Abstract:
Methods and apparatus for fluid flow velocity and flow rate measurement are provided. Fluid velocity is measured in optical method based on fluorescence photobleaching of a fluorescent dye. The invented method and apparatus requires a calibration relation between flow velocity and fluorescence signal and is easy to use. The invented method and apparatus can measure bulk flow velocity and flow rate inline, two and three components of flow velocity vector. It can also measure flow velocity in near wall region using evanescent wave. Since the invented method and apparatus uses molecular dye and calibration relation between flow velocity and fluorescence signal, it has ultra high spatial and temporal resolution. The invented method can be used not only to apparatuses and devices in conventional size, but also to that in MEMS and NEMS.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS  
       [0001]     Not Applicable  
       INCORPRATED-BY-REFERENCE OF METARIAL SUBMITTED ON A COMPACT DISC  
       [0002]     Not Applicable  
       BACKGROUND OF THE INVENTION  
       [0003]     1. Field of the Invention  
         [0004]     This invention relates to velocity of flow rate measurement of fluid flow. In particular, the invention involves the use of photobleaching of fluorescence dye to measure the fluid flow velocity, including direction, of the fluids in devices. The invention can be employed in a wide variety of applications including, but not limited to, the processes, apparatuses, devices and instrument in scales of conventional, millimeter, micro and nanometer. This invention is non-intrusive and capable of measuring flow velocity inline, point measurement, two dimensional and three dimensional flow fields with high temporal and spatial resolution.  
         [0005]     2. Description of Related Art  
         [0006]     The measurement of fluid velocity by fluorescence photobleaching is known in the art. Reviews of the measurement of fluid velocity with fluorescence photobleaching are presented in the following references: 
    C. A. Monnig and J. W. Jorgenson, Anal. Chem., 1991, 63, 802-807;     A. W. Moore and J. W. Jorgenson, Anal. Chem., 1993, 65, 3550-3560;     B. P. Mosier, J. I. Molho and J. G. Santiago, Exp. Fluids, 2002, 33, 545-554;     K. F. Schrum, J. M. Lancaster, S. E. Johnston and S. D. Gilman, Anal. Chem., 2000, 72, 4317-4321;     J. L. Pittman, K. F. Schrum and S. D. Gilman, Analyst, 2001, 126, 1240-1247;     J. L. Pittman, C. S. Henry and S. D. Gilman, Anal. Chem., 2003, 75, 361-370.     H. E. Fiedler and G. R. Wang, Deutsches Patent, 19838344.4, 1998, Germany;     J. Ricka, Exp. Fluids, 1987, 5, 381-384;     J. White and E. Stelzer, Trends Cell Biol., 1999, 9, 61-65;     B. Storrie, R. Pepperkok, E. Stelzer and T. E. Kreis, J. Cell Science, 1994, 107, 1309-1319.     G. R. Wang Laser-induced fluorescence photobleaching anemometer for microfluidic devices.  Lab on a Chip,  2005, 5, 450-456.    
 
