,EXP) ²(9)

In formula, wavelength at λ, liquid medium thickness L ₂time, the transmittance T of three layers of dielectric model setting _λ, T _λthe nutrient culture media transmittance T that corresponding spectrometer measurement obtains _{λ, EXP};

Wavelength at λ, liquid medium thickness L ' ₂time, the transmittance T of three layers of dielectric model setting _λ', T ' _λthe nutrient culture media transmittance T ' that corresponding spectrometer measurement obtains _{λ, EXP};

(2) adopt three layers of dielectric model simultaneous, two transmittance equations (3) and (4), utilize optimized algorithm and setting optimization object function f _λthe optical constant n of nutrient culture media is calculated by the genetic algorithm in Matlab Optimization Toolbox ₂and κ ₂.

5. the union measuring method of a kind of micro-algae culture medium optical constant according to claim 1 and micro-algae Spectrum attenuation coefficient, is characterized in that: the attenuation coefficient β obtaining micro-algae in step 4 _pdetailed process is:

(1) when light is vertically through micro-algae suspending liquid, the interface of glass and micro-algae suspending liquid is equal to the interface of glass and culture medium solution, now micro-algae suspending liquid transmittance is T _λbe expressed as:

$T_{\mathrm{\λ}}=\left(\frac{t_{01}{t}_{12}{e}^{-{\mathrm{\α}}_1{L}_1}}{1-r_{10}{r}_{12}{e}^{-2{\mathrm{\α}}_1{L}_1}}\right)\left(\frac{t_{23}{t}_{34}{e}^{-{\mathrm{\α}}_3{L}_3}}{1-r_{32}{r}_{34}{e}^{-2{\mathrm{\α}}_3{L}_3}}\right)e^{-\mathrm{\β}{L}_2}[{1-(r_{23}+\frac{t_{23}{t}_{32}{r}_{34}{e}^{-2{\mathrm{\α}}_3{L}_3}}{1-r_{32}{r}_{34}{e}^{-2{\mathrm{\α}}_3{L}_3}})(r_{21}+\frac{t_{21}{t}_{12}{r}_{10}{e}^{-2{\mathrm{\α}}_1{L}_1}}{1-r_{12}{r}_{10}{e}^{-2{\mathrm{\α}}_1{L}_1}})e^{-2\mathrm{\β}{L}_2}]}^{-1}---\left(10\right)$

Obtain the attenuation coefficient β of micro-algae suspending liquid, wherein, the interface transmissivity t of adjacent two layer medium i and j _ij, i=0,1,2,3 and 4, j=0,1,2,3 and 4; 0 represents incident air medium, and 1 is incident glass medium, and 2 is liquid medium, and 3 is outgoing glass, and 4 is outgoing air;

(2) the attenuation coefficient β of micro-algae suspending liquid is represented by micro-algae and nutrient culture media attenuation coefficient sum:

β＝β _p+α _m(1)

In formula, β is total attenuation coefficient, cm ^-1; β _pfor micro-algae attenuation coefficient, cm ^-1; α _mfor nutrient culture media attenuation coefficient, cm ^-1;

(3) nutrient culture media attenuation coefficient α _mbe expressed as:

${\mathrm{\α}}_m=\frac{2\mathrm{\π}{\mathrm{\κ}}_m}{\mathrm{\λ}}---\left(2\right)$

In formula: κ _mfor the absorption index of nutrient culture media, m represents nutrient culture media; λ is incident light source wavelength;

(4) β deducts the attenuation coefficient α of nutrient culture media _mobtain the attenuation coefficient β of micro-algae _p.