Patent Publication Number: US-9897535-B2

Title: Optical reader systems and methods for microplate position detection

Description:
This application claims the benefit of priority under 35 U.S.C. § 119 of U.S. Provisional Application Ser. No. 61/445,266, filed on Feb. 22, 2011, the content of which is relied upon and incorporated herein by reference in its entirety. 
    
    
     FIELD 
     The present disclosure relates to label-independent optical reader systems, and in particular to optical reader systems and methods with accurate microplate position detection. 
     BACKGROUND 
     Manufacturers of optical reader systems seek to design a new and improved optical reader systems that can be used to interrogate a resonant waveguide grating biosensor to determine if a biomolecular binding event (e.g., binding of a drug to a protein) occurred on a surface of the biosensor. Of present interest are optical reader systems and methods with improved position detection of the microplate that supports the biosensors to reduce variations in readings of the biosensors. Such new and improved optical reader systems and methods that have such capability are the subject of the present disclosure. 
     SUMMARY 
     Aspects of the disclosure are directed to optical reader systems and methods with accurate microplate position detection capability. The optical reader systems having scanning optical systems configured to scan select position-detecting features on the microplate to accurately determine their respective positions. This in turn allows for the microplate position to be accurately defined relative to a reference of the optical system. Such accurate position detection of the microplate enables accurate scan paths over the biosensor, leading to increased accuracy in reading the biosensors 
     The measurement of the positions of the position-detecting features can also be used to calibrate the optical reader system to reduce or eliminate distortions and non-linearities that arise from one or more of the system components, including the scanning optical system. 
     In various examples, the scanning optical systems employ f-theta lenses. Also in examples, the scanning mirror devices include micro-electrical-mechanical system (MEMS) mirrors, which can have substantial non-linearity relative to the positioning requirements of scanning a biosensor. 
     The position-detecting systems and methods disclosed herein can provide fast and accurate determination of the position of features on the microplate surface using generally one-dimensional scan paths that include a general direction with an oscillating component generally perpendicular to the general scan path direction. The systems and methods benefit from the use of a photodetector configured for integrating the detected reflected light so that it measures an averaged result for sections of the oscillation component of the scan path. 
     The position-detecting systems and methods disclosed herein are useful where exhaustive two-dimensional mapping of a sample does not meet cycle time requirements because the photodetector is slow. Photodetector integration is combined with the oscillatory motion of the beam spot to slow-photodetector limitations. 
     These and other advantages of the disclosure will be further understood and appreciated by those skilled in the art by reference to the following written specification, claims and appended drawings. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       A more complete understanding of the present disclosure may be had by reference to the following detailed description when taken in conjunction with the accompanying drawings wherein: 
         FIG. 1  is a generalized schematic diagram of an optical reader system of the disclosure; 
         FIG. 2  shows an exemplary biosensor array operably supported in regions or “wells” of a microplate, which in turn is held by a microplate holder; 
         FIG. 3  is a plot of resonant wavelength λ R  (nm) vs. position (mm) across the biosensor; 
         FIG. 4  is a plot of the peak amplitude (photon counts) versus spectrometer pixel location, which corresponds to wavelength; 
         FIG. 5  is a detailed schematic diagram of a single-channel embodiment of a scanning optical reader system of the disclosure; 
         FIG. 6  is a close-up schematic diagram of an exemplary scanning optical system that includes a scanning mirror device, a fold mirror, and an f-theta focusing lens; 
         FIG. 7  is a schematic diagram that illustrates an example of a first position detection method for determining the position of microplate; 
         FIG. 8  is a schematic diagram of optical scanning system as used to measure the position of position-detecting features on the microplate; 
         FIGS. 9A and 9B  are schematic diagrams that illustrate two different scan paths having different oscillation amplitudes; 
         FIG. 10A  and  FIG. 10B  illustrate an example position detection method where the scan path oscillation amplitude is made relatively large; 
         FIG. 11  illustrates an example of the position detection method that employs a shaped feature; 
         FIG. 12  illustrates an example position detection method where multiple scans of the object can be used to create multiple measured feature profiles that are combined to produce a final measured feature profile having optimal shape (maximum signal) relative to the actual feature profile; 
         FIG. 13A  and  FIG. 13B  illustrate an example position detection method that is useful for coarse initial detection of position-detecting features when the location of the sample feature is only generally known. 
         FIG. 14A  and  FIG. 14B  are schematic diagrams that illustrate an example array of position-detecting features, with some features being near the microplate corners, in connection with an example position detection method that first measures a corner feature; 
         FIG. 15  is a schematic diagram of an example scanning optical system similar to that shown in  FIG. 5 ; 
         FIG. 16  is a schematic diagram of an example control system where a control variable in the controller (e.g., microplate positions in controller coordinates) is converted to an actual measured parameter (e.g., microplate positions in microplate coordinates); 
         FIG. 17A  and  FIG. 17B  are schematic diagrams of the position-detecting features plotted along with the beam spot scan paths, illustrating how non-linearities in the optical reader can give rise to spatial distortion of the microplate coordinates; 
         FIG. 18A  and  FIG. 18B  example scan paths that account for the distortion of the microplate coordinates; and 
         FIG. 19A  and  FIG. 19B  are schematic diagrams that illustrate two related calibration techniques based on driving a beam spot with a sine-wave driving frequency. 
     
    
    
