DE102005036106B4 - Method and apparatus for determining the velocity profile of a dilute suspension in a microfluidic channel - Google Patents

Abstract

Method for determining a velocity profile of a suspension flowing in a microfluidic channel, comprising the following steps:
Obtaining image data by photographing the interior of the microfluidic channel through a fluorescence microscope (a);
Obtaining image data represented by a color of the light by filtering the image data to eliminate two colors among three primary colors of the light (b);
Forming a matrix by processing the color intensity in the selected part of the corresponding color stripe image from raw image data (c);
numerically differentiating the matrix along the flow direction of a particle (d);
Forming an average of each element in the numerically differentiated matrix to eliminate the noise (e);
Calculating a color stripe length between a pixel point having a maximum value and a pixel point having a minimum value, which values are noise reduced values (f); and
Determine the velocity of the particle from the calculated color stripe length (g).

Description

BACKGROUND OF THE INVENTION

Field of the invention

The The present invention relates to a method for determining a velocity profile of one in a microfluidic channel flowing Suspension.

Description of the state of technology

Information about that Speed profile of a suspension when designing a Microfluidic chips or lab-on-a-chip, taking into account a flow control according to the solution properties, the dispersion of the solution, the for the separation and analysis ability relevant, and an efficient flow distribution in each channel very strongly involved. In particular, the patterns of the velocity profile, the dependent from the properties of the canal walls be changed according to the material of the chip, a basis for understanding the Relationship between the material properties of the chip and the releasability create.

For example has the fluid contained in a microfluidic channel of a polydimethylsiloxane (PDMS) glass microfluidic chip, the one from a PDMS copy and a glass cover flows, because of the hydrophobic property PDMS and the hydrophilic property of glass an uneven expression. consequently shows the flow the suspension flowing in the microfluidic channel asymmetric speed profile. On the surface of the PDMS wall is true the boundary condition of sliding, while on the surface of the Glass wall, the boundary condition of non-slip applies. Therefore, it is required when designing a microfluidic channel a velocity profile of particles according to the solution environments to determine that for an analyzer (eg chromatography or electrophoresis) or used to control the flow in the microfluidic chip becomes.

It is through the MEMS process and the micromachining technology readily possible, to produce the microfluidic channel with a desired channel width. Lab-on-a-chip technology with one on the MEMS process and micromachining technology based category of the microfluidic chip enables the realization of the Micro Total Analysis Systems (micro total analysis system, μ-TAS) and the high throughput system (HTS). Just the tiny size of the lab-on-a-chip creates a portability, which makes it possible to immediately analyze a sample in the field [D.R. Reyes, D. Iossifidis, P.-A. Auroux, A. Manz "Micro Total Analysis Systems. 1. Indroduction, Theory, and Technology "Anal. Chem., 74, 2623-2636, 2002; P.-A Auroux, D. Iossifidis, D.R. Reyes, A. Manz "Micro Total Analysis System. 2. Analytical Standard Operations and Applications "Anal. Chem., 74," 2637-2652, 2002].

To Mid-1990s are microfluidic devices by application developed a silicon-based micromachining technology Service. In the second half In the 1990s, micromachining technology was the building blocks made with disposable plastic, successful. thanks to this Micromachining technology, the block can easily in Mass production to be copied. Among these technologies is that most widely used method of preparation with the PDMS and the photoresistant SU8. To generate the PDMS microfluidic channels, is first on a wafer corresponding to the microfluidic channel cast structure generated. Then a PDMS solution poured onto a wafer, followed by curing. Thereafter, the cured PDMS peeled and tailored to the right size. After that it is bonded to a glass cover. After the investigation of McDonald and Whitesides [J.C. McDonald, G.M. Whitesides "poly (dimethylsiloxanes) as a Material for Fabricating Microfluidic Devices "Acc. Chem. Res. 35 (7), 491-498, 2002], this method is more convenient at low cost than the conventional one Method of etching a wafer in the manufacture of many microfluidic chips.

Significant to the microflow flow visualization study was that Taylor and Yeung applied particle stretch velocimetry (PSV) to both the electrokinetic flow and pressure controlled flow of a fluorescent particle whose size was less than 1 micron , [Ya Taylor, ES Yeung [Imaging of Hydrodynamic and Electro-kinetic Flow Profiles in Capillaries ", Anal. Chem., 65, 2929-2932, 1993].) In the velocity profile of the pressure-controlled flow, a parabolic basic shape was obtained electrokinetic flow had an electroosmotic velocity on the wall, but occurred in the middle of the capillary because of a particle introduced by the electrophoretic motion viscous recoil a small speed error. Since the stripe length of a floating particle has been obtained without accurate numerical processing for the color stripe image, it is necessary to improve the accuracy of the result of the velocity profile.