         [0018]     For instance, fluorescence recovery after photobleaching (FRAP), which can be used to measure very low flow velocity near the region of Brownian motion, requires two laser beams at the detection point. One is high laser beam intensity to cause the photobleaching and the other one is used to measure the recovered fluorescence intensity due to molecular diffusion for a long time period. The new method can measure the flow velocity instantaneously with only one beam and the velocity range measured is higher than the molecular difusion. The two points based method in Pittman et al. (2003) also requires two laser beams and the first one bleaches the fluorescence and the second one has to be in the downstream of the first one in a distance to measure the bleached fluorescence signal. This method can only measure the bulk flow velocity and the temporal resolution is limited, since it has to wait for the bleached dye plug to translate from the first laser beam to the second one. Photobleached Fluorescence Visualization in Mosier (2002) requires camera to monitor images of flow field at different downstream positions to calculation flow velocity. Also a diffusion model is required. Rick (1987) published only qualitative method to visualize flow velocity, where the optical setup, detector and flow used for calibration were different from the real detector and flow. This make the quantitative measurement impossible, since the calibration relationship between velocity and fluorescence intensity is different from that in real flow. Also the calibration was linear. All these method cannot measure spatial velocity distribution, neither can they measure velocity vector, i.e. transverse velocity components.  
         [0019]     The present invented method is a different way from aforementioned methods to measure flow velocity, although it also use photobleaching. In the invented method, the optical setup, detector and the flow are all the same to be able to carry out precise quantitative measurement. The calibration relationship in current method is not necessarily linear, but can also be a polynomial or exponential relation. Since the invented method is based on a single point measurement, it can measure velocity distribution in the transverse direction with high spatial resolution; the velocity is directly measured with the calibration relationship between flow velocity and fluorescence intensity, and thus the invented method has high temporal resolution and can be used for inline or online flow velocity or flow rate monitoring. Since molecular tracer dye is used for the invented method, the spatial resolution is high and can be used to devices of Micro-Electro-Mechanical Systems (MEMS) and Nano-Electro-Mechanical Systems (NEMS) compared with Particle Image Velocimetry (PIV), Laser Doppler Anemometry (LDA) and Ultrasound Velocimetry. Using evanescent wave guide, the invented method can also measure flow velocity in near solid wall region.  
       BRIEF SUMMARY OF THE INVENTION  
       [0020]     The present invention represents an advance in the art of flow velocity measurement, specifically the fluid flow velocity measurement based on photobleaching in single spatial point. The single spatial point means that the flow velocity is measured directly at point of the excitation light beam and no wait is required either for the bleached signal to come at another beam downstream of the first one, or for the fluorescence intensity to recover. The invented method cannot measure molecular diffusion, but convection flow velocity, i.e. much higher than molecular diffusion. The flow velocity is measured and calculated by measured fluorescence intensity, based on a calibrated relationship between flow velocity and fluorescence intensity. The mechanism behind the calibration relationship is the fluorescent dye can photobleach when excited by excitation light: the higher the flow velocity is, the less bleaching time for the dye is, and thus, the higher the fluorescence signal at the detection point of the illuminated region. The fluorescence signal is detected by an optical detector and the velocity is calculated in the light of the calibrated relationship. The spatial resolution is determined by the excitation light dimension, and the pinhole size in frontal of the detector. The sensitivity depends on the laser power, dye concentration, dye property, beam size, PH value and temperature. The temporal resolution depends on the laser power at the detection volume, dye property (bleaching time constant), PH value and temperature.  
         [0021]     The present invented apparatus and method allow realtime resolution, inline velocity measurement, three dimensional velocity components measurement, high spatial and temporal resolution velocity measurement, and velocity distribution measurement in a flow field. The present invention can also measure near wall velocity with evanescent wave.  
         [0022]     The invention is described in more detail below. Those skilled in the art will recognize that the examples and embodiments described are not limiting and that the invention can be practiced in many ways without deviating from the inventive concept. 
     
