Patent Publication Number: US-7212701-B2

Title: Methods for measuring optical absorbance of a sample medium

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
   This application is a divisional of U.S. Ser. No. 11/077,834 filed Mar. 11, 2005, now U.S. Pat. No. 7,177,492 which claims priority under 35 U.S.C. 119 to the provisional application Ser. No. 60/552,158, filed Mar. 11, 2004, entitled “SYSTEM FOR COLORIMETRIC TESTING OF FLUIDIC SAMPLES”, which is hereby expressly incorporated herein by reference in its entirety. 

   FIELD OF THE INVENTION 
   DESCRIPTION OF THE PRIOR ART 
   Traditional systems for colorimetric testing generally provide a fixed length, single pass, optical path through a sample of liquid medium. Such traditional systems typically utilize a capillary micro-cuvette to contain the sample of liquid medium. In these traditional systems the sample of liquid medium is drawn into the capillary micro-cuvette to form a water core waveguide and, in operation, light from a light source propagates inside the water core waveguide before being received by a reader. One of the principal limitations of such traditional systems is the inconvenience of introducing and removing the sample of liquid medium from the system and the system&#39;s inability to conveniently support continuous flow monitoring. 
   Reducing the required volume of the sample of liquid medium needed to perform colorimetric testing reduces the cost of performing the colorimetric testing. Sample volume reduction in traditional systems is currently obtained by enhancing the path length to volume ratio by dimensional extension along the optical path and dimensional reduction perpendicular to the optical path. An improved way of enhancing the path length to sample volume ratio would lead to, among other things, reduced cost of sample analysis. 
   Therefore, there exists a need in the field of colorimetric testing for a system which would provide for convenient sample volume introduction and removal, an ability to support continuous flow monitoring and improved sample volume to path length ratios. It is to such a system that the present invention is directed. 

   
     BRIEF DESCRIPTION OF THE DRAWINGS 
       FIG. 1  is a schematic view of a system for colorimetric testing. 
       FIG. 2  is a side elevational view of probe for colorimetric testing. 
       FIG. 2A  is a perspective view of a probe for colorimetric testing. 
       FIG. 2B  is a detailed view of the probe tip. 
       FIG. 3  is a side elevational view of an inline sample cell. 
       FIG. 4  shows an exemplary spherical coordinate system. 
       FIG. 5  shows the radial distance dependence of a Whispering Gallery Mode. 
       FIG. 6  shows the Whispering Gallery Mode field potential of a 5-μm spheroid of optically transparent material shown in decibels at the equatorial (x-y) plane. 
       FIG. 7  shows Whispering Gallery Mode high radial mode with L=M=50, q=2. 
       FIG. 8  shows a normalized power distribution of the Whispering Gallery Mode potential along the latitudinal angle. 
       FIG. 9  shows a Whispering Gallery Mode field potential of a 5/μm spheroid of optically transparent material with a mode index L−M=6 shown in decibels at the vertical (x-z) plane. 
       FIG. 10  shows the spacing of Whispering Gallery Modes in a 0.75 mm quartz spheroid of optically transparent material relative to a typical visible absorbance spectrum. 
       FIG. 11  shows the evanescent field coupling between a prism and a spheroid of optically transparent material. 
       FIGS. 12A and 12B  depict the geometry of an angle polished fiber coupling and total internal reflection and evanescent wave. 
       FIG. 13  shows a first optical fiber having an angle polished first end in communication with a spheroid of optically transparent material and a second optical fiber having an angle polished first end in communication with the spheroid of optically transparent material. 
       FIG. 14  shows the arrangement of a fiber coupled 632 nm absorption experiment. 
       FIG. 15  shows the arrangement of the spheroid of optically transparent material and the tapered first ends of the first and second optical fibers. 
       FIG. 16  shows the spheroid of optically transparent material in a liquid envelope. 
       FIG. 17  shows a delrin trough. 
       FIG. 18  shows 790 nm WGM transmission for three concentrations of Indocyanine green (ICG); spheroid of optically transparent material diameter equals 350 μm; scan range equals 1 GHz; wavelength equals 790 nm. 
       FIG. 19  shows Whispering Gallery Mode height versus ICG concentration. 
       FIG. 20  depicts another 790 nm WGM transmission for three concentrations of ICG absorption; spheroid of optically transparent material diameter equals 350 μm; scan range equals 1 GHz; wavelength equals 790 nm. 
       FIG. 21  depicts Whispering Gallery Mode height versus ICG concentration. 
       FIG. 22  shows arrangement of fiber coupled broadband absorption experiment. 
       FIG. 23  shows broadband absorption of crystal violet. 
       FIG. 24  shows microsphere fabrication method for (a) large sphere and (b) small sphere by CO 2  laser. 
       FIG. 25  shows commercially available 2.3 cm microvette, with attached vacuum pump to facilitate sample removable. 
       FIG. 26  is a perspective view of a microfluidic chip constructed in accordance with the present invention, a perspective inset view is shown of a microfluidic channel formed in the microfluidic chip. 
       FIG. 27  is a cross-sectional view of a microresonator positioned within the microfluidic channel of the microfluidic chip of  FIG. 26 . 
   