     DETAILED DESCRIPTION 
     Reference is now made to embodiments of the disclosure, exemplary embodiments of which are illustrated in the accompanying drawings. 
     In the discussion below, in certain descriptions, the angle θ is a “deflection angle” and refers to the angle of incident optical beams  134 I relative to optical axis A 1  as these optical beams leave scanning mirror device  260 . Also in certain descriptions, the angle φ refers to an “incidence angle” that the incident optical beams  134 I make relative to the surface normal N of microplate  170 . Microplate  170  is assumed to lie in an X-Y plane thereby defining deflection angles θ X  and θ Y  and incident angles φ X  and φ Y  associated with incident optical beam(s)  134 I. In certain descriptions, the angle θ is used in place of angle φ as described above, and one skilled in the art will understand from the context of the discussion the meaning of the particular symbol used for a given angle. 
     Optical Reader System 
       FIG. 1  is a generalized schematic diagram of an optical reader system (“system”)  100  of the present disclosure and used to interrogate one or more biosensors  102  each having a surface  103  to determine if a biological substance  104  is present on the biosensor. Inset A shows a close-up of an exemplary biosensor  102 . Biosensor  102  may be, for example, a resonant waveguide grating (RWG) biosensor, a surface plasmon resonance (SPR) biosensor, or like biosensor. U.S. Pat. No. 4,815,843 describes example biosensors  102 . 
       FIG. 2  shows an exemplary configuration where biosensors  102  are arranged in an array  102 A and operably supported in regions or “wells” W of a microplate  170  having a surface  171 . An exemplary biosensor array  102 A has a 4.5 mm pitch for biosensors  102  that are 2 mm square, and includes 16 biosensors per column and 24 biosensors in each row. In an example, microplate  170  includes fiducials  169  on microplate surface  171  that can be used to position, align, or both the microplate  170  in system  100  relative to a reference location. A microplate holder  174  is also shown holding microplate  170 . Many different types of plate holders can be used as microplate holder  174 . U.S. Pat. No. 5,738,825 describes example microplates  170 . 
     With reference again to  FIG. 1 , optical reader system  100  includes a light source assembly  106  (e.g., lamp, laser, diode, filters, attenuators, etc.) that generates light  120 . Light  120  is directed by a coupling device  126  (e.g., a circulator, optical switch, fiber splitter or the like) to a scanning optical system  130  that has an associated optical axis A 1  and that transforms light  120  into an incident optical beam  134 I, which forms a beam spot  135  at biosensor  102  (see inset B). Incident optical beam  134 I (and thus beam spot  135 ) is scanned over the biosensor  102  by the operation of scanning optical system  130 . In an example, the biosensor  102  is moved (i.e., by moving microplate  170 ) so that the incident optical beam can be scanned across the biosensor  102 . Also in an example, the incident optical beam  134 I is scanned across a stationary biosensor  102  using scanning optical system  130 , as described further below. In another example, both scanning and microplate movement can be employed. 
     Incident optical beam  134 I reflects from biosensor  102 , thereby forming a reflected optical beam  134 R. Reflected optical beam  134 R is received by scanning optical system  130  and light  136  therefrom (hereinafter, “guided light signal”) is directed by coupling device  126  to a spectrometer unit  140 , which generates an electrical signal S 140  representative of the spectra of the reflected optical beam. In embodiments, a controller  150  having a processor unit (“processor”)  152  and a memory unit (“memory”)  154  then receives electrical signal S 140  and stores in the memory the raw spectral data, which is a function of a position (and possibly time) on biosensor  102 . 
     Thereafter, processor  152  analyzes the raw spectral data based on instructions stored therein or in memory  152 . The result is a spatial map of resonant wavelength (λ R ) data such as shown by way of illustration in  FIG. 3 , which shows the calculated resonance centroid as a function of the position of the scanning spot across the sensor for a number of different scans. The variation of the resonance wavelength indicates if a chemical or biological reaction happened for a specific sensor. 
     In embodiments, controller  150  includes or is operably connected to a display unit  156  that displays measurement information such as spectra plots, resonant wavelength plots, and other measurement results, as well as system status and performance parameters. In another embodiment, spectra can be processed immediately so that only the wavelength centroid is stored in memory  154 . 
     Also in example, system  10  includes a photodetector  160  used to detect the intensity of reflected optical beam  134 R without the reflected optical beam passing to spectrometer  140 . This configuration is useful when performing diagnostic measurements or for determining the position of microplate  170  using the positioning methods described in greater detail below. In an example, photodetector  160  is operably connected to a second circulator  126 ′ located between first circulator  126  and spectrometer  140 . Photodetector  160  generates a photodetector signals S 160  that is provided to controller  150  and is processed using, for example, processor  152  therein. Intensity data from photodetector  160  can also be stored in memory  154 . 
     Biosensors 
     Example biosensors  102  make use of changes in the refractive index at sensor surface  103  that affect the waveguide coupling properties of incident optical beam  134 I and reflected optical beam  134 R to enable label-free detection of biological substance  104  (e.g., cell, molecule, protein, drug, chemical compound, nucleic acid, peptide, carbohydrate) on the biosensor. Biological substance  104  may be located within a bulk fluid deposited on biosensor surface  103 , and the presence of this biological substance alters the index of refraction at the biosensor surface. 
     To detect biological substance  104 , biosensor  102  can be probed with incident optical beam  134 I while reflected optical beam  134 R is received at spectrometer unit  140 . Controller  150  can be configured (e.g., processor  152  can be programmed) to determine if there are any changes (e.g., 1 part per million) in the biosensor refractive index caused by the presence of biological substance  104 . In embodiments, biosensor surface  103  can be coated with, for example, biochemical compounds (not shown) that only allow surface attachment of specific complementary biological substances  104 , thereby enabling biosensor  102  to be both highly sensitive and highly specific. In this way, system  100  and biosensor  102  can be used to detect a wide variety of biological substances  104 . Likewise, biosensor  102  can be used to detect the movements or changes in cells immobilized to biosensor surface  103 , for example, when the cells move relative to the biosensor or when they incorporate or eject material a refractive index change occurs. 
     If multiple biosensors  102  are operably supported as an array  102 A in wells W of microplate  170 , which in turn is supported by microplate holder  174 , then they can be used to enable high-throughput drug or chemical screening studies. For a more detailed discussion about the detection of a biological substance  104  (or a biomolecular binding event) using scanning optical reader systems, reference is made to U.S. Patent Application Publication No. 2006/0141611. Other optical reader systems are described in U.S. Pat. No. 7,424,187 and U.S. Patent Application Publications No. 2006/0205058 and 2007/0202543. 
     Spectral Interrogation 