The longest is microparticle image velocity measurement (micro-particle image velocimetry, μ-PIV) to obtain the velocity profile in microfluidic channels. In the μ-PIV For example, the method of obtaining a velocity profile from Particles from a particle image classified into two types of process will: the pattern match speed measurement (pattern matching velocimetry, PMV) and the particle tracking velocity measurement (particle tracking velocimetry, PTV).

First, an initial image I ₁ containing no particle streaks by embedding a light layer in a channel in which particles flow. Then, a second image I ₂ containing no particle streaks by embedding a light layer after a certain period of time. Î ₁ and Î ₂ are the results of the fast Fourier transform for the respective images. Thereafter, a conjugate complex multiplication is performed on the result at the same local position with respect to Î ₁ and Î ₂ . Thereafter, cross-correlation data is obtained by performing the inverse fast Fourier transform.

The exact speed of each particle can be determined by using the Data will be obtained. However, the above process requires substantial Amount of numerical processing, as it is for each local position both performs the Fourier transform as well as the inverse Fourier transform [M. Raffel, C.E. Willert, J. Kompenhans, "Particle Image Velocimetry: A Practical Guide "Springer, Berlin, 1998; J.G. Santiago, S.T. Wereley, C.D. Meinhardt, D.J. Beebe, R.J. Adrian "A Particle Image Velocimetry System for Microdfluidics "Exp. Fluids, 25, 316-319, 1998].

From the patent US Pat. No. 6,653,651 B1 For example, a method is known for determining a velocity profile of a suspension flowing in a microfluidic channel, in which image data are obtained by photographing the interior of the microfluidic channel through a fluorescence microscope. In addition, from the cited patent a microfluidic chip is known, in which a microfluidic channel ("rectangular capillary") is formed and which consists of a lower substrate ("slide glass") and an upper substrate ("cover slip") Further, the substrate has a thickness that allows optical access for flow imaging.

The patent US 5,979,245 describes that a fluorescent substance containing tracer particles are supplied to a liquid, the particles are irradiated with light and observed by the light-excited fluorescence emission is observed by the tracer particles. In this way an accurate speed measurement of the liquid or a visualization of the liquid distribution is possible.

From the patent US 5,140,071 For example, microscopic, neutrally buoyant particles containing a fluorescent substance are used as tracers in the speed measurement of large scale turbulence liquids.

The patent US 5,491,642 describes a device for measuring velocities in a liquid into which light-reflecting particles have been introduced. For this purpose, the device has components for irradiating the particles with light and for receiving patterns formed by the illuminated particles.

From the publication DE 36 03 905 A1 For example, a flow cell is known in which a microfluidic channel is formed and which consists of a substrate which further has an inlet hole and an outlet hole. Further, the substrate has a thickness that allows optical access for flow imaging.

From the publication US 2003/0054558 A1 is known a two-part microfluidic chip.

From the publication JP 2003279471 A For example, a microfluidic chip is known that consists of a lower substrate and an upper substrate. The upper substrate has a ⊓⁣-shaped microfluidic channel with an inlet hole and an outlet hole. Further, the upper substrate is made of glass and has a thickness that allows optical access for flow imaging.

The patent US Pat. No. 6,309,886 B1 describes a device for imaging multiply fluorescent sample particles. The device comprises a flow channel, which specifies a direction of flow of the particles, a laser beam for illuminating the particles in an observation plane perpendicular to the flow direction, optical elements for imaging the observation plane and a camera for taking pictures.

From the publication US 2002/0137052 A1 discloses a system for the simultaneous analysis of a variety of analytes anchored to microparticles.

From the patent US 5,488,469 is a device known for analyzing cells. The device includes a flow cell through which a sample flows. A light source illuminates cells in the sample. Light information provided by the illuminated cells is recorded, further processed and evaluated.

Nearly all solutions treated by the Lab-on-a-Chip are a suspension, wherein particles with dilute Concentration are dispersed. In this case it is more effective instead of the PIV, which is a big one Amount of data processing and expensive tools needed Apply PSV. However, it is necessary when applying the PSV, the Improve accuracy by numerically processing the experimental data.

