CN101660992B - Method for quickly detecting settling velocity of alga cells - Google Patents

Abstract

The invention relates to a method for quickly detecting settling velocity of alga cells. The method includes the following steps: adopting volumetric solution with known concentration and corresponding particle number to calibrate the virtual sampling volume and virtual sampling volume height corresponding to a microscope objective, specifically, taking alga cell solution in exponential phase to conduct centrifugal weight suspension; continuously diluting the alga cell solution obtained in step b at equal multiplying power to obtain a plurality of samples; using an inverted microscope to conduct imaging observation on the bottom of all holes bearing the alga cell solution and selecting a settling column for detecting the settling velocity; continuously imaging the bottom of the settling column at an interval of equal time to obtain a plurality of images; adopting a digital image analysis method to calculate and obtain the number of alga cells in each image; and adopting a linear regression method to analyze the corresponding relation between the number of alga cells and settling time and using the calibrated virtual sampling volume height to calculate the settling velocity of alga cells. The method has the advantages of simple method and device, economical and practical properties, high detection accuracy, simple and convenient operation, etc.

Description

A kind of method of fast detecting settling velocity of alga cells

Technical field

The invention belongs to the detection technique field of phytoplankton settling velocity, especially relate to the method for settling velocity that a kind of inverted microscope that adopts imaging continuous time and digital image analysis detects the alga cells of 5-50um diameter.

Background technology

Settling velocity directly reflects physiological status and the nourishment of phytoplankton, so phytoplankton cell settlement speed is significant on phytoplankton ecology.Yet because the frustule size is less, settling velocity is slow, there is very big technical difficulty in the settling velocity of directly measuring frustule.In the current measuring methods, simple and efficient method measuring accuracy is limited, and its checkout equipment complexity of method that precision is high, and product-free product can not satisfy the research work demand.

Existing frustule settling velocity detection method mainly adopts settling leg to detect, and is broadly divided into two class methods according to the difference of detection position, is respectively that the settling leg fixed position is detected and the settling leg entire scan.The fixed position is the bottom of settling leg normally, the inverted microscope method that typical method has Smayda and Boleyn to adopt, this method preparation concentration known algae liquid sedimentation in settling leg, use the quantity of inverted microscope naked eyes counting settling leg bottom frustule, with the cut off of 80% frustule sedimentation as sedimentation, this method has been ignored as yet not the sedimentation cell to the influence of bulk settling speed; People such as Eppley adopt the fluorescence signal of photofluorometer monitoring settling leg bottom to change the settling velocity that detects alga cells; Bienfang has invented ' SETCOL ' method, the settling leg that this method uses a top and bottom all to be provided with sample tap is carried out sedimentation experiment, after a set time, analyze the settling velocity that frustule is asked in the distribution of frustule concentration in settling leg by hand sampling.

The entire scan method is divided into continuous sweep and interrupted scanning two classes again.Walsby and Xypolyta have invented and have a kind ofly determined the method for time from the detection alga cells sedimentation of the diverse location sample analysis of settling leg; Walsby and Holland combining laser scanning and Percoll gradient solution have been invented a kind of frustule settling velocity detection method of continuous sweep.There is following weak point in these methods: 1) manually participate in morely, can not guarantee the accuracy and the consistance of testing result; 2) the equipment complexity of most methods uses, and criteria of right and wrong instrument is unfavorable for the laboratory study application.

Summary of the invention

For overcoming prior art in the weak point that exists aspect the detection frustule settling velocity, a kind of method of fast detecting settling velocity of alga cells is proposed, this method has advantages such as square law device is simple, economical and practical, accuracy of detection is high, easy and simple to handle based on imaging continuous time of inverted microscope and the detection method of digital image analysis.The common lab that disposes the inverted microscope of imaging device just can be carried out the settling velocity detection of frustule.