   DESCRIPTION OF THE PREFERRED EMBODIMENTS 
   Referring now to  FIG. 1  shown therein is a system  10  for the colorimetric testing of a sample constructed in accordance with the present invention. The system  10  includes a light source  12 , a reader  14  and a probe  16 . The probe  16  includes a resonator  18 , a first coupler  20  and a second coupler  22 . The probe  16  also can be provided with a handle  24  ( FIG. 2A ) for facilitating enclosure of and handling of at least a portion of the resonator  18 , the first coupler  20  and the second coupler  22 . 
   The light source  12  for the system  10  can be, for instance, a broad line width laser or an incoherent light source. For general purpose colorimetric testing, light sources  12  that can be utilized and are readily available commercially include, but are not limited to, tungsten quartz halogen bulbs. For colorimetric testing where a single spectral band provides sufficient information, the light source  12  can be a light emitting diode. 
   The reader  14  can be any reader capable of performing colorimetric testing, such as a spectrometer or an Si detector. A spectrometer which can be employed as the reader  14  is an Ocean Optics USB 2000, commercially available from Ocean Optics Inc., Dunedin, Fla. 
   The first and second couplers  20  and  22  are devices capable of transmitting light such as, optical fibers, planar waveguides, prisms or gratings capable of simultaneously directing a multitude of optical resonances. The first coupler  20 , and the second coupler  22  of the probe  16  are coupled to the resonator  18 . The first coupler  20  receives and couples at least a portion of the light from the light source  12  and directs the light coupled from the light source  12  into the resonator  18 . The second coupler  22  of the probe  16  receives and couples at least a portion of the light coupled into the resonator  18  from the first coupler  20  and directs the light coupled from the resonator  18  to the reader  14 . The portion of the light coupled from the resonator  18  and directed to the reader  14  is then analyzed by the reader  14 . 
   The resonator  18  is constructed of optically transparent material shaped so as to constrain light to a repeating path and simultaneously support a multitude of optical resonances. For example, the resonator  18  can be a disc, torus, ring, cylinder, bloated cylinder or spheroid of optically transparent material capable of supporting a multitude of optical resonances. The resonator  18  can be constructed from silica, glass, quartz, gallium, silicon, or combinations and derivations thereof. 
   Light coupled into the resonator  18  is constrained to bands of frequencies known as Whispering Gallery Modes (WGM). Frequencies of light outside of the WGM&#39;s will not couple into the resonator  18 . For use with broadband illumination, it is desirable to employ resonators supporting WGM resonances that are more tightly spaced than the resolution of the reader  14  utilized to measure the light exiting the resonator  18 . This is achieved by using resonators generally larger than 50 μm in diameter. 
   Light from the light source  12  that is coupled into the resonator  18  propagates around the perimeter of the resonator  18  in WGM resonances by continuous internal reflection. As the light travels around the perimeter of the resonator  18 , an evanescent field of photons of light extends slightly beyond the resonator  18 . The evanescent field of photons interacts with a sample to be tested that preferably substantially envelops the resonator  18 . The evanescent field of photons which extends slightly beyond the resonator  18  travels into the sample to be tested which is disposed in close proximity to the resonator  18 . The photons of light within the evanescent field interact or react with the molecules of the sample many times as the light propagates around the perimeter of the resonator  18 . In the resulting interaction or reaction between the evanescent field of photons and the sample, absorption may occur which is then measured by the reader  14 . 
   The relationship between the concentration of an absorbing material in the sample and the absorbance measured by the reader  14  at a particular wavelength is given by Beer&#39;s Law:
 
 A =−log  T =−log( I÷I   o )= εbc 
 
   Where T is the transmittance through the sample and is equal to the ratio of the intensity of the light exiting the sample, I, and the intensity of the light entering the sample, I o . The three quantities on the right side of Beer&#39;s law are the extinction coefficient of the absorbing material, ε; the path length through the material, b; and the concentration of the absorbing material, c. For a given absorber species, ε is fixed. The minimum measurable absorbance value, A, is also fixed. The path length through the sample determines the minimum detection level. 
   Referring now to  FIGS. 2 ,  2 A and  2 B, shown therein in more detail is the probe  16 . In this embodiment of the probe  16 , the resonator  18  is configured as a spheroid of optically transparent material  26 , sometimes referred to as the sphere  26 , the first coupler  20  is configured as a first optical fiber  28  and the second coupler  22  is configured as a second optical fiber  30 . 
   The processes for fabricating spheroids of optically transparent material and optical fiber are well known in the industry and will not be discussed in detail herein. One advantage of constructing the first and second couplers  20  and  22  of optical fiber is that optical fiber can be economically terminated to provide low-cost connections that are compatible with available light sources and readers. 
   Another advantage of the system  10  over traditional systems for colorimetric testing is that the minimum sample needed to perform a colorimetric test utilizing the probe  16  is set by the amount of the sample required to envelop the evanescent field of the photons substantially surrounding the resonator  18 . For example, if the resonator  18  is configured as a spheroid, 1 μL of a defined sample would cover the surface of a 1 mm diameter spheroid of optically transparent material to an approximate depth of 200 μm which is many times the evanescent field penetration. 
   In the present embodiment, the first optical fiber  28  has a length  35 , a first end  36 , and a second end  38 . The first end  36  of the first optical fiber  28  is positioned adjacent the resonator  18 . The second end  38  of the first optical fiber  28  is in communication with the light source  12 . The probe  16  can also include a first connector  39  connected to the second end  38  of the first optical fiber  28  compatible with the light source  12  for connecting the first optical fiber  28  to the light source  12 . The process for making and using connectors for optical fiber is well known in the industry and will not be discussed herein. The first end  36  is angled and polished so as to facilitate the transfer of light from the first optical fiber  28  to the resonator  18 . The first end  36  of the first optical fiber  28  serves the role of a prism. When the first end  36  of the first optical fiber  28  is brought to the surface of the spheroid of optically transparent material  26 , overlapping evanescent fields of the first end  36  of the first optical fiber  28  and of the spheroid of optically transparent material  26  form an evanescent tunnel where light can couple between the first optical fiber  28  and the spheroid of optically transparent material  26 . The first end  36  and the second end  38  of the first optical fiber  28  are in communication with each other and the light source  12  and spheroid of optically transparent material  26  such that at least a portion of the light from the light source  12  is directed from the light source  12  to the second end  38  of the first optical fiber  28 , through the length  35  of the first optical fiber  28 , to the first end  36  and coupled from the first end  36  into the spheroid of optically transparent material  26 . 
   The second optical fiber  30  has a length  35   a , first end  40  and a second end  42 . The first end  40  is positioned within the evanescent field produced by the spheroid of optically transparent material  26 . The first end  40  of the second optical fiber  30  also acts as a prism. The second end  42  of the second optical fiber  30  is in communication with the reader  14 . The probe  16  can also include a second connector  43  connected to the second end  42  of the second optical fiber  30  compatible with the reader  14  for connecting the second optical fiber  30  to the reader  14 . The first end  36  of the second optical fiber  30  is coupled to the spheroid of optically transparent material  26  preferably opposite the side of the spheroid of optically transparent material  26  from where the first end  36  of the first optical fiber  28  was coupled to the spheroid of optically transparent material  26 . When the first end  40  of the second optical fiber  30  is brought to the surface of the spheroid of optically transparent material  26 , overlapping evanescent fields of the first end  40  of the second optical fiber  30  and the spheroid of optically transparent material  26  form an evanescent tunnel where the light can couple between the first end  40  of the second optical fiber  30  and the spheroid of optically transparent material  26 . The first end  40  of the second optical fiber  30  and the second end  42  of the second optical fiber  30  are in communication with the spheroid of optically transparent material  26  and the reader  14  respectively such that at least a portion of light from the spheroid of optically transparent material  26  is coupled from the spheroid of optically transparent material  26  to the first end  36  of the second optical fiber  30 , through the length  35   a  of the second optical fiber  30 , to the second end  42  of the second optical fiber  30  and to the reader  14  allowing the light to be exploited for chemical analysis through absorption spectroscopy for instance. The appropriate angle to polish the first ends  36  and  40  of the first and second optical fiber  28  and  30  will be discussed in more detail later. 
   The system  10  will greatly improve the range of colorimetric testing through improved portability, enhanced cost-effectiveness, reduced consumption and waste of the sample, simplification of operation and better sensitivity in smaller sample volumes. These characteristics will make colorimetric testing practical in long term monitoring applications and for in process systems such as remediation and recovery operations. 
   An advantage of the probe  16  is that fluidic connections for the probe  16  can be made compatible with standard fittings (such as commercially available mixing T&#39;s) that are used in high performance chromatography, flow injection analysis and similar automated analysis systems. 
   Referring now to  FIG. 3 , shown therein is an inline sample cell  44 . In this configuration the sample flows past the resonator  18  of the probe  16  in a standard T connection. The inline sample cell  44  includes the probe  16 , a mixing T  46  having an input  47  for receiving an inflow line  48 , an output  49  for receiving an outflow line  50 , and a plurality of connectors  52  for connecting the probe  16 , the inflow line  48 , and the outflow line  50  to the mixing T  46 . The probe  16 , the inflow line  48  and outflow line  50  are connected to the mixing T  46  using the plurality of connectors  52 , such that, in operation the sample to be tested flows from the inflow line  48  into the mixing T  46  and past the resonator  18  of the probe  16  in such a manner that the sample substantially envelops the evanescent field of photons surrounding the resonator  18 , interacts or reacts with the evanescent field of photons and thereafter the sample discharges out of the output  49  of the mixing T  46  and to the outflow line  50 . The interaction or reaction of the evanescent field of photons and the sample is analyzed by the reader  14  as previously described. Although the mixing T  46  is described and depicted as having a single input  47  connected to the inflow line  48  and a single output  49  connected to the outflow line  50 , those skilled in the art will readily recognize and understand that any plurality of inputs and outputs could be connected to the inflow and outflow lines  48  and  50 , respectively, and integrated into the mixing T  46 , or a similar mixing apparatus of appropriate configuration taking into consideration the number of inflow and outflow lines  48  and  50 . It will also be recognized and understood by those skilled in the art that a plurality of probes  16  could be included in the sample cell  44  for providing redundancy. 
   In order to calculate the correct angle to polish the first ends  36  and  40  of the first and second optical fibers  28  and  30 ; and in order to determine the WGM resonance frequencies of the spheroid of optically transparent material  26 , a number of factors need to be taken into consideration, such as, the energy keeping or frequency selecting capability of the spheroid of optically transparent material  26 , the quality factor of the spheroid of optically transparent material  26 , the resonant electromagnetic field components inside the spheroid of optically transparent material  26 , Free Spectral Range (FSR), which is the frequency spacing between adjacent major modes, and the effective refractive indices of the spheroid of optically transparent material  26 , of the first and second optical fibers  28  and  30 , and of the enveloping sample. 
   The energy keeping or frequency selecting capability of the spheroid of optically transparent material  26  is characterized by its Quality factor (Q). A high Q factor provides for a long effective path length. A long effective path length means that the light can make numerous trips around the spheroid of optically transparent material  26  so that the photons of light in the evanescent field can interact with the molecules of the substance to be tested a number of times. 
   The Q factor of the spheroid of optically transparent material  26  is determined by several loss factors such as radiation loss due to curvature, scattering loss because of inhomogeneity of the spheroid of optically transparent material  26 , absorption by the spheroid of optically transparent material  26 , and absorption by surface contamination or surrounding material. The unloaded Q factor (with no coupler present) is expressed by the unloaded Q factor equation:
 