Therefore it is necessary to have a method for determining an accurate velocity profile the microflow in the microfluidic channel by means of effective numerical processing to develop.

SUMMARY OF THE INVENTION

The The present invention has been made to overcome the above-mentioned problems, which occur in the prior art to solve. The inventors form the Determining the velocity profile of a suspension a one-dimensional flow using a PDMS glass lab-on-a-chip with slit-like channels, wherein the channel depth is much larger than is the channel width, processes the color stripe image data obtained from experiments of fluorescent particles of a model and obtain the velocity profile more accurately and faster than the conventional one Method. The numerical calculation in the data processing is by implementing the MATLAB program (Mathworks, MA). From the particle color stripes obtained by fluorescence microscopic observation can the asymmetric velocity profile depending on be obtained from the suspension conditions by the speed of fluorescent particles along the lateral position of the Center of the microfluidic channel up to the wall areas determined becomes.

Therefore The object of the invention is a method for determining the velocity profile to create a suspension flowing in the microfluidic channel.

The present invention relates to a method for determining the velocity profile of a suspension flowing in the microfluidic channel. More specifically, the present invention includes the following steps:
Obtaining image data by photographing the interior of the microfluidic channel with a fluorescence microscope (a);
Obtaining image data represented by a color of the light by filtering the image data to eliminate two colors among three primary colors of the light (b);
Forming a matrix by processing the color intensity in the selected part of the corresponding color stripe image from raw image data (c);
numerically differentiating the matrix along the flow direction of the particle (d);
Forming an average of each element in the numerically differentiated matrix to eliminate the noise (e);
Calculating a color stripe length between a pixel point having a maximum value and a pixel point having a minimum value, which values are noise reduced values (f);
Determine the velocity of the particle from the calculated color stripe length (g).

In the method for determining the velocity profile of a particle suspension is preferably a microfluidic chip consisting of an upper Substrate and a lower substrate used. The upper substrate is with an inlet hole through which the suspension is introduced becomes, a ⊓⁣-shape the microfluidic channel in which the suspension flows, and an outlet hole from which the suspension flows out, provided.

In the microfluidic chip, the lateral sidewall of the upper substrate, which becomes a lid of the microfluidic slot-like channel, is made with a proper thickness to provide optical access for the microfluidic channel Allow flow imaging.

Such a microfluidic chip can be produced by a manufacturing method comprising the following steps:
Depositing a photoresist on a substrate;
Patterning the substrate on which a photoresist is deposited by exposing it to ultraviolet (UV) radiation according to a mask pattern;
Forming a base mold by developing the substrate and cutting out the substrate parallel to the lateral side of the photoresist pattern and at a point some distance away from that side;
Probing a glass as a side wall on the surface where the substrate has been cut out;
Pouring PDMS into the base mold in which the glass side wall has been bonded, and peeling the cast PDMS out of the base mold; and
Rinse off the PDMS copy and a glass cover, and bond them together by using reactive ion etching (RIE).

BRIEF DESCRIPTION OF THE DRAWING

The above and other objects, features and advantages of the present invention Invention will become more apparent from the following detailed description, in which which was created in conjunction with the accompanying drawing is, in which:

1 shows a one-dimensional flow and velocity profile of a particle suspension in the microfluidic channel;

2 an image data processing procedure for particle color stripes according to the present invention;

3 is a mask layout of a microfluidic channel;

the 4a to 4g Cross-sectional views for making a microfluidic chip are;

5 Figure 3 is a perspective view of a microfluidic chip;

6 Fig. 10 is a structural diagram of an experimental setup for determining a velocity profile using a microfluidic chip;

7 Fig. 11 is an image showing fluorescent light illuminating the microfluidic channel structure at the observation station of a fluorescence microscope;

8th Image data containing a particle color stripe photographed by the fluorescence microscope in the microfluidic channel;

the 9a to 9d Experimental results are showing velocity profiles of a particle suspension in the microfluidic channel obtained by applying the method according to the present invention.

1

Microfluidic chip

2

object lens a fluorescence microscope

3

digital CCD camera

4

Fluorescent light source

5

optical filter

6

syringe pump

8th

pressure gauge

9

electronic Libra

10

computer

100

silicon wafer

200

photoresist

300

introductory pipeline

350

inflow hole

360

outflow hole

400

Glass side wall

500

PDMS Copy

600

glass cover

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

in the Following are the embodiments of the method for determining a velocity profile of a Particle suspension flowing in a microfluidic channel, under With reference to the accompanying drawings described in detail. however For example, the present invention is not limited to these embodiments.