     The most commonly used technique for measuring biochemical or cell assay events occurring on RWG-based biosensors  102  is spectral interrogation. Spectral interrogation entails illuminating biosensor  102  with a multi-wavelength or broadband beam of light (incident optical beam  134 I), collecting the reflected light (reflected optical beam  134 R), and analyzing the reflected spectrum with spectrometer unit  140 . 
     An exemplary reflection spectrum from an example spectrometer unit  140  is shown in  FIG. 4 , where the “peak amplitude” is the number of photon counts as determined by an analog-to-digital (A/D) converter in the spectrometer. When chemical binding occurs at biosensor surface  103 , the resonance shifts slightly in wavelength as indicated by the double arrow, and this shift can be detected by spectrometer unit  140 . 
     While the general concept of spectral interrogation of biosensor  102  is straightforward, the implementation details of how light can be delivered to and collected from the biosensor can have a major impact on the quality of the data and practical utility of system  100 . For example, due to inevitable non-homogeneity of the resonant wavelength λ R  across biosensors  102 , the measured resonant wavelength λ R  is extremely sensitive to the position of incident optical beam  134 I over the biosensor. 
     Further, variation in absolute readings between microplates  170  is large compared with the wavelength shift. There can be significant differences between absolute readings of biosensors  102  on the same microplate, between microplates, and between readings of the same microplate taken by two different optical readers. Optical readers currently need at least two readings to detect activity. The reported measurement is a wavelength shift between the current condition and an earlier “baseline” condition, such as before the addition of biological substance  104 , such as cells or proteins. Typically, microplate  170  needs to be removed and repositioned between some measurements to add reagents or so that other microplates  170  can be measured while the reagents take effect. This typically leads to errors in plate position, which in turn causes errors in the reading of biosensors  102 . 
     Biosensors  102  are also inherently non-homogeneous due to manufacturing processes used to make them. For example, there is typically a variation in the absolute resonant wavelength within each biosensor. Consequently, any wavelength shift between readings of a biosensor can only be attributed to biochemical change if the same point(s) on the biosensor are measured. An error of 0.01 mm in the position of beam spot  135  on biosensor  102  can cause a wavelength shift large enough to be mistaken for biological activity. Optical readers thus need to account for this non-homogeneity in order for the measurements to be repeatable. It is therefore desirable that microplate positioning within the optical reader be as accurate as possible. This requires both position detection capability and positioning capability, which are discussed in greater detail below. 
     Single-Channel Scanning Optical Reader System 
       FIG. 5  is a detailed schematic diagram of an example single-channel embodiment of system  100 . Cartesian X-Y-Z coordinates are shown for reference. An exemplary light source assembly  106  comprises a light source  106 A, a variable optical attenuator  106 B, a polarization scrambler  106 C and an optical isolator  106 D. Polarization scrambler  106 C serves to randomize the polarization of light  120 , and optical isolator  106 D serves to prevent scattered or reflected light from returning to light source  106 A. 
     An exemplary light source  106 A includes a wide spectrum source such as a superluminous diode (SLD). Light source assembly  106  is optically connected by a first optical fiber section  202  to coupling device  126 , which in the present embodiment is a 1×2 fiber splitter. Spectrometer unit  140  comprises a spectrometer, such as an HR-2000 spectrometer, available from Ocean Optics, Dunedin, Fla. Spectrometer unit  140  can be connected by a second optical fiber section  204  to coupling device  126 . A third optical fiber section  206  can be connected at one end  206 A to coupling device  126 , while the other end portion  206 B can be mounted on a X-Y-Z translation stage  220 . 
     Also mounted on translation stage  220  can be a focusing lens  230  having a focal length f 2 , a linear polarizer  234  and a quarter-wave plate  238 . Note that focusing lens  230  may comprise one or more optical elements. Fiber section end  206 A, focusing lens  230 , linear polarizer  234  and quarter-wave plate  238  constitute an adjustable beam-forming optical system  250  that shares the aforementioned optical axis A 1 . 
     In embodiments, translation using translation state  220  can be accomplished manually, while in other embodiments can be accomplished automatically under the control of controller  150  via a control signal  5220 . In an exemplary embodiment, the first, second, and third fiber sections  202 ,  204  and  206  can be single-mode (SM) fiber sections. 
     System  100  includes a scanning mirror device  260  arranged along optical axis A 1  adjacent beam-forming optical system  250 .  FIG. 6  is a close-up schematic diagram of an exemplary scanning optical system  130  that includes beam-forming optical system  250  and scanning mirror device  260 . Scanning mirror device  260  can be, for example, a micro-electro-mechanical system-(MEMS)-based mirror, such as is available from Mirrorcle Technologies, Inc., Albany, Calif., or from Texas Instruments, Dallas, Tex., as model TALP 1011, for example. Other exemplary embodiments of scanning mirror device  260  can include a scanning galvanometer, a flexure-based scanning mirror, an oscillating plane mirror, a rotating multifaceted mirror, and a piezo-electric-driven mirror. 
     Scanning mirror device  260  can be adapted to scan in at least one dimension (1D) and preferably two-dimensions (2D) (i.e., along axes X and Y, thereby defining associated scanning angles θ X  and θ Y ). Scanning mirror device  260  can be operably connected to a mirror device driver  264 , which may be based on voltage or current depending on the nature of scanning mirror device  260 . In embodiments, scanning mirror device  260  can be mounted on translation stage  220 . 
     A field lens  280  can be arranged along optical axis A 1  adjacent scanning mirror device  260  and opposite beam-forming optical system  250 . In embodiments, field lens  280  has an F-theta configuration wherein light from any angle θ is directed substantially parallel to optical axis A 1  (i.e., φ˜0°). Suitable F-theta field lenses  280  are commercially available from optics suppliers, such as Edmund Optics, Barrington, N.J. Field lens  280  has a focal length f 1  and comprises at least one optical element. In embodiments, field lens  280  comprises multiple optical elements, including at least one mirror, or at least one lens, or a combination of at least one mirror and at least one lens. In an exemplary embodiment, field lens  280  includes one or more aspherical surfaces. 
     System  100  also includes the aforementioned microplate holder  174  configured to operably support microplate  170 , which in turn is configured to operably support an array of biosensors  102 . In an exemplary embodiment, the position of microplate holder  174  is adjustable so that the position of microplate  170  can be adjusted relative to optical axis A 1 . Scanning mirror device  260  is located at the focus of field lens  280 , i.e., at a distance f 1  from the field lens. 
     System  100  of  FIG. 5  is also configured in an example with aforementioned photodetector  160  optically connected to circulator  126 . 