 Q   −1   =Q   rad   −1   +Q   scat   −1   +Q   mat   −1   +Q   surf   −1 
 
   Each of the terms on the right side of the above equation may be examined independently to determine its effect upon the total Q of the spheroid of optically transparent material  26 . 
   Radiation loss reduces rapidly with increasing sphere size of the spheroid of optically transparent material  26 . For diameters greater than 15λ, Q rad  is greater than 10 11 , where λ is the vacuum wavelength of the entrapped light. The scattering Q factor has been generally reported in the literature as having a limit of 10 10 . 
   The material absorption presents a fixed limitation regardless of the size of the spheroid of optically transparent material  26 . The material Q factor can be expressed as
 
 Q   mat =2 πn/αλ, 
 
where a is the loss per meter traveled. For example, the silica used in optical fiber has attenuation of about 7 dB/km at 633 nm (5-dB bulk Rayleigh scattering loss and 2-dB absorption), which translates to a=1.588×10 −3  and a maximum total Q factor of about 0.9×10 10 . Experimentally, the Q factor can be found by measuring the energy damping time τ=Q/(2πf). This examination indicates that measured Q&#39;s below 10 10  can be attributed to coupling losses (not yet discussed) or absorption by surface contaminants or surrounding media.
 
   Since the system  10  utilizes the evanescent field outside the spheroid of optically transparent material  26  to interact with the analyzed material and because the evanescent field contains only a portion of the total energy within the spheroid of optically transparent material  26  and its intensity drops exponentially, it is intuitive to assume the sensitivity will be less than that of direct absorption spectroscopy. However, the sensitivity of the system  10  can be optimized to be even greater than that of a traditional direct absorption spectroscopy system which utilizes an open path beam. 
   The energy distribution of a WGM mode can be described by the resonant electromagnetic field components inside the sphere of optically transparent material  26 :
 
E r =0
 
             E   θ     ∝         j   l     ⁡     (   kr   )       ⁢       P   l   m       sin   ⁢           ⁢   θ       ⁢       (     cos   ⁢           ⁢   θ     )               ⁢       ⅇ     im   ϕ       ⁡     (     TE   ⁢           ⁢   mode     )                       E   ϕ     ∝         j   l     ⁡     (   kr   )       ⁢       ∂     P   l   m         ∂   θ       ⁢       (     cos   ⁢           ⁢   θ     )               ⁢     ⅇ     im   ϕ               
where j 1  and P l   m  are the spherical Bessel function and associated Legendre function, and k is the wave number inside the sphere. Outside the sphere of optically transparent material  26 , the spherical Bessel function j l  is replaced by the spherical Hankel function of the 1 st  kind h l   (1) .
 