First becomes the particle color strip velocity measurement based on the present invention (PSV) explained.

A coordinate of the maximum point M _x and a coordinate of the minimum point m _{x of} the particle color stripe in the flow direction of the fluid (ie in the x direction) for the particles flowing in the microfluidic channel are determined, the channel depth being H, the channel width W is and the channel length is L. The ratio of the actual distance to the number of pixels is γ, the particle radius is R _p and the exposure time of the camera is t _E. Then, the particle velocity v _{x can be} obtained by the following equation 1:

When the color stripe of particles in the microfluidic channel is observed as such, it is preferable to observe the movement of particles in the region unaffected by the top wall of the microfluidic channel (ideally at position H / 2). The coordinate in the vertical direction of the maximum point M _x in the flow direction of the fluid represents the particle position in the channel.

2 Fig. 10 shows an image data processing procedure for the particle color strip according to the present invention.

First are photographed by photographing the interior of the microfluidic channel with a Fluorescence microscope received the image data. (1) in the figure the raw image data, which is a particle color band and its surrounding area among the whole photographed by the fluorescence microscope Show image data. These image data contain n × m pixels depending on their size.

After that turns the color stripe image into three primary colors of light, red (R), green (G) and blue (B), divided. (2) in the figure is a graph that the intensity the green Color component among the three primary colors. Pictures can be up Computers with a desired one Color can be shown by the intensity of the three primary colors of the Light is set and the basic colors are mixed. The Image data will also be added through the respective intensity data for the red, the green and the blue color for every pixel stored. Therefore, you can the intensity data the red, the green and the blue color are separated. This intensity data the red, the green and the blue color can through an n × m matrix be reproduced.

Of the on the right side and the left side of (2) shown graph gives the intensity of red light or blue light resulting from the image data of (1) removed was again. Since red is a background color, it does not become Calculate the color stripe length used by particles. Because the blue color through a fluorescent filter is screened out, the intensity of blue light is always zero.

Therefore, in the present invention, those for determining a velocity profile of particles correspond only to the green color image data (2). The n × m matrix representing data for the green intensity of (2) pixel by pixel is denoted by G. The intensity of the green color for each pixel is denoted by g _ij , where i and j represent row and column indices. In the section where the particle color stripes start, the intensity of green color increases. In the section where the particle color stripes end, the intensity of green color decreases.

Therefore when differentiating the matrix G along the flow direction of particles the differentiated value at the point where the intensity of the light increases, maximizes and at the point where the intensity of light decreases, minimizes.

It is assumed that the flow direction of the particle x is the row direction of the G matrix. The value subtracting an element g _ij-1 from g _ij (ie, g _{ij -g ij-1} ) is a numerical differentiation value in the row direction.

(3) in 2 ist der Graph von G'_x. Die linke Seite zeigt ihn dreidimensional und die rechte Seite zweidimensional, von einer Seite betrachtet.In order to obtain numerical differentiation for the entire matrix G, the backward difference operator X which performs the calculation by subtracting each element from the right element (ie, a matrix subtracting the left column vector from a right column vector) can be defined. Thereafter, the differentiation of G can be obtained by using Equation 2:

(3) in 2 is the graph of G ' _x . The left side shows it in three dimensions and the right side in two dimensions, viewed from one side.

In the differentiation value has noise in a local area. The noise creates an undesirable Distortion in the picture. Consequently, the noise should be local Points are minimized by local averaging.

The local matrix G ' _{L (ij)} containing nine elements for a specified point g' _ij can be expressed by Equation 3:

Further, a new noise-reduced dot g ' _{N (ij) is} obtained by estimating the mean value around the point g' _ij according to Equation 4:

When the above averaging process has been performed for all elements of G ' _x , G' _x becomes a new noise reduced matrix composed of g ' _{N (ij)} . (4) in 2 Returns the values resulting from the local averaging procedure.

The intensity of noise in specific areas is relatively stronger than that of neighboring areas. However, the averaging process does of the noise in the specific areas and the neighboring ones Areas the intensity weaker as the section of the color strip in which the light intensity is strong is. According to this principle, the graph of (4) as a result of Noise reduction process in the specific areas. In (4) is the maximum point the starting point of the particle color band and the minimum point of the End point of the color strip. Based on this result, the length of the particle color strip and the particle velocity can be calculated.