     System  100  of  FIG. 6  illustrates an exemplary scanning optical system  130  shown optically coupled to beam-forming optical system  250  and that includes scanning mirror device  260 , a fold mirror M 1 , and f-theta field lens  280 . Also shown is microplate holder  174  with microplate  170  supported thereby. Fold mirror M 1  can be used to fold optical axis A 1  and thus fold the optical path to make scanning optical system  130  more compact. In embodiments, focusing lens  230  has a focal length f 2 =10 mm and field lens  280  has a focal length f 1 =200 mm with an aperture of 72 mm. This particular configuration for scanning optical system  130  fits within dimensions L 1 ×L 2 =140 mm×140 mm and thus has a relatively compact form factor. In embodiments, beam-forming optical system  250  can be included in scanning optical system  130 . 
     The size of the microplate  170  that can be scanned by scanning mirror device  260  is given by the tangent of the mirror deflection multiplied by the focal length of the field lens  280 . So, with +/−10 degrees of optical deflection and a 200 mm focal length field lens  280 , a 72 mm area can be scanned in both the X- and Y-directions. 
     An exemplary scanning optical system  130  of  FIG. 6  is capable of interrogating a single microplate column of biosensors  102  when configured in a standard microplate format of sixteen wells per column on a 4.5 mm pitch, or about a 72 mm total distance. An exemplary nominal size of beam spot  135  formed by incident optical beam  134 I at microplate  170  is 0.1 mm at 1/e 2  (diameter) and an exemplary beam diameter of the incident optical beam at scanning mirror device  260  is 2 mm at 1/e 2 .  FIG. 6  illustrates incident optical beam  134 I at three different scan positions (angles). The central ray of incident optical beam  134 I is denoted  134 C. Note the incident optical beam  134 I is a converging beam at microplate  170 , with the central rays  134 C being parallel to optical axis A 1  at the microplate. 
     As discussed above, exemplary scanning mirror device  260  is a MEMS-based mirror (such as the aforementioned TALP1011 from Texas Instruments), which in an example has a clear aperture of 3.2 mm×3.6 mm and optical scanning angles θ X  and θ Y  of +/−10°. The variation of incidence angle φ of incident optical beam  134 R over microplate  170  due to aberrations in an exemplary field lens  280  was found in one example system  100  to be less than 0.3 mRd. 
     Controller  150  is operably connected to light source assembly  106 , spectrometer unit  140  and mirror device driver  264 , and is configured (e.g., via software embodied in a computer readable medium such as in processor  152  or memory  154 ) to control the operation of system  100  as described below. In embodiments, controller  150  can be configured with a General Purpose Interface Bus (GPIB) and the devices to which the controller is operably connected can be configured to communicate with the controller using the GPIB. 
     With reference again to  FIG. 5 , in the general operation of system  100 , controller  150  sends a light source control signal S 106  to light source assembly  106  to cause the light source assembly to generate light  120 , which is coupled into first fiber section  202  as guided light. Light  120  travels down first fiber section  202  and to third fiber section  206  via coupling device  126 . Light  120  is then processed by beam-forming optical system  250 , which forms incident optical beam  134 I. Incident optical beam  134 I is then selectively deflected by scanning mirror device  260  under the operation of a control signal S 260  from mirror device driver  264 , which in turn is activated by a control signals S 264  from controller  150 . 
     Because scanning mirror device  260  is located at the focus of field lens  280 , in the region between the field lens and microplate, the incident optical beam  134 I (or, more precisely, the central ray  134 C of this beam) is parallel to optical axis A 1  for all deflection angles. System  100  can be adjusted so that incident optical beam  134 I remains substantially normal to microplate  170  as the beam scans the microplate. 
     Incident optical beam  134 I scans over biosensor  104  as described below and reflects therefrom at substantially normal incidence to form reflected optical beam  134 R. Reflected optical beam  134 R thus travels substantially the reverse optical path of incident optical beam  134 I and is coupled back via beam-forming optical system  250  into third fiber section  206  at end portion  206 B and becomes guided light signal  136 . Guided light signal  136  then travels through third optical fiber section  206  to second optical fiber section  204  via coupling device  126 , where it is received and spectrally decomposed by spectrometer unit  140 . Spectrometer unit  140  provides electrical signal S 140  representative of the spectral information in reflected optical beam  134 R to controller  150  and to memory  154  therein. Memory  154  stores the spectral information as a function of the scanning angles (θ X , θ Y ). In embodiments, memory  154  stores and processor  152  runs analysis software for analyzing and visualizing the spectral information, such as Matlab, available from Mathworks, Inc., Natick, Mass. 
     In embodiments, memory  154  stores a number (e.g., 50) of spectra for each biosensor  102 , and processor  152  sums the spectra to obtain a total spectra, and then calculates the centroid to determine resonant wavelength λ R . In embodiments, tens, hundreds, or thousands of spectra can be saved in memory  154  for processing by processor  152 . Spectra measurements can be divided up by, for example, individual biosensors  102  or by columns or rows of biosensors. 
     Biosensor Scanning 
     One method of scanning using system  100  is to operate scanning mirror device  260  to scan one or more biosensors  102  in a single scanning direction. However, a shortcoming of this approach is that the resonance wavelength λ R  varies significantly as a function of the position of beam spot  135  across biosensor  102 . Accordingly, in this approach the position of beam spot  135  needs to be monitored closely to avoid introducing measurement bias. 
     A preferred method of operating system  100  involves scanning biosensors  102  with incident optical beam  134 I in two dimensions X and Y to obtain an integrated measurement of each scanned biosensor. Because a MEMS-based mirror scanning device can be driven at a relative high frequency (e.g., ≧100 Hz), it is possible to rapidly perform such a two dimensional scan of a sensor. In one example, biosensor  102  is scanned by moving optical beam  134 I (and thus beam spot  135 ) faster in one of the two dimensions to obtain a zig-zag or sinusoidal scan path. 
     In embodiments, system  100  can be configured so that the position of field lens  280  is adjustable relative to scanning mirror device  260  and beam-forming optical system  250 . In embodiments, the relative positions of field lens axis A 280 , scanning mirror device  260  and focusing lens axis A 230  are adjustable, i.e., one or more of these elements is displaceable relative to optical axis A 1 . In embodiments, this adjustability is provided by translation stage  220 . The angle of incidence φ of incident optical beam  134 I relative to microplate  170  is defined by the vector joining the center of the incident optical beam at focusing lens  230  and the apex of field lens  280 . 
     Thus, in embodiments, incidence angle φ of incident optical beam  134 I can be adjusted by adjusting the relative position of lenses  230  and  280 . Such adjustment can be made in embodiments by adjusting translation stage  220  that includes scanning mirror device  260  and focusing lens  230 . This operation does not require translation stage  220  to have high precision. By way of example, for a field lens  280  having a focal length f 1 =200 mm, the alignment precision only needs to be in the order of 0.2 mm to insure that the precision of incidence angle φ is within 1 mrad. This adjustability makes system  100  substantially insensitive to microplate misalignment. 
     Microplate Position Detection 