     FIG. 4  illustrates the spherical coordinate system and orthogonal unit vectors. The potential function has a radial (r) dependence defined by the spherical Bessel function inside the spheroid of optically transparent material  26  and the spherical Hankel function of the 1 st  kind outside the spheroid of optically transparent material  26 , a latitudinal angle dependence (θ) is specified by the associated Legendre function, and azimuthal angle (φ) dependence described by the complex exponential function. 
   The tangential components of the electric and magnetic fields need to be continuous across the surface of the spheroid of optically transparent material  26 . Such boundary condition can be expressed by the characteristic equation. 
                   [       k   1     ⁢       Rj   l     ⁡     (       k   1     ⁢   R     )         ]     ′         j   l     ⁡     (       k   1     ⁢   R     )         =         [       k   2     ⁢       Rh   l     (   1   )       ⁡     (       k   2     ⁢   R     )         ]     ′         h   l     (   1   )       ⁡     (       k   2     ⁢   R     )           ,         
where
 
             k   1     =           2   ⁢           ⁢   π   ⁢           ⁢     n   1       λ     ⁢           ⁢   and   ⁢           ⁢     k   2       =       2   ⁢           ⁢   π   ⁢           ⁢     n   2       λ             
are the wave number inside and outside the spheroid of optically transparent material  26 , and the derivatives are with respect to k 1,2 R, respectively.
 
   By solving the boundary equation, the resonance frequency of the spheroid of optically transparent material  26  can be determined, with the resonance frequency depending on the mode number l. If l is sufficiently large, multiple solutions to the Boundary condition equation will exist. These are denoted by a secondary index, q. The root giving the lowest mode (i.e., lowest resonance frequency) is denoted by mode index q=1, with q=2, 3, . . . for the successively higher modes. A third index, m, is also needed to fully describe the structure of a WGM resonance. 
     FIG. 5  illustrates the radial dependence of a WGM in the spheroid of optically transparent material  26  with radius of five wavelengths. The top figure is TE l=50,m,q=1  mode, which has one major energy peak close to the surface. The bottom figure is TE l=50,m,q=2  mode, showing that the first peak moves toward the center of the spheroid of optically transparent material  26  while the second peak maintains the evanescent field. 
   The azimuthal angle dependence is specified by a traveling sinusoidal wave, which can be visualized as a simple cos(mφ) distribution around the spheroid of optically transparent material  26 . Referring to  FIG. 6 , the radial distance and azimuthal angle dependence are combined into a contour plot. This two-dimensional simulation of the WGM energy at the θ=90° equatorial plane (x-y plane) has been shown in dB. The spheroid of optically transparent material  26  boundary is marked by a circle. The evanescent field quickly drops down outside the boundary. 
   Referring to  FIG. 7  plotted therein is the higher radial mode with q=2 combined with azimuthal dependence (m=50). The major ring with higher energy goes toward the center of the spheroid of optically transparent material  26  with an additional weaker ring appearing beneath the surface. Such higher modes may not exist for smaller mode number l-the R/λ ratio must exceed a threshold for the higher modes to be present. 
   When index m equals l, most of the energy concentrates at the equator. As mode index l differs from m, the WGM energy spreads symmetrically in the latitudinal directions, and the “ring” distribution becomes a wider “band” distribution. 
     FIG. 8  illustrates that (l−m+1) lobes exist, symmetrically-spaced with respect to the equator of the spheroid of optically transparent material  26 . The maximum spread angle is cos(m/l). 
   The combination of radial distance and latitudinal angle dependence can also be visualized in a contour plot. A snapshot of the WGM potential with l−m=6 at φ=90° vertical plane (x-z plane) is shown in  FIG. 9 . There are l−m+1 lobes appearing symmetrically with respect to the equator of the spheroid of optically transparent material  26 . If the coupling conditions are right, more than one mode with index l≠m may be excited inside the spheroid of optically transparent material  26 . 
   The major WGM is the mode with l=m and q=1. Since the ray is propagating almost exactly on the equator of the spheroid of optically transparent material  26  and the propagation phase angle has to match around the circle, the mode number is l=m ≈2πRn/λ where R is the radius—so that the mode index m equals the number of wavelengths around the equator. This gives a simplified relation between the resonance frequency and mode number. The free spectral range (FSR), which is the frequency spacing between adjacent major modes (successive l), can then be estimated by 
           FSR   =     c     2   ⁢           ⁢   π   ⁢           ⁢   Rn             
where c is the vacuum speed of light.
 
   Referring now to  FIG. 10 , shown therein is a comparison of the WGM major mode resonance spacing in a 0.75 mm diameter spheroid of optically transparent material  26  and the dimensions of a typical visible absorbance spectrum. Although these major modes shift with temperature and pressure applied to the spheroid of optically transparent material  26 , the result of such shifts is averaged out by the modest resolution of the analysis spectrometer and does not affect the resulting spectrum. 
   The resonance frequencies of a perfect spheroid of optically transparent material  26  are mainly decided by the mode numbers l—and q, if such modes exist. Modes with the same mode number l but different m—although separated spatially—will have the same resonance frequency. This is called mode degeneracy. However, the fabrication process will inevitably introduce a certain degree of eccentricity, ε, to the spheroid of optically transparent material  26 , splitting the degenerate modes. 
   The resonance frequency can be approximated by 
               f   qlm   i     ≈       c     2   ⁢           ⁢   π   ⁢           ⁢   Rn       ⁡     [     l   +     1   /   2     +         a   q     ⁡     (       (     l   +     1   /   2       )     /   2     )         1   /   3       -     Δ   i       ]         ;             when               m   =   l     ,         
where i denotes TE or TM, a q  is the q th  zero of the Airy function (a 1 =2.338, a 2 =4.088, a 3 =5.521, etc.), R is the sphere radius,
 Δ TE   =n/√{square root over (n 2 −1,)} and Δ   TM   =[n√{square root over (n 2 −1] −1 )}.   
and the frequency shifting due to eccentricity can be approximated by
 
                 Δ   f     f     =     ±         ɛ   2     ⁡     (       l   2     -     m   2       )         4   ⁢     l   2             ,         
where the oblate spheroid of optically transparent material  26  has the positive sign and the stretched spheroid of optically transparent material  26  has the negative one. The spheroid of optically transparent material  26  then simultaneously supports multiple WGM resonances at closely-spaced frequencies around each major mode. With a coupling method that supports excitation of these modes, sufficient optical throughput is obtained for performing long path length spectroscopy using broadband illumination.
 