This process is applied repetitively to each particle in image data obtained by photographing the inside of the microfluidic channel with a fluorescence microscope. Then the velocity profile of emergent particles can be obtained in a one-dimensional plane become.

in the The following will be incorporated in the method for determining a velocity profile preferably used microfluidic chip and its manufacturing method explained.

3 is a photomask layout required to fabricate the microfluidic channel. The circular arcs on the left and right sides indicate the inlet hole and the fluid outlet hole. The microfluidic channel is designed in the form of a ⊓⁣ to allow a minimum thickness of the lid in the microfluidic channel. Consequently, the focal plane of the fluorescence microscope can be adjusted so that the image is placed in the correct position within the microfluidic channel, ie in the main mass region not affected by the overhead wall (ideally at height H / 2).

In this embodiment is the length of the microfluidic channel is set to 3 cm. This length is enough to allow the fluid in the channel to become fully developed after the channel has been bent to the "L" shape flow can maintain from the inlet hole. The channel width is up 100 μm fixed and the channel depth to 1000 microns.

The 4a to 4g FIG. 15 are cross-sectional views for producing a microfluidic chip preferably used in the method for determining a velocity profile.

First, a SU-8 negative photoresist (at a height of about 100 microns by rotating at the speed of 1150 min ^-1 by means of a rotary coater 200 ) on the silicon wafer ( 100 ) ( 4a ).

After that, this is applied to the wafer ( 100 ) applied photoresist ( 200 ) at 65 ° C for 10 minutes and preheated at 95 ° C for 30 minutes. In this way, the solvent of the photoresist evaporates. Thereafter, the wafer is made using the photomask, as in 4 exposed to UV radiation to crosslink the desired portion of the photoresist polymer. Since the exposed part is constantly crosslinked by heat, it is heated at 65 ° C for 1 minute and at 95 ° C for 10 minutes. After removal of the unexposed portions by means of a developer solution, the mold containing the negatively patterned photoresist remains. ( 4b ).

Thereafter, the introductory pipeline ( 300 ) are glued and placed on the corresponding inlet hole and the corresponding outlet hole before pouring ( 4c ).

Thereafter, the wafer ( 100 ) at a position at a certain distance (eg 0.5 mm to 2 mm, preferably 1 mm) from the lateral side of the photoresist pattern (FIG. 200 ), in which the microfluidic channel is formed, removed and cut out parallel to this. Then the glass side wall ( 400 ) placed vertically on the cut surface ( 4d ). 4e is a plane figure showing the manufactured mold. 4d is one along the line AA 'in 4e recorded cross-sectional view.

After that, PDMS solution ( 500 ) (Dow Corning Sylgard-184, MI) in the volume ratio of 10: 1 with a curing agent and poured into the mold. Thereafter, the PDMS is cured by heating for 2 hours at 80 ° C ( 4f ).

Thereafter, the cured PDMS is peeled out of the mold and cut to the correct size. Then the PDMS copy ( 500 ) using ion etching (RIE) (Plasmatherm 760 , USA) with a glass cover ( 600 ) (Marienfeld Micro Slides, Germany) ( 4f ). In this process, charged oxygen is added to the PDMS and the glass cover. In this way, the surfaces of the material become unstable so that they can be readily joined together to become stable.

After that, as in 5 is shown, the main pipeline by means of an epoxy adhesive to the inlet hole ( 350 ) and the outlet hole ( 360 ) glued. Thereafter, it is firmly bonded by means of a special rapid adhesive (SILY, Korea Altecho) for silicon. 5 is a perspective view of the complete microfluidic chip as such. In the present invention, it is also possible to use other transparent plastics instead of the PDMS.

The method for determining a velocity profile may be performed using the fabricated microfluidic chip. 6 is a structural diagram of a test setup for Bestim a velocity profile of the microfluidic chip according to the present invention.

Using a syringe pump ( 6 ) (Cole-Parmer 7600 series, IL) a suspension in which polystyrene latex particles (Sigma L-5280, MO) are dispersed with a diameter of 2 microns, under constant pressure (the pressure can be determined by the high-precision pressure gauge ( 8th ) can be measured) through the inlet hole of the microfluidic chip ( 1 ).