    
     BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS  
       [0023]      FIG. 1  is a typical calibration relation between fluorescence intensity and flow velocity.  
         [0024]      FIG. 2  is a schematic of the measurement system of the average bulk flow rate in a channel.  
         [0025]      FIG. 3  illustrates the point measurement for measuring velocity distribution of a flow field in a flow chamber.  
         [0026]      FIG. 4  is a schematic of measurement of two-dimension flow field with periodic illumination of the field.  
         [0027]      FIG. 5  represent a case in, but not limited to, an epi-fluorescecne microscope.  
         [0028]      FIG. 6  is a different optical arrangement of  FIGS. 1 and 2 . It does not require the axial direction of excitation light to be orthogonal to that of the detection system.  
         [0029]      FIG. 7  shows multi-beams with an angle between 10-350° for the velocity vector components and vorticity measurement.  
         [0030]      FIG. 8  illustrates the two dimensional velocity measurement in a light sheet with arrangement of selected orthogonal pixel lines from a detector, such as, but not limited to a camera.  
         [0031]      FIG. 9  illustrates the two dimensional velocity measurement in a light sheet with arrangement of selected parallel pixel lines from a detector, such as, but not limited to a camera.  
         [0032]      FIG. 10  depicts an example of near wall velocity measurement with evanescent wave. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0033]     The invented method requires a calibration relationship between the flow velocity and fluorescent intensity.  FIG. 1  is a typical calibration relation between fluorescence intensity and flow velocity, where the fluorescence intensity increases linearly with flow velocity when the flow velocity is small (but larger than molecular diffusion). With the increase of the velocity, the fluorescence intensity increases with the velocity nonlinearly. With further increase of the velocity, the fluorescence intensity saturate with the velocity. The velocity to be measured should be within the calibration range. There are several methods that can carry out the calibration. An easy way is to measure the fluorescent signal at the detection point for a given optical setup with a given flow velocity. For example, the bulk average flow velocity can be calculated once the flow rate is known. The flow rate can be obtained from, but not limited to, the pump flow rate reading. Record each fluorescent signal for different flow rates to obtain the calibration relation. Another method is the flush time measurement. One example is to generate a dye plug in the channel, or an interface between dye solution and non-dye solution at a given position for a given flow velocity. Then monitor the time trace of the fluorescent signal. The traveling time of the plug or interface from the position where it is injected (generated), to the detection point can be used to calculate the flow velocity since the distance between the injection position to the detection point is known. The calibration can also be measured by traveling the flow channel on a translation stage while the flow is at rest. The flow velocity is regarded as the velocity of the translation stage. The calibration relationship can be obtained through measuring the fluorescence signals at a detection point corresponding to different translate stage velocity.  
         [0034]     To increase the accuracy of the measurement, aspect ratio of the excitation beam can be increased, when the transverse velocity component that is in parallel to the excitation light propagation direction, is not negligible. This can reduce the influence of the dye molecules that transport from the cross surface of the excitation light beam into the detection volume.  
         [0035]     The invented method measures the flow velocity through measuring the fluorescent signal. Since the fluorescence intensity changes with source light power, which can fluctuate, relative fluorescence intensity could be used to reduce the error caused by source light power fluctuation. The relative fluorescence intensity is the ratio of fluorescent intensity to a reference source light power, which can, but not limited to, be a fraction of source light signal, measured with a photodetector through a beam splitter or a mirror.  
         [0036]     In the first embodiment of the invention as shown in  FIG. 2 , an exciting light beam  004  from light source  001  is required to illustrate and passes through a detection point  005  of the flow field in transverse direction of a flow channel  011  for measuring bulk flow velocity. The light intensity should be sufficiently high to cause photobleachinhg of a fluorescent trace dye. The light source can be, but not limited to, a laser, mercury lamp, halogen lamp, xenon lamp, LED and etc. A fluorescent trace dye will be used to generate fluorescence signal. The stronger the photobleaching of the dye, the higher the sensitivity of the method. There is a photodetector  009  to receive the fluorescence signal. The detector can be, but not limited to a CCD camera, CMOS camera, photodiode, avalanche, photomultiplier tube (PMT) and etc.  
         [0037]     The following methods can be applied to improve the sensitivity and signal to noise ratio, spatial and temporal resolution. A lens  003  can be used to manipulate or focus the exciting light onto the detection point  005  to increase the exciting light intensity there and an optical filter  002  can be used between the exciting light source and the detection point to filter away all other wavelength light and only pass the exciting wavelength light. A collection lens  006  can be located between the detection point and detector to image the signal to the detector. A pinhole  008  can be used between the detector and detection point to image the signal to the detector. An optical filter  007  can be placed between the detection point and the detector to filter away all noise and only pass the fluorescence signal. The output signal from the detector can be sent to a data processor  010 , for example, a computer for data procedure to calculate flow velocity or flow rate.  
         [0038]     In a second embodiment in  FIG. 3 , the invention comprises similar optical and detector components to the first embodiment, except that the purpose here is to measure different spatial point in transverse direction. The light from the source  020  is focused to a small beam point  023  as detection point in the flow channel  024  to measure flow velocity at this particular spatial point. With the change of the focus point in transverse direction, the flow field in the transverse direction can be measured. Similar to the first embodiment, there are also optical filter  021  and lens  022  for manipulating the light source, and lens  025 , optical filter  026  and pinhole  027  for manipulating fluorescent signal to the detector  028 , whose output signal is transferred to data processor  029  for velocity calculation.  
         [0039]      FIG. 4  is a third embodiment for measuring two dimensional flow field. The light beam from light source  030  is spread with a cylindrical lens  032  to generate a light sheet  033 . There is a light screen  034  which can periodically block the sheet light spatially to generate periodic light sheet  035  in the flow field of the flow channel  040  to be measured. The signal from the periodic light sheet  035  is recorded to the detector  038  and then sent to the data processor  039  for velocity calculation. To improve the measuring system, optical filter  031  and  037  can be used for excitation light and emission signal light respectively, and a collection lens can be used to image the signal to the detector, as in  FIG. 2 . Since fluorescence intensity increases with the increase of flow velocity, two-dimensional light sheet without the screen can also be used to measure flow velocity.  
         [0040]     The embodiment in  FIG. 5  is with an epi-fluorescence microscope for miniaturized (nano- and microfluidics) systems. The source light  050  pass through an optical filter  051 , a dichroic  052 , and a lens  053  and is focused to the detection point in the flow channel  058  of a device, such as, but not limited to a chip  057 . The fluorescent signal is collected through the lens  053  and the dichroic  052  and an optical filter  054 , and is imaged to the detector  055 , and sent to data processor  056 .  
         [0041]     In cases where the sidewalls of the flow channel is not optically transparent, the excitation light cannot illuminate the detection point through sidewall, but can enter the detection point from top or bottom of the channel with a angle between 10-350° to the top surface. The collection lens of the fluorescence signal of the detection system can have an angle between 10-350° to the excitation light.  
         [0042]     In the embodiment of  FIG. 6 , there is an angle α 1    070  between exiting light and axial direction of collection lens  064 . The angle can be in the range of 10-350°. There can also be, but not limited to, an optical filter  062  and a focusing lens  063  to focus the excitation light beam  071  to the detection point  073  in a flow channel  068  of a device or a chip  069 . The exciting light beam  071  from source light  061  can also be a sheet at detection point, if a cylindrical lens  072  is added. For detection of the fluorescent signal, a collection lens  064 , an optical filter  065 , a pinhole  066  and a detector  067  are used.  
         [0043]     In order to measure velocity vector components, multi excitation light beams with an angle between 10-350° from each other are required. The orthogonal light beams from each other are more preferred as shown in  FIG. 7 . The effective velocity V A,eff , V B,eff , and V C,eff  of flow velocity V for light beam A  080 , light beam B  081  and light beam C  082  is
 