   Electromagnetic energy can be transferred to or extracted from a spheroid of optically transparent material  26  through evanescent wave coupling with minimal impact upon the WGM resonance frequencies. Reflection from an internal prism face that is located near the surface of the spheroid of optically transparent material  26  overlaps the prism&#39;s and the spheroid of optically transparent material  26  evanescent fields and provides coupling,  FIG. 11 . 
   Although a prism coupler is flexible to use, it is also bulky and awkward because of its requirement for collimation optics and alignment. Angle-polished fibers provide a hybrid coupling method, which preserves the benefits of prism coupling while eliminating the collimating optics. Optical fiber conveniently delivers guided light to its angle-polished tip, which serves the role of a prism,  FIG. 11 . 
   When guided light strikes a spheroid of optically transparent material  26  optical fiber interface, as shown in  FIG. 12   a , at an angle greater than the critical angle, total internal reflection occurs, and an evanescent wave propagates parallel to the interface,  FIG. 12   b . The intensity of this evanescent wave decays exponentially with respect to the distance from the interface. 
   In order to support excitation of these modes and couple light from the light source  16  to the spheroid of optically transparent material  26  and from the spheroid of optically transparent material  26  to the reader  14 , the first end  36  of the first optical fiber  28  and the first end  40  of the second optical fiber  30  must be polished to an angle and aligned at an angle with the spheroid of optically transparent material  26 . 
   In order to polish and align the first and second optical fibers  28  and  30  correctly, the effective refractive indices of the spheroid of optically transparent material  26  and the first and second optical fiber  28  and  30  need to be found. The effective refractive index for the spheroid of optically transparent material  26  can be approximated by the equation: 
               n   sphere     =     l     2   ⁢           ⁢   π   ⁢           ⁢     R   /     λ   0             ,         
where λ 0  is the vacuum wavelength of the light to be coupled, l is the WGM mode number, and R is the radius of the spheroid of optically transparent material  26 .
 
   The effective refractive index for the first and second optical fibers  28  and  30  is non-trivial to find analytically, but may be approximated by the ratio of the fiber propagation constant (β) to the free-space propagation constant (k): 
   
     
       
         
           
             
               n 
               fiber 
             
             = 
             
               β 
               k 
             
           
           , 
           
               
           
           ⁢ 
           
             
 
           
           ⁢ 
           
             
               where 
               ⁢ 
               
                   
               
               ⁢ 
               k 
             
             = 
             
               
                 2 
                 ⁢ 
                 
                     
                 
                 ⁢ 
                 π 
               
               λ 
             
           
         
       
     
   
   The propagation constant of the fundamental LP 01  mode inside the first and second optical fibers  28  and  30  can be approximated by the equation 
             β   ≈       kn   2     ⁡     [     1   +           n   1     -     n   2         n   1       ⁢       (     α   V     )     2         ]         ,         
where n 1  and n 2  are the refractive index of a fiber core of the first and second optical fibers  28  and  30  and cladding, respectively, a is the normalized transverse decay constant, and V is the normalized frequency.
 
   After finding the effective refractive indices of the first and second optical fibers  28  and  30  and spheroid of optically transparent material  26 , the fiber polishing and alignment angle is calculated by the equation 
             θ   c     =       sin     -   1       ⁡     (       n   sphere       n   fiber       )             
where the n sphere  and n fiber  are the effective refractive indices for spheroid of optically transparent material  26  and the first and second optical fibers  28  and  30 .
 
   Two angle polished optical fibers can couple light into and out of the spheroid of optically transparent material  26  as shown in  FIG. 13 . With a single angle polished optical fiber coupling efficiencies of up to 60% have been demonstrated (˜2.1-dB insertion loss). Maximum fiber to fiber transmission at resonance of about 23% has also been demonstrated in dual fiber arrangement. 
   Although the reported coupling efficiency obtained from angle-polished fibers (60%) is lower than prisms (80%) or tapered fibers (&gt;90%), it is adequate and can be compensated for by employing a brighter light source  12 . The important figure of merit for this system  10  is its long effective path length, not its coupling efficiency. So, angle-polished optical fibers, with their desirable mechanical advantages, are the preferred coupling method for this system  10 . 
   As an alternative to first and second optical fibers  28  and  30  having angled polished first ends, the first and second optical fibers  28  and  30  can have first ends  36  and  40  tapered. In practice, a section of the first and second optical fibers  28  and  30  are heated and stretched to form a waist with a much smaller radius. The shape of the fiber radius reduction can be linear or curved. There are procedures to fabricate the tapered fiber with desired shape of tapering and excellent repeatability. 
   The key factor for efficient coupling using tapered fibers is to match the propagation constant of the light inside the spheroid of optically transparent material  26  and the first and second optical fibers. At the lowest mode of WGM resonance, the mode number l is approximately equal to the number of wavelengths around the spheroid of optically transparent material  26  waist. Therefore, the propagation constant of the spheroid resonance can be approximated by: 
               β   sphere     ≈       N   sphere     ⁢   k       =       (     kl     2   ⁢           ⁢   π   ⁢           ⁢     R   /     λ   0           )     =     L   R             
where k and λ 0  are the free space propagation constant and wavelength, respectively.
 
   For a fiber radius tapered down to a few micrometers, the propagation constant inside the taper can be expressed as 
               β   fiber     =         k   2     ⁢     n   2       -       (     2.405   ρ     )     2         ,         
where ρ is the waist radius. By matching propagation constants, tapered fibers have reached 90–99.8% coupling efficiency, which is the highest of any coupling method.
 