     The use of a MEMS-based scanning mirror device  260  provides certain performance and size advantages for optical reader system  100 . However, such a scanning mirror device is not particularly precise. MEMS-based scanning mirror devices can have significant part-to part variations, are sensitive to temperature, and are only linear to about 5%. Where MEMS-based scanning mirror devices have positional feedback, its positioning ability can be considered coarse relative to the positioning capability needed for optical readers. However, a given MEMS-based scanning mirror devices can provide very repeatable positioning at a given temperature. 
     As discussed above, scanning mirror device  260  scans biosensor columns (or rows) by moving incident beam  134 I and the attendant beam spot  135  nominally through the center of biosensors  102 . The lack of precision of a MEMS-based scanning mirror device  260  is compensated by rapidly oscillating beam spot  135  in a direction normal to the scan direction so that substantially the entire biosensor  102  is covered. Typically, the spectrometer obtains an integrated (i.e. summed) response from one or more complete passes of the oscillation. The passes represent (beam-width) cross-sections that are numerically integrated. 
     This scanning method reduces but does not eliminate the above-described positional sensitivity issues. To substantially reduce or eliminate positional sensitivity, system  100  needs to measure an equal contribution from each point on the biosensor. To accomplish this, a number of conditions are satisfied. 
     The first condition is that velocity of beam spot  135  needs to be constant, as different dwell times on bio sensor  102  result in different measurement contributions from the different points on biosensor. 
     The second condition is that there be no changes in the relative illumination of beam spot  135  in between passes. For a beam spot  135  with uniform intensity, this condition can be achieved with perfectly vertical passes that just touch each other without overlap and without any gaps. 
     Where the scan path of beam spot  135  over biosensor  102  has a sine-wave (oscillating) pattern, the beam spot velocity is not constant. Also, spacing between measurements in the scan direction is larger between measurements near the top of the scan path than at the center. In addition, the intensity of beam spot  135  is not uniform and typically has a Gaussian intensity profile, so that a scan path with vertical sections and performed at a perfectly constant velocity cannot be properly spaced to give the desired uniform illumination. 
     For system  100  to overcome the positional errors of microplate  170  using the above-described scanning approach, scanning mirror device  260  would need to oscillate far faster (e.g., at least 50 times faster), and the spectrometer would need to sample far faster (e.g., over 5000 times faster). In addition, spatial information obtained by an area scanner in the direction of oscillation is applicable to the MEMS mirror response at that frequency, and since the frequency response of MEMS mirrors is usually not flat, the information cannot be used to move the beam to a static level in that direction. Thus, in short, the above-described light-spot scanning approach has its limitations in achieving improved measurement resolution in the face of the aforementioned microplate positioning sensitivities. 
     Accordingly, an aspect of the disclosure includes system  100  being configured for fast and accurate position detection of microplate  170 . The position detection system and methods described herein generally include searching for and detecting select position-detecting features (“features”)  300  on microplate  170  using scanned beam spot  135 . The reflected light is detected, and the detected signal allows the features to be distinguished from the background. In an example, the detected features are the biosensors  102  and the background is the otherwise flat microplate surface  171 . In another example, features  300  include fiducials  160 . 
     Since features  300  are accurately placed on microplate  170  (i.e., their positions are by definition very accurately known), the relative position of microplate  170  can be accurately determined by measuring the locations of one or more features  300 . This in turn allows for accurate scanning of beam spot  135  over the biosensor, to increase the performance of system  100 . 
     The position detection methods disclosed herein can fall into one or both of two general categories: Those that use a scan path having an oscillating component added to a generally one-dimensional component to enhance the otherwise one-dimensional feature scan, and those that use the results of a previous search to define a new feature scan. 
       FIG. 7  is a schematic diagram that illustrates an example of a first position detection method for determining the position of microplate  170 . Shown in  FIG. 7  is a feature  300  located on microplate  170 . Feature  300  can be, for example, biosensor  102  or the aforementioned fiducial  169 . Using for example incident light beam  134 I, beam spot  135  is positioned at a location on microplate surface  171  using for example scanning optical system  130 . Beam spot  135  is moved in a general scan path direction (i.e., linear component)  320 . As beam spot  135  is moved in the general scan path direction, it is oscillated in the perpendicular direction to impart an oscillatory component in forming the overall scan path  324 . The oscillatory component can be sinusoidal, as shown. As scan path  324  crosses feature  300 , reflected light  134 R therefrom is directed to photodetector  160  to obtain a measured feature profile. In an example, the measured feature profile is established by relating the detected intensity in reflected light  134 R to the location of beam spot  135  as determined by the corresponding mirror orientation of scanning mirror device  260 . The plot in  FIG. 7  illustrates an example of a measured feature profile for a feature  300  having substantially uniform reflectivity at the wavelength of incident and reflected light beams  134 I and  134 R. 
     In an example, multiple scans along the same direction can be performed using, for example, different oscillation amplitudes in the oscillation component of the scan path. In an example, photodetector  160  integrates by sampling reflected light beam  134 R at a select sampling interval. The dashed lines accompanying the measured profile plot in  FIG. 7  show where the edges of feature  300  reside, with the plot showing the integrated measurement between one time point to the next time point for the corresponding portion of the oscillating scan path  324 . The measured profile of feature  300  can be further defined by taking readings from one (or more) oscillation passes, e.g., over different sections of the feature. 
     In an example, the center of feature  300  can be found by determining the feature edge locations and taking the half-way point, or by finding the center of the signal as represented by the plot of the measured profile, e.g., by measuring the centroid or using a similar center-finding technique. 
       FIG. 8  is a schematic diagram of optical scanning system  130  as used to measure the position of features  300  on microplate  170 . As described above in connection with the operation of system  100  in reading biosensors  102 , now system  100  is reading features  300 , which can be the biosensors, fiducials  169 , or any other type of reference features that can provide accurate position information about microplate  170  relative to a reference location of system  100 . Thus, as described above, incident beam  134 I from light source  106  is directed by scanning mirror device  260  generally to microplate  170  to be incident up microplate surface  171 . 