 V   A,eff   =V  cos α  (1)
 
 V   B,eff   =V  cos β  (2)
 
 V   C,eff   =V  cos γ  (3)
 
 respectively, where α, β, and γ is the angle between beam A  080  and velocity V,  083 , beam B  081  and velocity V,  084  and beam C  082  and velocity V,  085  respectively. One example of calculating flow velocity is, but not limited to, to apply following equations:  
               V     A   ,   eff     2     =       V   2     ⁡     (         sin   2     ⁢   α     +       b   2     ⁢     cos   2     ⁢   α       )               (   4   )                 V     B   ,   eff     2     =       V   2     ⁡     (         sin   2     ⁢   β     +     b     2   ⁢           ⁢     cos   2     ⁢   β         )               (   5   )                 V     C   ,   eff     2     =       V   2     ⁡     (         sin   2     ⁢   γ     +       b   2     ⁢     cos   2     ⁢   γ       )               (   6   )                 V   2     =         V   A   2     +     V   B   2     +     V   c   2         2   +     b   2                 (   7   )             
 
 where b is a constant. If only two dimensional velocity components are required, only the first two light beams will be used. 
 
         [0044]      FIG. 7  is an embodiment for measurement of three components of velocity vector. Two or three orthogonal (but not limited to orthogonal) excitation light beams A  080 , beam B  081  and beam C  082  are used in the detection point. Each excitation light can have its own light source or they can share one light source through beam splitters. Each excitation light beam has its own detection system and detector components. The optical components for each excitation light path, and the optical components for detection path are similar to their counterparts in  FIG. 3 . In order to reduce noise due to cross talk of the multi light beams, the excitation light can be different in wavelength for each beam, and each excitation light beam can has its own dye, which has different emission light wavelength from that of the other light beams. Each detector will have a corresponding optical filter to filter away the noise from the other beams. With one detector, such as a camera, the two fluorescence signals can also be measured and distinguished. This can be realized by partition of pixels into two regions, and each region measure one beam fluorescence signal.  
         [0045]      FIG. 8  is a different embodiment for the measurement of velocity vector components. This is a two-dimensional image of fluorescent signal in a two-dimensional flow field. In  FIG. 8 , two orthogonal pixel lines (pixel array) consisting of multi-pixels are selected.  FIG. 8  shows two examples (but not limited to) of pixel lines, used for calculation of the velocity components. The pixel line functions as the excitation light beam in  FIG. 7 . The fluorescent signal from a pixel line is similar to that of a beam in  FIG. 7 . In  FIG. 8   a , the orthogonal pixel array lines  091  and  092  are consisted of multi pixels, which are connected through the vertex. In  FIG. 8   b , the orthogonal pixel line  092  and  093  are consisted of multi pixels, which are connected through two side of a pixel.  
         [0046]      FIG. 9  is a different embodiment for the measurement of velocity vector components from that in  FIG. 8 . The main difference in  FIG. 8  and  FIG. 9  is that the pixel lines in  FIG. 8  are in cross, while the pixel lines  FIG. 9  are in parallel. The signal in  FIG. 9  can be used to measure not only velocity vector component, but also vorticity.  FIG. 9  shows two examples (but not limited to) of pixel lines, used for calculation of the velocity components. In  FIG. 9   a , the parallel pixel array lines  100  and  101  are consisted of multi pixels similar to the pixel line  092  in  FIG. 8 . These pixel lines can be used to measure velocity component orthogonal to them. In  FIG. 9   b , two pairs of parallel pixel lines  102  and  104 ,  103  and  105  are used to measure two orthogonal velocity components and corresponding vorticity.  
         [0047]     The invented method can also measure flow velocity at near wall region with ultra high spatial resolution, since it is a molecular dye tracer based method. To achieve a very thin excitation light, we can use evanescent wave illumination as shown in  FIG. 10 . In the embodiment of  FIG. 10 , the source light  110  pass through a prism  111  to generate a evanescent wave illumination  112 , which can be normally very thin, in the order of 200 nm thick. The evanescent wave illumination is generated on the solid surface of a flow channel  113 .  114  is an example of the fluid flow direction. The light can be a sheet or a line. The collection system include a collection lens, filter, pinhole  117 , detector  118  and data processor  119 .