The method of a kind of fast detecting settling velocity of alga cells of the present invention is characterized in that, may further comprise the steps:

The volumetric solution of a, employing concentration known and corresponding amounts of particles is demarcated micro objective corresponding virtual sampling volume and virtual sampling volume height Δ H:

Wherein the floorage S of virtual sampling volume is:

Virtual sampling volume height Δ H is: Δ H=K ₁/ C ₀S; K in the formula ₁Corresponding to C in the definite virtual sampling volume of linear regression method ₀The endocorpuscular quantity of calibration solution of concentration;

B, get the frustule solution that is in exponential phase, use low speed centrifuge to carry out centrifugal resuspended with centrifuge tube;

C, with etc. the frustule solution that obtains of multiplying power serial dilution b step, obtain to be not less than three samples, place continuous several holes of 24 orifice plates respectively;

D, with inverted microscope imaging is carried out in each hole that is loaded with frustule solution and observed, the imaging observation place is the bottom in hole, and selecting initial cell quantity in the objective angular field is that 50-200 hole is as the settling leg of detection settling velocity;

E, to settling leg with interval equal time, settling leg bottom continuous imaging more than 3 times, is obtained 3 width of cloth altogether with epigraph;

F, based on the image that e step obtains, adopt method of digital image analysis to calculate the quantity of frustule in every width of cloth image;

The quantity of g, the frustule that calculates based on f step adopts linear regression method to analyze frustule quantity and the corresponding relation of settling time, and with a settling velocity that the virtual sampling volume height Δ H substitution of demarcation can calculate frustule in the step is:

V _a＝K ₂ΔH/b ₂，

K in the formula ₂And b ₂Be respectively to increase frustule quantity and time t=0 moment frustule quantity (being initial cell quantity) by the unit interval in the definite virtual sampling volume of linear regression method.

Characteristics of the present invention and effect:

1, compared with the prior art, this method has good exploitativeness and higher precision, and device therefor all has configuration in general laboratory, can obtain high-precision frustule settling velocity under simple condition.

2, this method is introduced the notion of virtual sampling volume, only needs to demarcate once for a kind of microcobjective, does not need repetition in follow-up test, effectively reduces the workload of measuring.

3, this method adopts method of digital image analysis that continuous imaging is carried out in the frustule sedimentation, adopts linear regression method analysis of cells subsidence rate, has improved the robotization of testing process and the stability of testing result.

Description of drawings

Fig. 1 is the structural representation of the employed device of the inventive method.

Fig. 2 is the synoptic diagram of virtual sampling volume and settling leg.

Fig. 3 is the distribution schematic diagram of volumetric solution sample in 24 orifice plates of six kinds of concentration in the inventive method.

The method of digital image analysis FB(flow block) that Fig. 4 adopts for the present invention.

Embodiment

The method of a kind of fast detecting settling velocity of alga cells that the present invention proposes reaches embodiment in conjunction with the accompanying drawings and is described in detail as follows:

The pick-up unit that the present invention adopts as shown in Figure 1, this pick-up unit comprises light source 1, by being successively set on the inverted microscope that the light source below is formed with the condenser on the optical axis 2, article carrying platform 4, micro objective 5; Also comprise CCD camera 7, be arranged on the reflective mirror 6 between micro objective 5 and the CCD camera 7, reflective mirror 6 is used to make CCD camera 7 can receive the emergent light of micro objective 5; And multi-purpose computer 9, the data transmission interface 8 of connection multi-purpose computer 9 and CCD camera 7.This device all adopts the conventional universal product to assemble.

Among the embodiment of this device: inverted microscope is standard configuration Olympus X71, and light source 1 adopts 100W transmission light beam, and micro objective 5 differs object lens for Olympus long reach achromatism, numerical aperture 0.4, and enlargement ratio is 20X; It is 1392x 1040 that CCD camera 7 adopts the CCD camera resolution of Qimaging, and pixel size is 6.45um; Data transmission interface 8 is the IEEE-1394 interface, and this interface is finished view data and the transmission of control command between multi-purpose computer 9 and CCD camera 7.

