Abstract:
Light emitting diodes (LEDs) are mounted in an array to an upper structure overlying a lower structure with a plurality of light detectors thereon. Each LED is configured to overlie a separate detector. Each LED emits light at a frequency relevant for measuring optical density of a specimen. LEDs having different frequencies are included within the LED array. A corresponding array of detectors is also provided, mounted to the lower structure. Spacing between adjacent LEDs and between adjacent detectors match a spacing between wells in a microtiter plate. Spacing between the lower structure and the upper structure supporting the LEDs is sufficient for the microtiter plate to fit between. Circuitry sequentially fires individual LEDs and gathers optical density data through the detectors for specimens in the wells of the microtiter plate. The structures are then moved to a next adjacent well position on the microtiter plate and the process repeated.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application claims benefit of the earlier filing dates associated with International Patent Application No. PCT/US2010/002012 filed on Jul. 16, 2010, which designates the United States and other countries; and BE 2009/0434 filed on Jul. 16, 2009 which was claimed for priority in the above-identified international application. 
     
    
     FIELD OF THE INVENTION 
       [0002]    The following invention relates to densitometers for measuring the optical density of fluids, such as in the medical diagnostic field. More particularly, this invention relates to densitometers which measure optical density of multiple specimens, such as those within wells of a microtiter plate (MTP) at more than one wavelength, such as for Enzyme Immuno Assay (EIA) tests or Enzyme Linked Immuno Sorbent/Immuno-Sorption Assay (ELISA) tests. 
       BACKGROUND OF THE INVENTION 
       [0003]    Densitometry is a field where the optical density of a fluid specimen is measured by providing a light source and a detector with the detector detecting the amount of light transmitted through the specimen. In more advanced forms of densitometry, the light is limited to a narrow band of light frequencies. Multiple densitometer readings are obtained at different light frequencies to learn more about the specimen being examined. 
         [0004]    To efficiently analyze multiple specimens as quickly and reliably as possible, a microtiter plate is often utilized with a plurality of wells therein arranged in rows and columns. The microtiter plate is substantially planar and is typically oriented substantially horizontally to keep the specimens within the wells of the microtiter plate MTP. One such arrangement of microtiter plate is shown in  FIG. 2 . 
         [0005]    Furthermore, to efficiently analyze specimens within separate wells of the microtiter plate, a single light source can be divided through known prior art equipment into separate light sources, such as through the use of fiber optic light conductors each feeding a single light source to multiple separate wells. Using known prior art equipment, such as that depicted in  FIG. 1 , a single light source such as a halogen lamp  2  is provided. An infrared filter  4  can be utilized to remove infrared portions of the spectrum, to remove heat containing portions of the spectrum from the light being emitted from the lamp  2 . An interference filter  6  is utilized to absorb all light frequencies other than those particularly desired for a measurement being conducted by the densitometer. In some more advanced systems, multiple interference filters  6  are provided which can be selectively positioned in line with the lamp  2  so that different frequencies of light are allowed to pass onto the light conductors  8 . 
         [0006]    Detectors  9  are provided below the wells in the microtiter plate MTP so that the liquid L contained within the wells W is separately measured by the detectors  9 . Typically, the interference filter  6  limits the wavelength to −5 to +5 nanometers of the desired wavelength. Depending on the nature of the liquid L, more or less of the light is absorbed. Under the well W, the non-absorbed light is captured and conducted to the detector  9 . The luminous intensity is measured and the absorption can be calculated. Multiple wells W are measured simultaneously with such prior art densitometers, so that the densitometer can act multiple times faster depending on the number of light conductors  8  utilized and the size of the microtiter plate MTP. 
         [0007]    While generally effective, one problem with such densitometers is the potential for light from adjacent light conductors to have some influence on the luminous intensity read by each detector, such that otherwise properly calibrated detectors might detect a greater intensity than is actually passing through the specimen because it is hitting the detector at least partially from one of the other light conductors. Furthermore, with the prior art multiple interference filters are required and mechanisms for adjusting the interference filters so that proper frequency light is utilized for the particular densitometer test being conducted. These details add complexity to the densitometer and slow down the process Accordingly, a need exists for further enhancement of the efficiency and reliability of densitometer systems and methods. 
       SUMMARY OF THE INVENTION 
       [0008]    With this invention a densitometer is provided which can perform multiple densitometer readings both at singular light frequencies and at different light frequencies for each position of the densitometer relative to a specimen support such as a microtiter plate (MTP). The densitometer includes a lower structure such as in the form of a lower plate and an upper structure such as in the form of a housing. One of these structures includes a plurality of detectors thereon with the other of these structures including a plurality of light emitting diodes (LEDs) thereon. Preferably the lower structure is in the form of a detector plate with a plurality of detectors thereon and the upper structure is in the form of the housing with the plurality of LEDs thereon. 