   A larger spheroid of optically transparent material  26  (&gt;50μm in diameter) or one that supports high order WGM&#39;s due to eccentricity will provide tightly spaced resonances. Small rings provide more separated resonances, but are convenient for integrated planar applications. 
   The system  10  provides technical benefits such as long optical path lengths in microvolume liquids, convenient optical and fluidic interfaces, inexpensive illumination and detection requirements and a migration pathway to integrated lab-on-a-chip applications. Longer optical path lengths correspond to higher sensitivities. The system  10  enables long-path length absorbance spectroscopy in liquids at wavelengths from the ultraviolet to near infrared in microvolume samples. The system  10  permits long path length absorbance spectroscopy in 96- and 384-well plates, with extension to 1536-well plates possible. The system  10  could also be useful at a lab bench as a convenient method for examining single samples in microcentrifuge vials or test tubes. 
   Adoption of a component or technology is greatly aided by its compatibility with installed systems and expertise. Furthermore, fluidic connections for the system  10 , always a problem with microfluidic components, will be compatible with standard ¼-28 fittings that are used in high performance liquid chromatography (HPLC), flow injection analysis (FIA), and similar automated analysis systems. 
   The system&#39;s  10  ability to transmit broadband light allows it to be used in moderate resolution absorbance spectroscopy. Light sources such as tungsten quartz halogen bulbs and spectrometers are readily available that permit the system  10  to be economical. For colorimetric applications where a single spectral band provides sufficient information, an LED source and Si detector could be utilized. Spheroids of optically transparent material  26  are only one type of resonator that could be used to enable long path length spectroscopy in liquids. Planar silicon-based micro-ring and micro-disk resonators could be designed into the channels of microfluidic systems and utilized for integrated optical measurements. Optical coupling to integrated waveguides could be accomplished using angle-polished optical fibers similar to those planned for use in the inline sample cell  44 . The system  10  is also expected to provide environmental, health, and economic benefits, such as, reduced cost of sample analysis, improved drug discovery and expansion of point-of-care diagnosis. 
   By reducing the required reagents and generated waste, the system  10  will reduce the cost of performing lab-based liquid sample analyses using absorption spectroscopy. Such testing is widely used for environmental compliance and medical diagnosis. An expected effect of the system  10  would be the increased use of such testing as the per-test costs drop, perhaps catching diseases at an earlier stage where they can be more effectively treated. 
   Absorbance spectroscopy is difficult to implement in massively paralleled samples that are analyzed during drug discovery, due to the small sample sizes that reagent costs and waste disposal require. The system  10  allows absorbance spectroscopy to be widely applied to this area, reducing development costs. 
   When combined with lab-on-a-chip technology, an integrated resonator  18  will permit the transition of many lab-based analyses to the doctor&#39;s office. This will improve the level of care received by patients and enable more rapid diagnosis and treatment of ailments. This system  10  will be especially useful when hospital-based facilities are not readily available, such as in rural locations and military deployments. 
   Referring to  FIG. 14 , absorption measurements were initially performed using a HeNe laser (632 nm) and methylene blue dye with a spheroid of optically transparent material  26  and first and second optical fibers  28  and  30  have tapered first ends  36  and  40 . 
   A 632 nm light from a 10 mW helium neon laser was focused by a microscope objective onto 200/225 multimode optical fiber. Fiber chucks held the first and second optical fibers  28  and  30  on Thorlabs XYZ translation stages. A 700 μm diameter spheroid of optically transparent material  26  was held by the spheroid&#39;s stem in a fiber chuck, which, in turn, was held by a 5-axis fiber positioner. The first and second optical fibers  28  and  30  were positioned on opposite sides of the spheroid of optically transparent material  26  as shown in  FIG. 15 . Light passing through the spheroid of optically transparent material  26  was detected with a fiber-coupled spectrometer, and data was collected and viewed on a computer. The first and second optical fibers  28  and  30  and spheroid of optically transparent material  26  were visually aligned with a microscope. 
   Initially the system  10  was aligned to obtain maximum through-coupling with the HeNe, then 15 μL of water were placed on the glass slide. The surface tension of the water disturbed the first and second optical fibers  28  and  30 , so the first and second optical fibers of  28  and  30  and the spheroid of optically transparent material  26  were realigned in the drop. Next, 5.2 μL of 0.005% methylene blue was added to the drop, followed by 20 μL of water without further realignment. 
   Referring now to Table 1, when methylene blue was added to the drop, the through-coupling decreased. The measured effective path lengths, 9.1 cm and 13.6 cm, argue against reflection or lensing effects as valid explanations for the coupling between first and second optical fibers  28  and  30 . When more water was added, the through-coupling actually increased as the concentration of methylene blue was lowered. The observation of decreased absorption with the addition of water to the water and methylene blue liquid envelope in both trials argued against misalignment of the first and second optical fibers  28  and  30  as an explanation of decreased throughput. 
   
     
       
         
             
           
             
               TABLE 1 
             
           
          
             
                 
             
             
               Methylene blue absorption 
             
          
         
         
             
             
             
          
             
                 
               Trial 1 
               Trial 2 
             
             
                 
                 
             
          
         
         
             
             
             
             
          
             
                 
               Intensity with 15 μL water 
               948 Arb. Units 
               960 Arb. Units 
             
             
                 
               Intensity with 5 μL blue 
               643 Arb. Units 
               537 Arb. Units 
             
             
                 
               Intensity with 20 μL water 
               799 Arb. Units 
               653 Arb. Units 
             
             
                 
               Percent of drop regained 
               51.1% 
               27.4% 
             
             
                 
               Effective Path length 
               9.1 cm 
               13.6 cm 
             
             
                 
                 
             
          
         
       
     
   
   To further validate WGM coupling as the cause of the long measured path lengths, the experiment was repeated using a tunable Ti:sapphire laser. Laser light was coupled into the WGMs of a fused-silica spheroid of optically transparent material  26  using a dual-tapered first optical fiber. A tapered second optical fiber, on the opposite side of the spheroid of optically transparent material  26 , coupled light out of the WGMs. The power transmitted through the first and second optical fiber  28  and  30  was measured; the excitation of a WGM was identified by a dip in the power continuing down the first optical fiber  28  and by a peak in the power coming out of the second optical fiber  30 . The spheroid of optically transparent material  26  and first and second optical fibers  28  and  30  tapers were then immersed in a weak solution of indocyanine green dye (ICG) in methanol. 
   Light of wavelength 790 nm, near the absorption peak of micromolar ICG in methanol, was produced by a cw Ti:sapphire laser pumped by a diode-pumped solid-state green laser. The laser light was focused with a high-quality short-focal-length lens into a single-mode optical fiber. A polarizer and quarter-wave plate before the lens reduced back-reflection into the laser from the injection face of the fiber. A fiber polarization controller before the coupling region then allowed conversion to linear polarization for selection of TE or TM WGMs. The first ends  36  and  40  of the first and second optical fiber  28  and  30  taper were produced by heating and stretching the first and second optical fibers  28  and  30  to produce regions, approximately 1 cm in length and 5 um in diameter, that could be brought into contact with the spheroid of optically transparent material  26  for coupling, with the optical fiber returning to normal size following the taper. Each coupling fiber was mounted on a holder so that the tapered section, bent into the shape of a flat-bottomed U, could be inserted into the sample solution while keeping the glue, which fastened the fiber to the holder, out of the solution. Three-axis positioning stages were used to carefully position the first and second optical fibers  28  and  30  in the proper positions and orientations next to the spheroid of optically transparent material  26 . As the laser&#39;s frequency was tuned, detectors monitored the power on the first optical fiber  28  reflected from the spheroid of optically transparent material  26  and second optical fiber  30  transmitted through the spheroid of optically transparent material  26  via WGMs. 
   Positioning of the first and second optical fibers  28  and  30  was done by maximizing reflection dips and transmission peaks in air with the trough empty. When the WGM resonance signatures were clear, the trough was filled with a precisely measured volume of methanol and the first and second optical fibers  28  and  30  re-positioned, if necessary. Various concentrations of ICG were added by pipette, and changes in the dips and peaks observed simultaneously. 
   Reflection resonance dips in the reflected optical fiber output could be used to measure absorbance but were not used here primarily because these results could not be compared to similar results using a broadband source. With a broadband source, individual resonance dips are not resolved, which means that the reflected optical fiber output will consist of a very small change on a large background. 
   Two sequences of traces were identified using different modes. In both data sets, a sequence of scans over 1 GHz in frequency is presented, with the reliably identified mode at the center of the scan. By measuring the line width Δv of the resonance peak, we can estimate the quality factor of the mode (Q=v/Δv). Using the pure methanol (zero concentration) trace, Q was estimated to be approximately 10 7 -in both trials. This relatively high value of the loaded Q (including the effect of coupling, which reduces the intrinsic value of Q) for a fiber-coupled microsphere is a result of the fact that the refractive index of methanol, nm=1.33, is fairly close to the index of the spheroid (n=1.45) and so has the effect of “smoothing out” the surface roughness and reducing scatter. This increases the intrinsic Q (without coupling, so not measured) of the modes. Immersing the spheroid of optically transparent material  26  in methanol also increases the evanescent volume fraction. It is the intrinsic Q, along with the evanescent volume fraction of a WGM, that determines absorbance sensitivity. The maximum transmitted power was approximately 1 μW. 
   Referring now to  FIG. 18 , in the first data set, three traces at concentrations of 0, 0.476 μM, and 0.909 μM are shown. The broadening of the peak with increasing concentration, as absorption reduces the measured value of Q, should be noted. 
   Decreasing peak height corresponds to increasing concentration. Fitting these heights to an exponential, T(a)=T(0)exp(−a L eff ), gives an effective absorption path length of L eff =8.8 cm. 
   