Adopt the method for the foregoing description device fast detecting settling velocity of alga cells may further comprise the steps:

A, demarcation micro objective corresponding virtual sampling volume, determine the floorage S and the height Δ H of virtual sampling volume: said virtual sampling volume, as shown in Figure 2, right cylinder is a settling leg among the figure, cubic space is virtual sampling volume, this cubical floorage S is a CCD camera apparent field, can directly calculate according to the correlation parameter of object lens and CCD camera; It is relevant that clear degree falls in the image that causes owing to out of focus in virtual sampling volume height Δ H and the microcobjective depth of field and the imaging process;

The inventive method specifically may further comprise the steps:

1) the floorage S of the virtual sampling volume of calculating:

In the present embodiment: S=1392 * 1040 * 6.45 * 6.45/20 ²=150568um ²(1);

2) adopting diameter is the polystyrene standard spheric grain configuration volumetric solution 2mL of 5-30um (for example 10um), the volumetric solution concentration C ₀10 ⁵-10 ⁷Granules of polystyrene/mL;

3) this volumetric solution is carried out 2 multiplying powers dilutions continuously, obtain and comprise original volumetric solution totally 6 samples, each sample concentration is C _i=C ₀/ 2 ⁱ, wherein i=0-5 is the mark mark of sample, get the 1mL sample respectively and place continuous 6 holes of 24 orifice plates with delegation, as shown in Figure 2, C ₀Sample is at A-1 position, C ₁Sample is at A-2 position, C ₂Sample is at A-3 position, C ₃Sample is at A-4 position, C ₄Sample is at A-5 position, C ₅Sample is in the A-6 position;

4) use inverted microscope that the orifice plate bottom granules of polystyrene that sample respectively is housed is carried out imaging, present embodiment needs solution in the hole is no less than 5 piping and druming before imaging, move horizontally micro objective the diverse location imaging of each bottom surface, hole three times, add up the quantity of granules of polystyrene in every width of cloth image, obtain N after averaging _i, N _iCorresponding C _iThe quantity of the granules of polystyrene of concentration granules of polystyrene solution, wherein i=0-5;

5) calculate virtual sampling volume height Δ H:

The quantity N of granules of polystyrene in each hole in theory _iHas following relation with i

$N_i=C_0\×\mathrm{\ΔH}\×S\×\frac{1}{2^i}---\left(2\right)$

Wherein S is the floorage of above-mentioned virtual sampling volume; In practical operation, adopt the first-order linear homing method to 6 groups of N _iWith 1/2 ⁱCarry out regretional analysis, can obtain a linear relationship

$N_i=K_1\×\frac{1}{2^i}+b_1---\left(3\right)$

K wherein ₁C in the corresponding virtual sampling volume ₀The quantity of concentration granules of polystyrene, b ₁Since the error that factors such as operation are introduced, K ₁, b ₁It is real number; Suitable operation in granules of polystyrene solution allocation process (being that each sample concentration is accurate in the step 3)), b ₁With respect to N _iBe to ignore event, therefore, can make

K ₁＝C ₀×ΔH×S (4)

With S and C ₀, K ₁Go into this formula, can try to achieve Δ H; The relative position relation in Δ H and hole (being right cylinder among the figure) as shown in Figure 3, Δ H is the height of virtual sampling volume (among the figure for cuboid);

B, get the chlorella that is in exponential phase to be detected (this algae mature cell diameter is about 10um) frustule solution 1mL with the 1.5mL centrifuge tube, this solution is added resuspended liquid uses low speed centrifuge to carry out centrifugal resuspended 2 times, last resuspension is a 2mL frustule solution, centrifuge speed 3000rpm wherein, centrifugation time is 1min, and resuspended liquid adopts deionized water;

C, the frustule solution that obtains with 2 multiplying power serial dilution b steps obtain three samples of 1mL, and sample concentration is respectively C _a, C _a/ 2, C _a/ 4; Respectively get every kind of frustule solution example and place continuous three holes of 24 orifice plates, with pipettor sample well is blown and beaten gently, the piping and druming number of times is no less than 5 times;

D, with inverted microscope imaging is carried out in each hole that is loaded with frustule solution and observed numeration, the imaging observation place is the bottom in hole, and selecting initial cell quantity in the objective angular field is that 50-200 hole is as the settling leg of detection settling velocity;

E, to settling leg to settling leg bottom imaging once with interval equal time (for example every 5min) with the CCD camera, continuous imaging 10 times obtains 10 width of cloth images altogether;