         [0009]    The detectors and LEDs are preferably provided in arrays which match each other with a similar number of rows and columns of LEDs and detectors. Each LED is aligned with a corresponding detector substantially vertically. Spacing between adjacent LEDs and spacing between adjacent detectors is preferably similar to spacing between wells in a microtiter plate (MTP) so that a plurality of wells can be interposed between sets of LEDs and detectors for substantially simultaneous detection, or rapid sequential detection without movement of the microtiter plate (MTP) relative to the upper and lower structures of the densitometer. 
         [0010]    Some of the LEDs such as each LED in a common column, preferably have a common frequency of light emitted thereby. Preferably, LEDs in separate columns emit light at differing frequencies, such that within a given row, each LED has a different frequency of light being emitted. The detectors can be similar to each other or, if needed, can be optimized for particular light frequencies and hence be different from each other. 
         [0011]    The housing or other upper structure and detector plate or other lower structure are fixed together and spaced apart by a distance sufficient to allow a specimen supporting structure such as the microtiter plate (MTP) to pass between the upper structure and lower structure of the densitometer. The entire densitometer is carried in a movable fashion such as by being mounted to a carriage sliding upon a rail, so that the densitometer can move relative to the microtiter plate (MTP). Optionally, the microtiter plate can also be configured to move. 
         [0012]    When using the invention, the densitometer would first be positioned with the various LEDs aligned over wells in the microtiter plate. Then, individual LEDs would be sequentially caused to emit light and associated detectors would measure luminous intensity transmitted through specimens in associated wells of the microtiter plate. Such data would then be processed, such as by storage in a database and correlated to the particular well in the microtiter plate. After each of the LEDs has been illuminated and an appropriate detection made by detectors associated with each LED, the entire densitometer can then be moved one well over and the complete process repeated. In this way, each well in the microtiter plate (MTP) is exposed to a different frequency of light and appropriate data is collected. A number of LEDs in each column determines how many samples can be simultaneously processed. The number of rows in the array of LEDs determines the number of separate frequencies of light which can provide separate data for the densitometer analysis. 
       OBJECTS OF THE INVENTION 
       [0013]    Accordingly, a primary object of the present invention is to provide a densitometer which can measure optical density rapidly for a large number of specimens and provide accurate optical density data at a variety of different frequencies. 
         [0014]    Another object of the present invention is to provide an optical density densitometer which works with a microtiter plate (MTP) and which exhibits a high duty cycle. 
         [0015]    Another object of the present invention is to provide a method for measuring optical density of a plurality of liquid specimens in a rapid and reliable manner 
         [0016]    Another object of the present invention is to provide a densitometer which has a simplified configuration and only requires a single degree of freedom in movement to measure a large number of specimens. 
         [0017]    Another object of the present invention is to provide a densitometer which only has a single light source illuminated adjacent a single specimen at a given time for maximum reliability of optical density measurement. 
         [0018]    Another object of the present invention is to provide a densitometer which simultaneously measures optical density of a large number of specimens and correlates optical density data for the plurality of specimens in an automatic fashion back to the individual specimens being measured. 
         [0019]    Other further objects of the present invention will become apparent from a careful reading of the included drawing figures, the claims and detailed description of the invention. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0020]      FIG. 1  is a schematic of a typical prior art densitometer system. 
           [0021]      FIG. 2  is a perspective view of the densitometer of this invention utilizing LEDs and in use with a microtiter plate (MTP) containing multiple specimens within wells thereof. 
           [0022]      FIG. 3  is a perspective view of the LED densitometer of this invention shown alone, and with one wing portion thereof removed to illustrate interior details of the densitometer. 
           [0023]      FIG. 4  is a perspective view of an alternative densitometer according to this invention. 
           [0024]      FIG. 5  is a perspective view of that which is shown in  FIG. 4 , and with portions cut away to reveal interior details. 
           [0025]      FIG. 6  is a detail of a portion of that which is shown in  FIG. 5  illustrating details along a light path of the densitometer of the alternative embodiment of  FIGS. 4 and 5 . 
       
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENT 
       [0026]    Referring to the drawings, wherein like reference numerals represent like parts throughout the various drawing figures, reference numeral  10  ( FIGS. 2 and 3 ) is directed to an LED densitometer for use with a microtiter plate MTP or other specimen supporting structure. The densitometer  10  uses LEDs  44  some of which can emit light at a common frequency and others which emit light at differing frequencies, the different frequencies being selected to match frequencies desired in conducting optical density measurements according to known densitometry techniques. 
         [0027]    In essence, and with particular reference to  FIG. 3 , basic details of the densitometer of this invention are described, according to a most preferred embodiment. In this embodiment, a mount wall  20  supports various portions of the densitometer  10  in a preferably substantially fixed manner. The mount wall  20  itself is movably carried through a carriage  14  upon a rail  12  ( FIG. 2 ) so that the densitometer  10  has one degree of freedom of movement horizontally along a length of the rail  12 , and relative to the microtiter plate MTP. 