     
       
         
             
           
             
               TABLE 2 
             
           
          
             
                 
             
             
               Absorption data 
             
          
         
         
             
             
             
             
             
          
             
                 
               Concentration 
               Absorption 
                 
                 
             
             
                 
               (μM) 
               (1/mm) 
               Transmission 
               Ln(T) 
             
             
                 
                 
             
          
         
         
             
             
             
             
             
          
             
                 
               0 
               0 
               7.1 
               1.960095 
             
             
                 
               0.476 
               0.019 
               1.4 
               0.336472 
             
             
                 
               0.909 
               0.036 
               0.3 
               −1.20397 
             
             
                 
                 
             
          
         
       
     
   
   Referring now to  FIG. 19 , the exponential fit to the data is very good. Referring now to  FIG. 20 , the second data set consists of five traces. The second-highest peak is for zero concentration and is not considered because a jump in the scan seems to have cut it off below its true height (note the asymmetry and narrow width of the peak). The trend in peak width is not consistent here, but the heights are not affected by the scan slowing that broadens the peak. Referring now to Table 3, the other four traces are for concentrations of 0.043 μM, 0.0826 μM, 0.118 μM, and 0.152 μM. 
   
     
       
         
             
           
             
               TABLE 3 
             
           
          
             
                 
             
             
               Absorption Data 
             
          
         
         
             
             
          
             
               Trial 1 
               Trial 2 
             
          
         
         
             
             
             
             
          
             
               Concentration (μM) 
               Absorption (1/mm) 
               Transmission 
               Ln (T) 
             
             
                 
             
          
         
         
             
             
             
             
          
             
               0.043 
               0.00172 
               6 
               1.791759 
             
             
               0.0826 
               0.003304 
               4.2 
               1.435085 
             
             
               0.118 
               0.00472 
               3.2 
               1.163151 
             
             
               0.152 
               0.00608 
               2 
               0.693147 
             
             
                 
             
          
         
       
     
   
   Referring now to  FIG. 21 , the transmission is fit reasonably well by an exponential, giving an effective absorption path length of L eff =25 cm. 
   The circumference of the spheroid is 1.1 mm, and the evanescent volume fraction of a typical mode is probably on the order of f≅5%. With L eff =25 cm, this means that the WGM intrinsic Q=πnL eff /λf≅3×10 7 . The other observed value of L eff =8.8 cm then implies an intrinsic Q≅1×10 7 . Different WGMs will have different intrinsic Q values because of their different spatial field distributions. A range of a factor of three is well within normal expectations. 
   Referring now to  FIG. 22 , in an experimental procedure, broadband light from a 360 W tungsten bulb was focused by a large fresnel lens and a fiber collimator into 200/225 multimode optical fiber. The first and second optical fibers were positioned on opposite sides of the spheroid of optically transparent material as in  FIG. 15 . Fiber chucks held the first and second optical single-tapered fibers on Thorlabs XYZ translation stages. The spheroid of optically transparent material was held by its stem in a fiber chuck, which in turn was held by a 5-axis fiber positioner. The spheroid of optically transparent material was approximately 750 μM in diameter. 
   The light that passed through the spheroid of optically transparent material and coupled into the second optical fiber was detected by a fiber-coupled spectrometer. The data was stored and viewed on a computer. The system was used to measure the absorption of crystal violet using the same methods as those previously discussed. Low concentrations of crystal violet were added to a freestanding volume of water surrounding the spheroid of optically transparent material. First, through-coupling was achieved with the broadband light source. 20 μL of water was added, and the coupling was realigned for the last time. Next, 5 μL of 1 ppm crystal violet was added to the water. The solution was then diluted by adding 20 μL of water to the drop. Lastly, 10 μL of 500 ppm crystal violet was added. 
   Referring now to  FIG. 23  shown therein in a representation, the spectra obtained during the experiment. These spectra are:
         Direct Source Measurement: This data was taken by holding the spectrometer near the Light Source lamp. No optical fiber was used for collection. The amplitude is arbitrary.   Dry Sphere Through-Coupling: This data was taken through the tapered fibers and spheroid of optically transparent material  26  in air. Integration time: 1 sec. 207 pW was the maximum power coupled.   Coupling in Water: This data was taken after the spheroid of optically transparent material was submerged in 20 μL of deionized 18 megaohm-cm water. The coupling alignment was adjusted after the water was added.   Integration time: 1 sec.; Coupled power: 3.   0.2 Crystal Violet: This data was taken about 30 sec after 5 μL of 1 ppm crystal violet solution was added to the 20 μL of water around the spheroid of optically transparent material.   Integration time: 1 sec.   0.11 Crystal Violet: This data was taken about 30 sec after 5 μL of water was added to the 0.2 ppm solution around the spheroid of optically transparent material  26 . This data shows an increase in absorption instead of an expected decrease. It is suspected that that this was caused by poor mixing in the drop—water added to the edge of the drop pushed higher concentration crystal violet solution toward the spheroid of optically transparent material  26  as the added water took its place at the drop perimeter. Integration time: 1 sec.   91 ppm Crystal Violet: This data was taken after 10 μL of 500 ppm crystal violet was added to the drop. Integration time: 1 sec.       