F, based on 10 width of cloth images that e step obtains, adopt method of digital image analysis to calculate the quantity of frustule in every width of cloth image; Method of digital image analysis flow process in the present embodiment as shown in Figure 4, comprise: at first carry out the medium filtering noise reduction process for the original image of gathering, image adopts OSTU (big Tianjin threshold method) method to extract the image segmentation threshold value behind the noise reduction, to cut apart the background that obtains adopts pixel to adopt 0 gray scale to fill, image after the filling adopts corrosion and expanding method to remove the noise spot that produces in the image segmentation, adopts the quantity of the method counting frustule of connected component labeling at last; The embodiment of this method is the known technology of this area;

The quantity of g, 10 width of cloth image frustules that calculate based on f step adopts linear regression method to analyze frustule quantity and the corresponding relation of settling time, and with a in the step the virtual sampling volume height Δ H substitution of demarcation can calculate the settling velocity of frustule;

Theoretical derivation is the result show, for the uniform frustule solution of initial concentration, frustule quantity and time have linear relationship in the microscopic fields of view, and this point has also obtained affirmation from the regretional analysis of experimental result; By mathematical derivation as can be known, the quantity N of frustule in the virtual sampling volume of t Δ H height during the time _{Δ H}(t) available following formulate:

$N_{\mathrm{\ΔH}}\left(t\right)=\frac{N_{t}S}{\mathrm{Al}}\×(\mathrm{\ΔH}+V_{a}t)---\left(5\right)$

N in the formula _tBe the total quantity of frustule in the hole, A is the floorage in hole, and l is the liquid level of frustule solution in sedimentation hole, V _aSettling velocity for frustule; The physical significance of following formula clearly, wherein

Be the highly inherent t=0 of Δ H frustule quantity constantly, Be the frustule quantity that increases in the Δ H height in the time at t; By the first-order linear regretional analysis, can obtain N _{Δ H}(t) has following relation with time t

N _ΔH(t)＝K ₂t+b ₂(6)

Order $K_{}$ ${}_2=\frac{N_{t}S}{\mathrm{Al}}\×V_a,$ $b_2=\frac{N_{t}S}{\mathrm{Al}}\×\mathrm{\ΔH},$ Have thus

K ₂/b ₂＝V _a/ΔH (7)

Just the Δ H substitution following formula that obtains of step a can obtain V _a:

V _a＝K ₂ΔH/b ₂＝K ₁K ₂/C ₀Sb ₂ (8)。

Claims (1)

1. the method for a fast detecting settling velocity of alga cells is characterized in that, may further comprise the steps:

A, adopt known C ₀The volumetric solution of concentration and corresponding amounts of particles is demarcated micro objective corresponding virtual sampling volume and virtual sampling volume height Δ H:

Wherein the floorage S of virtual sampling volume is:

Virtual sampling volume height Δ H is: Δ H=K ₁/ C ₀S; K in the formula ₁Corresponding to C in the definite virtual sampling volume of linear regression method ₀The endocorpuscular quantity of the volumetric solution of concentration;

B, get the frustule solution that is in exponential phase, use low speed centrifuge to carry out centrifugal resuspended with centrifuge tube;

C, with etc. the frustule solution that obtains of multiplying power serial dilution b step, obtain to be not less than three samples, place continuous several holes of 24 orifice plates respectively;

D, with inverted microscope imaging is carried out in each hole that is loaded with frustule solution and observed, the imaging observation place is the bottom in hole, and selecting initial cell quantity in the objective angular field is that 50-200 hole is as the settling leg of detection settling velocity;

E, to settling leg with interval equal time, settling leg bottom continuous imaging more than 3 times, is obtained 3 width of cloth altogether with epigraph;

F, based on the image that e step obtains, adopt method of digital image analysis to calculate the quantity of frustule in every width of cloth image;

The quantity of g, the frustule that calculates based on f step adopts linear regression method to analyze frustule quantity and the corresponding relation of settling time, and with a settling velocity that the virtual sampling volume height Δ H substitution of demarcation can calculate frustule in the step is:

V _a＝K ₂ΔH/b ₂，

K in the formula ₂And b ₂Be respectively to increase frustule quantity and time t=0 moment frustule quantity (being initial cell quantity) by the unit interval in the definite virtual sampling volume of linear regression method.

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