         [0028]    A detector plate  30  extends from the mount wall  20  beneath a gap in which the microtiter plate MTP is adapted to be positioned. This detector plate  30  includes a plurality of detectors  32  thereon. A housing  40  is provided parallel with the detector plate  30  and above the detector plate  30 , and also mounted to the mount wall  20 , such as through a pair of wings  22  (only one of which is shown in  FIG. 3  for clarity, with both shown in  FIG. 2 ). This housing  40  supports a plurality of LEDs  44  therein facing downwardly towards the detectors  32  of the detector plate  30 . Each LED  44  is aligned with a detector  32 . Spacing between the LEDs  44  and between the detectors  32  is similar to spacing between wells W in the microtiter plate MTP ( FIG. 2 ). 
         [0029]    Circuitry powers the LEDs  44  and the detectors  32  with the system programmed preferably to cause individual LEDs  44  to be sequentially powered to cause light emission at the desired frequency, and an associated measurement from an associated detector  32 , for each position of the densitometer  10  relative to the microtiter plate MTP. The microtiter plate MTP can then be moved a distance of one well W spacing and the process repeated with the LEDs  44  aligned with next adjacent well W in the microtiter plate MTP, such that each of the specimens can have optical density measurements taken at each frequency available from the separate LEDs  44 . 
         [0030]    More specifically, and with continuing reference to  FIGS. 2 and 3 , details of the support system for the overall densitometer  10  are described. The densitometer  10  is simplified so that movement is only required in a single direction, horizontally along a long axis of the rail  12 . This rail  12  is preferably a rigid substantially square elongate linearly extending structure which can be mounted to a wall or other support, preferably in a horizontal fashion. A carriage  14  is coupled to the rail  12  and is adapted to transit along a long axis of the rail  12  in a horizontal direction. Power supplied to the densitometer  10  would typically have appropriate slack and mounting to maintain electrical connection for all different positions of the densitometer  10  upon the rail  12 . If the microtiter plate MTP has more columns of wells W than the housing  40  has LEDs, the microtiter plate MTP can be supported in a manner allowing it to be more perpendicular to the rail  12  so that specimens in wells can all be measured. 
         [0031]    With continuing reference to  FIGS. 2 and 3 , details of the mount wall  20  are described according to a most preferred embodiment. The mount wall  20  is coupled to the carriage  14  and secures both the detector plate  30  or other lower structure with the housing  40  or other upper structure. This mount wall  20  is preferably a rigid mass secured to the carriage  14 . The mount wall  20  is configured to support the detector plate  30  and housing  40  rigidly to the mount wall  20 . For such attachment, most preferably a pair of wings  22  are mounted to edges of the mount wall  20 . These wings  22  transition into arms  48  overlying a gap between the upper structure and lower structure of the densitometer  10 . 
         [0032]    With continuing reference to  FIGS. 2 and 3 , details of the detector plate  30 , defining a preferred form of lower structure are described according to this preferred embodiment. The detector plate  30  is preferably in the form of a printed circuit board mounted directly to an underside of the mount wall  20  and to under portions of the wing  22  adjacent the mount wall  20 . The detector plate  30  extends substantially horizontally away from the mount wall  20 , with a long axis of the detector plate  30  substantially perpendicular to a long axis of the rail  12 . 
         [0033]    By configuring the detector plate  30  as a printed circuit board, the detectors  32  can be surface mounted on the printed circuit board and appropriate circuitry can be formed on the printed circuit board in the form of the detector plate  30 . Such a structure minimizes mass of the lower structure in the form of the detector plate  30 , minimizing forces associated with movement of the densitometer  10  along the rail  12 . 
         [0034]    The detectors  32  can be passive in that they do not require any power and generate a signal when light is incident upon the detectors  32 . Alternatively, the detectors  32  can be of a type which are continuously powered and send an appropriate signal correlating with an amount of light incident thereon during operation of the densitometer  10 . 
         [0035]    Each of the detectors  32  have a particular address. A controller, such as in the form of a microprocessor is able to receive a signal from each detector  32  correlating with light intensity and from which optical density of a specimen can be calculated for a particular wavelength associated with the LED  44  associated with each detector  32 . Such signals can be received from the detectors and then stored in a database associated with the microprocessor. This database can also include particular information relating to the specimen within the particular well of the microtiter plate MTP associated with the detector  32 . The densitometer  10  can thus load data into this database relating to optical density of a specimen or array of specimens being examined. 