   Using the absorbance of calibrated-concentration crystal violet solutions measured with a spectrophotometer at 591 nm, the 0.2 ppm measurement gives an effective absorption path length of 22.4 cm. The 0.11 ppm crystal violet measurement gives an effective absorption path length of 60.4 cm. The small changes in collected signal intensity at 750 nm and 450 nm indicate that only negligible misalignment could have occurred during the in-liquid measurements, changes in the coupling would have caused a wavelength-independent drop in transmission. 
   Spheroids of optically transparent material  26  have been fabricated from fused silica fiber using a hydrogen torch. Laser heating provides an alternative approach. Referring now to  FIG. 24(   a ), in one such process, a CO 2  laser beam and silica fiber are aligned, the laser beam is projected paraxial with the silica fiber and focused into a small focal point. Due to the extremely small size of focal point (˜60 μm in diameter) and the large vertical gradient of laser intensity, only a small amount of silica is fused. Spheroids as small as 60 μm in diameter can be fabricated by this method. 
   Referring now to  FIG. 24(   b ), smaller spheroids of optically transparent material  26  can be made by a modified method. The tip of the optical fiber can be pre-ground into a cone shape to produce a spheroid of optically transparent material  26  approximately 40 μm in diameter. The precision control of the CO 2  laser power also avoids the possibility of overheating of the spheroid of optically transparent material  26 , which may cause recrystallization of the silica. 
   According to Beer&#39;s Law, the sensitivity of the absorption spectroscopy is proportional to the optical path length. Extending the path length with a given amount of sample has been an important goal for many researchers. Referring now to  FIG. 25 , the capillary micro-cuvette is an example of an existing approach. In this water-core waveguide based device, liquid samples are drawn into a rigid capillary to form the water-core waveguide. The input light propagates inside the capillary before being received by the reader  14 . In order to prevent light escaping out of the water core, the material of the capillary is chosen so that the refractive index is lower than 1.33. In order to employ glass capillaries, a low refractive index polymer may be coated on the interior wall of the glass capillary. With the sample volume ranging from 4 to 15 microliters, the water core can be setup with a physical length between 2.3 cm and 10 cm. 
   Referring to  FIGS. 26–27 , another embodiment of a system for colorimetric testing is shown utilizing a microfluidic chip  60 . The microfluidic chip  60  is fabricated from a substrate of silicon, glass, or other material known in the art to construct the microfluidic chip, such as that used by Caliper Life Sciences. The microfluidic chip  60  is provided with at least one microfluidic channel  62 , a microresonator  64 , and at least one waveguide or surface  66 . The microfluidic channel  62  includes three microchannel walls  67 ,  68 , and  69  formed in the microfluidic chip  60 . Preferably, the microfluidic channel  62  is formed by etching the microfluidic channel  62  into the substrate of the microfluidic chip  60  or into a layer of material which has been coated onto the substrate. The etching process produces the microfluidic channel walls that are needed to form the microfluidic channel  62 . It is to be understood that the microfluidic channel  62  may be formed by any process known in the art. Further, the microfluidic channel  62  may be a microchannel flow cell. 
   A microresonator  64  is positioned within the microfluidic channel  62 . The microresonator  64  may include a stem  70 . The microresonator  64  is fixed within the microfluidic channel  62  either by its stem  70  or by trapping the microresonator  64  using structures within the microfluidic channel. The microresonator  64  is constructed of any of the following materials: silica, glass, quartz, silicon, GaAs, or materials containing gallium or silicon. The microresonator  64  can be constructed in various shapes, such as a sphere, spheroid, torus, disc, or cylinder. 
   At least one waveguide or surface is provided in the microfluidic chip  60 . In one preferred embodiment, as shown in  FIG. 27 , a flat plate  66  is positioned over the formed microfluidic channel  62  to form a fourth wall of the microfluidic channel  62 . (The flat plate  66  is removed from  FIG. 26  for the purpose of more clearing showing the microfluidic channel  62 .) The flat plate  66  is constructed of an optically transparent substrate. As shown in  FIGS. 26 and 27 , the flat plate  66  receives a light  76  having a frequency bandwidth greater than the spacing between the whispering gallery mode resonance frequencies. The light reaches the flat plate  66  via a prism (not shown) or other suitable optical coupler capable of producing an evanescent field at the inner surface of the flat plate  66 . The flat plate  66  is evanescently coupled to the microresonator  64  such that supported whispering gallery mode resonance frequencies are coupled from the flat plate  66  into the microresonator  64  and light at frequencies not resonant with the microresonator  64  are not coupled into the microresonator  64 . Further, light  78  is coupled out from the microresonator  64 . Preferably, the optically transparent plate  66  is bonded to the substrate of the microfluidic channel  62 . However, it should be understood that any other method or procedure known in the art for attaching the plate may be utilized for attaching the plate  74  to the substrate of the microfluidic chip  60 . Alternatively, at least one other surface of the microfluidic channel  62  may be constructed so that the light reaches the other surface via the prism, grating, or other suitable optical coupler capable of producing an evanescent field at the other inner surface. 
   In another embodiment (not shown), at least one waveguide is embedded in the microfluidic channel  62  in proximity to the microresonator  64 . The principal requirement for the preferred embodiments is that one of the surfaces that define the microfluidic channel  62  needs to support evanescent coupling to the microresonator  64 . The microfluidic channel  62  may be fabricated within a single block, as long as the process employed is able to produce at least one channel wall that supports evanescent coupling to the microresonator  64 . 
   In use of one preferred embodiment, a fluid (not shown) is transitioned into the microfluidic channel  62  of the microfluidic chip  60 . Light  76  from a light source (not shown) is coupled through the flat plate  66  by use of the external prism. The light source is a light emitting diode, blackbody light source, an arc discharge light source, or a laser. The light  76  strikes an inner surface of the plate  66 , where it is evanescently coupled into the microresonator  64 . Light coupled into the microresonator  64  undergoes precession, so that upon coupling back out of the microresonator  64 , it exits at a slightly different angle. The precession-based spatial separation of reflected and through-coupled beams allows the absorbance spectrum of the fluid in the microfluidic channel  62  to be measured. 
   Coupling into the microresonator  64  placed within a microfluidic channel  62  lends itself to parallelization to increase sample analysis throughput. Multiple microfluidic channels may be constructed and monitored by placing a microresonator  64  in each of the channels that need to be monitored. Light  76  is then coupled into each of the microresonators  64  using the same methods described above. 
   Changes may be made in the construction and the operation of the various components, elements and assemblies described herein or in the steps or the sequence of steps of the methods described herein without departing from the spirit and scope of the invention as defined in the following claims.