     The beam angular range is controlled by controlling scanning mirror device with mirror device driver  264  (see  FIG. 5 ), which can be programmed (or operated via controller  150 ) so that the mirror configuration (e.g., micro-mirror configuration of a MEMS-based mirror) correspond to positions on microplate  170 . Scanning mirror device  260  scans beam spot  135  over scan path  324 . Detector  160  can determine, via the detection of reflected light  134 R, whether beam spot  135  scans over a feature  300  or background, which here is assumed to be the planar microplate surface  171  with a different reflectivity than the feature. This allows for scanning optical system  130  to detect the location of features  300 , which leads to establishing an accurate microplate position. 
     An aspect of the positioning systems and methods disclosed herein account for variations in the reflectivity of feature  300  due to any number of reasons, including for example the presence of debris or a defect  301  in the feature.  FIGS. 9A and 9B  are schematic diagrams that illustrate two different scan paths  324  having different oscillation amplitudes. Note how increasing the scan path oscillation amplitude allows for feature  300  to be detected as a single feature rather than as two features despite the presence of defect  301 . Thus, the “hole” that was present in the plot of the “measurement value” (i.e., detected intensity) vs. mirror location (i.e., mirror configuration) of  FIG. 9A  shows up as a much smaller measurement variation that indicates a single feature  300  rather than two separate features. Thus, by finding the feature edges in the measured feature profile as described above, the center of the feature can be determined even though the feature has a defect or contaminant  301 . 
       FIG. 10A  and  FIG. 10B  illustrate an example position detection method where the scan path oscillation amplitude is made relatively large, i.e., about as large as or larger than the size of (i.e., at least one dimension of) feature  300 . This allows for the general scan path  320  to be loosely selected because the relatively large size of the scan path oscillations is more likely to overlap a feature  300  or a portion thereof. Thus, if scan path  324  only partially overlaps feature  300 , the measured feature profile can still approximately show the feature, and for certain features will allow for determining a center based on an edge-to-edge measurement. With respect to  FIG. 10A , the centroid of the peak measures the (one-dimensional) location of the circular feature  300 . The same can be done with features having other shapes, such as triangles (see  FIG. 10B ), diamonds, etc. 
     The position detection method using a relatively large scan path oscillation works particularly well for regularly shaped (e.g., symmetric) features  300  having known orientations, where the centroid measurement will provide sufficient information to locate the feature. Finding the centroid is more easily and accurately determined for features having a measurement value vs. mirror location plot that is more flat (e.g.,  FIG. 10A ) than for features where the plot has substantial variation (e.g.,  FIG. 10B ) due to the feature shape. Increasing the number of measurements (e.g., detector sampling) increases the measurement accuracy for determining the position of the center of feature  300 . 
     With reference to  FIG. 10B , note that oscillating scan path  324  completely covers feature  300  and the resulting measurement plot (measured feature profile) has a peak. The centroid of the peak measures the (one-dimensional) location of the feature far more precisely than any type of edge detection. In this instance, scanning in the y-direction (i.e., the addition of the oscillation to the scan path) is unnecessary. 
       FIG. 11  illustrates an example of the position detection method that employs a shaped feature  300 , which are most likely to be fiducials rather than biosensors or biosensor wells. Shaped features  300  such as triangles can provide information about how far scan path  324  is from the feature center in the direction perpendicular to the path. By examining the results from scan paths  1 ,  2  and  3 , the scan path that yields the feature center can be deduced. 
     In cases where dust or defects  301  on a feature  300  interfere with the scanning profile, an aggregate scanning profile can be formed by mathematically combining the results of one or more cross sectional measurements. An example of this approach is illustrated schematically in  FIG. 12 . 
       FIG. 13A  and  FIG. 13B  illustrate an example position detection method that is useful for coarse initial detection of features  300  when the location of the sample feature is only generally known. In the method, beam spot  135  is traced over an oscillating scan path  324  having a relatively small amplitude. Oscillating scan path  324  is shown as crossing feature  300  slightly center in  FIG. 13A  and in a general scan path  320  in the x-direction. When feature  300  is found, oscillating scan path  324  is automatically changed so that the oscillating scan path has a general scan path in the y-direction so that feature  300  is scanned in the (in-plane) perpendicular direction. 
     With reference to  FIG. 13B , another x-y scanning method of position detection is to first scan along the x-path with an oscillating scan path  324  having relative large oscillations. This type of oscillating scan path  324  has a high probability of hitting feature  300  but will not generally allow for finding the feature center location directly with any accuracy. However, once the presence and rough position of feature  300  has been identified, subsequent searches with less amplitude (or no amplitude) can be employed. As shown in  FIG. 13B , an oscillating scan path  324  having a medium-sized oscillation and a general scan path  320  in the x-direction passes relatively close to the feature center and finds the feature y-height with reasonable accurately. Then a finer x-scan can be performed using information from the previous scan and employing a smaller oscillation amplitude. 
     Another method of position detection involves detecting multiple features  300 .  FIG. 14A  and  FIG. 14B  are schematic diagrams of a microplate that includes an array of features  300 , including features near the microplate corners. Features  300  can be microplate wells W or fiducials  169  arranged on microplate surface  171  adjacent or in between adjacent wells. Features  300  are shown as being square by way of illustration. The method locates the position of microplate  170  and not just features  300 . If the dimensions of a rectangular or square microplate  170  are well known (which is typically the case), the method can establish the positions of the features  300  on the microplate. 
     With reference to the inset of  FIG. 14A , a first scan SX 1  and a second scan SX 2  in the x-direction are performed in the vicinity of one corner  170 C 1  of microplate  170 . These x-direction scans are separated from each other by a distance about ½ that of the feature diameter. In both scans SX 1  and SX 2 , the sought-after feature  300  was not found. Thus, a third scan SX 3  (and possibly subsequent scans) is performed based on the results of the earlier scans. Once feature  300  is found (say, in scan SX 3 ), this x-direction scanning can stop. At this point, the x-direction scan information is used to perform a scan SY in the y-direction that cuts through the middle of feature  300 . 
     Once microplate corner  170 C 1  is located (or, corner feature  300  closest to corner  170 C 1  is located), the other corners (or corner features) can be located. Once one of the corner features is located, the other features can be readily located since their relative positions are known. An exact fit of the following form can be used with at least three pieces of feature measurement data (e.g. x and y for one corner, x or y for another). 
     A best fit of the same form is also straightforward with more than three pieces of data, for example the centers of all four corners. 
     