That of the fluorescent light source ( 4 ) (Carl Zeiss M2 FL S, Germany) emitted fluorescent light is emitted through the fluorescence filter ( 5 ) (Carl Zeiss FITC fluorescence filter, Germany) to give wavelengths suitable for observing the polystyrene latex fluorescent particles. In this way, latex fluorescent particles emit light having an inherent wavelength. The emitted light is transmitted through the object lens ( 2 ) by a multiple of 2.5 of the fluorescence microscope (Carl Zeiss SV-11, Germany) and also by a digital CCD camera ( 3 ) (Carl Zeiss AxioCam HR, Germany). With the digital imaging software (Axio Vision 3.1, Carl Zeiss), the data from the camera ( 3 ) for image acquisition to a PC-based frame grabber ( 10 ). The weight of the suspension, which flows out through the outlet hole of the microfluidic channel, is transmitted through the electronic balance ( 9 ). In this way the mean velocity of the suspension can be estimated.

7 Fig. 11 is an image by photographing the shape of the fluorescent light-lit microfluidic chip.

8th is a digital image obtained by photographing the microchannel at a camera exposure time of 200 ms. The suspension, in which fluorescent latex particles are dispersed at an extremely dilute concentration of 0.48 ppm, flows in the microfluidic channel at a flow rate of 55 nl / s and a Reynolds number of 0.1. The color stripe, which traces the movement of particles, appears in the digital image. As described above, the velocity profile of each particle can be obtained by numerically processing the obtained image.

In the present invention, the velocity profile can be more accurately determined because the particle size is smaller compared to the channel width. Specifically, assuming that the particle _radius is R _p and the channel width is W, the velocity profile can be more accurately determined if 2R _p / W ≤ 0.1. Further, a more accurate velocity profile can be obtained when the present invention is applied to a dilute suspension.

The 9a to 9d show experimental results of determining the velocity profile according to the present invention.

9a is a result for a solution with KCl, 1.0 mM, pH 5. 9b is a result for a solution with KCl, 1.0 mM, pH 10. 9c is a result for a solution with KCl, 0.1 mM and pH 7. 9d is a result for a solution with KCl, 10 mM, pH 7.

KCl is used as electrolyte in every solution. The pH is adjusted by HCl and NaOH. In the 9a to 9d The left side and the right side correspond to the x-axis (equivalent to the channel width) of the glass wall and the PDMS wall, respectively. They show that the particle velocity at the glass surface, due to the hydrophilic property, converges to zero. However, the particle velocity at the PDMS surface, due to the hydrophobic property, has a finite sliding velocity value instead of zero.

According to the procedure For determining the velocity profile of the invention, it is possible to determine the velocity profile in the microfluidic channel without execution an excessive numerical Accurately and efficiently determine processing. The procedure for Determining the velocity profile can be used for flow control in dependence of fluid properties in the microfluidic channel and efficient flow distribution be applied.

Even though for illustrative purposes, preferred embodiments of the present invention Invention have been described to those skilled in the art clear that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention, as set forth in the accompanying drawings claims is disclosed departing.

Claims (7)

Method for determining a velocity profile a flowing in a microfluidic channel suspension, the following steps include: Obtaining image data by photographing of the interior of the microfluidic channel through a fluorescence microscope (A); Obtaining represented by a color of light Image data by filtering the image data to two colors under three To eliminate basic colors of light (b); Forming a matrix by processing the color intensity in the selected part the corresponding color stripe image from raw image data (c); numeric Differentiate the matrix longitudinally the flow direction a particle (d); Make an average of each element in the numerically differentiated matrix to eliminate the noise (E); Calculating a color stripe length between a pixel point with a maximum value and a pixel point with a minimum Value, where these values are noise-reduced values (f); and Determine the velocity of the particle from the calculated Paint stripe length (G). The method of claim 1, wherein the focal plane of Fluorescence microscope is adjusted so that during the Photographing the interior of the microfluidic channel in step (a) is located in the middle of the microfluidic channel height. The method of claim 1, wherein in step (b) the exclusively through the green Color represented Image data by filtering the image data to the red color and the to eliminate blue color among the three primary colors of light, to be obtained. The method of claim 1, wherein the parts in which in step (c) the light intensity is processed by the pixel, a particle color strip and its surrounding area among the entire image data. The method of claim 1, wherein each element in the numerically differentiated matrix in step (e) is averaged locally becomes. The method of claim 5, wherein the local mean each element is the mean of the element and its neighboring one Elements is. The method of claim 1, wherein steps (c) to (g) for repeats each particle color strip shown in the image data become.