       
         
           
             
               
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     If translation and rotation of microplate  170  and apparent translation and scaling of the microplate due to a scale change of scanning mirror device  260  are considered, there are five unknowns (translation from the two effects can be combined). A general equation to which data can be fit is as follows. Five unknowns require five pieces of information for an exact solution. The x and y locations of two opposing corners plus the x or y location of one of the others is sufficient for a solution. 
     
       
         
           
             
               
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     An alternative is to use the general linear form. Even if the equation above has six unknowns, solving it requires six pieces of information. The x and y locations of three corner features are a good choice. In an example, all four corners are found for a best fit using a linear regression. 
     In an example, the position detecting methods employ an f-theta optical configuration such as shown in  FIG. 5  and  FIG. 6 . The detector used may be photodetector  160 , or alternatively spectrometer  140  can be employed as the photodetector since this includes an array of integrating photodetectors that each sample a narrow bandwidth of light. The methods detailed herein are structured to make use of the spectrometer&#39;s integrating ability, and to overcome its speed limitations. Integration can be performed optically where possible, or numerically i.e., sampling rapidly with respect to the sine-wave scan path  324 , and combining the samples in an external system. 
     Examples of the position detecting methods described herein are capable of locating microplate  170  to within 0.025 mm in translation, rotation and linear deformation in approximately two seconds. This allows for scanning optical system  130  to accurately place beam spot  135  on biosensor  102 , and in particular accurately scan the beam spot over each biosensor in a very controlled manner to obtain accurate readings. 
     Optical Reader System Calibration 
     With reference again to  FIG. 8 , the position of a point p on microplate  170  relative to perpendicular is given by p=d·tan(θ) where θ now refers to the tilt angle of the mirror (scanning mirror device  26 ) and d is the distance from the mirror to the surface at the optical center (θ=0°) and where θ is a small angle, p≈k·θ. 
     This approximation worsens as angle θ increases, leading to distortion near the edges of the lens field. That is, a small change in angle θ results in a certain change in position on microplate  170 , but the same small change in angle at the extremities of the microplate causes a larger change in position. The result is that the size of features  300  appears to increase with distance away from the optical axis. This causes position errors of incident beam  134 I and thus error locations in the placement of beam spot  135  relative to biosensors  102  during the biosensor measurement process. It also leads to reduced accuracy in determining the position of microplate  170 . 
     Thus, an aspect of the disclosure is calibrating system  100  to account for the aforementioned distortion to correctly position incident beam  134 I and thus accurately position beam spot  135 . The calibration involves properly identifying the angle θ required to achieve a given position on microplate  170 . In particular, the angle θ is given by θ=arctan(p/d). Note that for an f-theta lens with a focal length f, the distance d=f. 
       FIG. 15  is a schematic diagram of an example scanning optical system  130  similar to that shown in  FIG. 5  and  FIG. 6 . An objective lens  280  (i.e., an f-theta lens) is placed between microplate  170  and scanning mirror device  260 , with the focal point of the objective lens located at the center of rotation of the mirror of scanning mirror device  260 . Scanning optical system  130  exhibits the aforementioned tangential distortion, with the beam position on the sample being p=f·tan(θ), where f is the focal length of the f-theta objective lens  280 . 
       FIG. 16  is a schematic diagram of an example control system where a control variable in the controller  150  (e.g., microplate positions in controller coordinates) is converted to an actual measured parameter (e.g., microplate positions in microplate coordinates). A digital control signal from software stored in controller  150  goes through a converter (e.g., mirror driver  264 ) to become analog power (i.e., an analog power signal) that drives the mirror of scanning mirror device  260  to a specific angle. The angle of input light beam  134 I is determined by the mirror angle. Input light beam  134 I goes through scanning optical system  130  and forms beam spot  135  at a corresponding position at microplate  170 . 
     Embodiments of scanning optical system  130  can also have feedback from the mirror. This feedback also goes through a second converter (dashed-line box) and presented to the controller  150  in a digital format and is processed by the aforementioned software (i.e., instructions embodied in a computer-readable medium in the controller). 
     In the control system illustrated in  FIG. 16 , there are potential non-linearities in every block shown, with some of the non-linearities being more significant than others. The non-linearities can be small in the converters, for example, depending on their design. For MEMS mirrors, the relationship between input and output beam angle can have substantial non-linearity, with example MEMS mirrors being linear only to above 5%. Additionally, in 2-dimensional mirrors, there may be interaction between axes so that axis calibration cannot be done separately. 
     Thus, even if optics were used to correct for the above-described geometric and optical distortions, the MEMS mirror would defeat the purpose. Consequently, short of making a custom lens matching every MEMS mirror, a hardware solution directed to substantially eliminating distortions in a MEMS-based optical reader is problematic. 
     To perform a software-based method of calibrating system  100 , the following functions are defined: (i) a control output function that specifies control output (X CTRL ,Y CTRL ) from desired static positions (X SAMP ,Y SAMP ), where X SAMP  and Y SAMP  are (x,y) positions at microplate  170 ; (ii) a control dither function that specifies control dither output (A CTRL , B CTRL ) from desired sample sizes (A SAMP , B SAMP ) at static points (X SAMP ,Y SAMP ) on microplate  170 . For multiple dither frequencies, multiple control dither functions—one for each frequency—are required. Here, “sample” means in one embodiment a microplate having one or more biosensors. 
     For the most accurate calibration to take place, the drive frequency of scanning mirror device  260  is accounted for because the calibration differs at different drive frequencies. 
     For effective calibration to occur, the relationship between the control quantities (X CRTL , Y CRTL ) (controller coordinates) and the sample quantities (X SAMP , Y SAMP ) (sample coordinates) (and also between the feedback at the controller (X FB , Y FB ) and sample coordinates if feedback is employed) need to be determined to create correction maps (functions). These functions are necessary in finding the control setting required to move to a particular position on the sample (F 1 ) and finding the position on the sample from the feedback (F 2 ) as denoted by the equation below.
 
( X   CRTL   ,Y   CRTL )= F   1 {( X   SAMP   ,Y   SAMP )}
 
( X   SAMP   ,Y   SAMP )= F   2 {( X   FB   ,Y   FB )}.
 
Function F 1  drives scanning mirror device  260  to obtain an accurate (non-distorted) location of beam spot  135  on microplate  170 . Function F 2  is used to calibrate the feature coordinates with feedback coordinates from scanning optical system  130  and in particular from scanning mirror device  260  and mirror driver  264 .
 
       FIG. 17A  and  FIG. 17B  are schematic diagrams of the position-detecting features plotted along with the beam spot scan paths, illustrating how non-linearities in the optical reader can give rise to spatial distortion with respect to the actual microplate coordinates.  FIG. 17A  also schematically illustrates how a calibration sample is derived in the controller&#39;s coordinates (X CRTL , Y CRTL ). If the distortion is not too large, the X centers of features  300  can readily be detected with 1-D horizontal scans as described above, and calibration can be performed directly. The X coordinate centers in  FIG. 17A  have been found, but because of the aforementioned non-linearities of system  100 , the calibration sample appears distorted. 
     If the distortion is too large, a straight line cannot go through all the features in one row without touching features in other rows.  FIG. 17B  is similar to  FIG. 17A  but with a larger amount of distortion. In the event of such large distortion, the scanning optical path  324  for locating the position of features  300  can be modified to find the border or part of the border.  FIG. 17B  scan paths  324  can be used to search in either the horizontal or diagonal directions where the amplitude of oscillation must be at least the size of the largest expected gap between features in the search region. This is to allow an estimated calculation of the sample&#39;s shape outline. 
     The calibration method corrects distortion observed in the X path (but not in the Y path) that is caused by the mirror tilt in scanning mirror device  260  when the input light beam  134 I turns 90°. The distorted image of  FIG. 17B  appears to shrink from left to right. With this pattern type, locating the outside dimension or centers of the corner features in Y is sufficient to find search paths that pass through all features in the top and bottom rows. The other rows are interpolated between the top and bottom. If the distortion is barreled in the center (as it is for X), the known location of a point half-way along the row is needed for curvilinear (e.g., parabolic) interpolation instead of linear interpolation. 
     This information is available from “windowing,” as shown in  FIG. 17B . The results are points (X CRTL , Y CRTL ) that match known locations X SAMP  such as edges or center points. At this stage, an intermediate calibration can be done since Y SAMP  is still unknown. An intermediate transformation equation is defined as X CRTL =G(X SAMP , Y CTRL ). 
       FIG. 18A  and  FIG. 18B  illustrate example scan paths that account for the distortion of the microplate coordinates.  FIG. 18A  shows an example of the interpolation from previously found y-center locations for the four corner calibration features. By using the intermediate equation, for any mirror location Y CTRL  one can move to the desired X location on the plate (X SAMP ) by finding X CRTL .  FIG. 18B  schematically illustrates another example calibration method that involves performing 1-D searches made to the y-centers where the x-centers are known. This works even if the sample is placed in a tilted holder. In this case, the method just interprets the distortion and corrects for it. 
     If the nominal locations of position-detecting features  300  follow some other pattern, one can still generate a path that goes through their approximate locations if Y CTRL  is found using the method in  FIG. 18A . The results are points (X CRTL , Y CRTL ) that match known locations (X SAMP , Y SAMP ) such as edges or center points. The calibration can now be completed. 
     It is noted that when there is feedback and (X FB , Y FB ) are known, these coordinates match known the locations (X SAMP , Y SAMP ) because they can be recorded every time an edge or a center is found. 
       FIG. 19A  and  FIG. 19B  are schematic diagrams that illustrate two related calibration techniques based on driving a beam spot with a sine-wave driving frequency.  FIG. 19A  illustrates an example calibration technique that involves scanning beam spot  135  over feature  300  with a sine-wave driving frequency provided to scanning mirror device  260 . The calibration scanning involves placing beam spot  135  in the center of the feature  300  of known size, and then scanning the beam spot in a given direction with varying amplitude until the return signal (reflected beam  134 R) starts to decrease. Since MEMS mirrors have non-linear dynamics a 1-volt change that moves the mirror by 1 mm does not guarantee a 1 volt sinusoid at say, 500 Hz, will result in a 1 mm oscillation. It could be much larger or smaller, depending on the mirror and the frequency. The mirror needs to be calibrated at each oscillation frequency at which the mirror will be driven. 
     With feedback, a sinusoidal control of known size is produced to observe the feedback. The feedback can then be converted to sample units, and the relationship for a sinusoid at that point is produced. This can be done in an X and Y grid, and with oscillation in both X and Y to complete a detailed map. Without feedback, features of known size are needed to perform the calibration. 
       FIG. 19B  shows another method for calibrating a sine-wave driving frequency. A feature  300  of known size was used. The sine-wave oscillation imparted to beam spot  135  needs to be larger than a dimension of the feature. Beam spot  135  is scanned in the same direction as the oscillation, resulting in a signal profile as a function of spot position that has two peaks, as shown in the “signal vs. P” plot. It is much easier to find the center of a peak than a point at which power decreases. 
     Once the two peaks are established, the difference in their positions is also the difference in the size of the sine wave versus the feature. If the feature size is known, the sine-wave size can be deduced. This should be done in X and Y at multiple locations to acquire enough data about how the sinusoid varies. Alternatively, this method can be carried out in a single location (e.g., at the feature center) if the scale does not change too much with position on the plate. 
     It will be apparent to those skilled in the art that various modifications to the preferred embodiment of the disclosure as described herein can be made without departing from the scope of the disclosure as defined in the appended claims. Thus, the disclosure covers the modifications and variations provided they come within the scope of the appended claims and the equivalents thereto.