         [0036]    While the lower structure is preferably in the form of a detector plate  30 , it is conceivable that the detector plate  30  and housing  40  could be swapped so that the lower structure would in fact be the housing  40  supporting the LEDs  44  thereon. As another alternative, some other form of structure could be provided supporting the LEDs  44  and functioning as the lower structure of the densitometer  10 . 
         [0037]    With continuing reference to  FIGS. 2 and 3 , details of the housing  40  as a preferred form of upper structure, are described to this preferred embodiment. The housing  40  resides between two arms  48  extending from the wings  22 . This housing  40  includes an upper plate  42  which is preferably in the form of a printed circuit board supporting the LEDs  44  thereon on an undersurface thereof. A support  46  is preferably provided which both holds the upper plate  42  at an upper portion of the housing  40 , and also supports the LEDs  44  below the upper plate  42  and within the housing  40 . This support  46  preferably has a plurality of openings therein with each opening aligned with one of the LEDs  44 , so that the LEDs  44  can shine light down through the support  46  and toward the detectors  32 . If desired, appropriate lenses or filters can be provided in these openings in the support  46  to focus the light and/or provide further filtering to keep the frequency of the light preferably between +5 and −5 nanometers of a desired frequency for measurement of optical density. 
         [0038]    Each LED preferably has a base  45  mounted to the upper plate  42  and a tip  47  opposite the base  45  which is adjacent holes in the support  46 . This base  45  of each LED  44  is preferably surface mounted on the upper plate  42  with the upper plate  42  in the form of a printed circuit board. Power to the printed circuit board and associated circuitry on the printed circuit board defining the upper plate  42  can send an appropriate driving signal to the LEDs  44 , preferably sequentially in a manner directed by a microprocessor controller associated with the LEDs  44 . 
         [0039]    In a most preferred form of the invention, a program is executed to drive the LEDs  44  so that the LEDs  44  do not fire simultaneously, but rather sequentially for each position of the densitometer  10  relative to the microtiter plate MTP. With this firing sequence for the LEDs  44 , optical density of each specimen is being read when only one LED  44  is illuminated and only one detector  32  associated with the illuminated LED  44  is generating an optical density correlated signal. In this way, the potential for a misreading due to light from other sources during detection is eliminated. 
         [0040]    In the form of the invention particularly shown herein, the upper plate  42  includes a 4×8 array of LEDs  44 . Eight LEDs  44  are provided in each column and with four rows of such columns. Each column of LEDs  44  preferably has a similar frequency of light emitted thereby. Each LED in a common row preferably has a different frequency of light emitted thereby. In this way, and with a 4×8 array of LEDs  44 , eight specimens within eight separate wells W of the microtiter plate MTP can be substantially simultaneously analyzed. Furthermore, a total of thirty-two specimens can be analyzed at four frequencies. The densitometer  10  can then be advanced along the rail  12  one well W position, and the process repeated. Three-fourths of the wells will receive an additional optical density reading at a different frequency and a new column of wells W on the microtiter plate MTP will have a first set of data generated correlating with optical density. The densitometer  10  can then move ahead another well W position and again repeat this process. 
         [0041]    At each position, the LEDs  44  and detectors  32  can rapidly detect optical density so that a data set is rapidly collected for a large number of specimens. The size of the microtiter plate MTP and the size of the arrays of LEDs  44  and detectors  32  could be adjusted up or down from this example. If the LEDs  44  have an undesirably long time required to illuminate and dissipate light from the LEDs, the firing pattern for the LEDs can be arranged so that LEDs  44  adjacent each other are not sequentially fired, but rather LEDs  44  having significant distance therebetween would be sequentially fired until all thirty-two LEDs  44  (in this example) have fired, so that the optical density readings can be obtained as quickly as possible while not affecting the quality of the optical density data being read. 
         [0042]    While the densitometer  10  of this embodiment is shown with movement in a single direction, it is conceivable that a carrier for the densitometer  10  could facilitate movement of the densitometer  10  in two degrees of freedom perpendicular to each other and for a lesser number of LEDs  44  to be provided and/or a greater number of wells W within the microtiter plate MTP to be provided, and merely move the densitometer  10  in two mutually perpendicular directions, or the densitometer  10  in one direction and the microtiter plate MTP in one opposing direction, to gather the readings required for each of the specimens within the microtiter plate MTP. 
         [0043]      FIGS. 4-6  show an alternative densitometer  110  provided according to this invention. With this alternative densitometer  110 , rather than providing a two-dimensional array of LEDs, most preferably a single LED  130  is provided for each frequency of interest while as few as a single LED could conceivably be provided. In this particular example densitometer  110 , five such LEDs  130  are provided. Details of the carriage of the alternative densitometer  110  are similar to those of the densitometer  10  described in detail above. However, the housing  40  ( FIGS. 1-3 ) is slightly larger and interior details are modified somewhat. In particular, and within this larger housing  40 , providing a preferred form of first structure for the densitometer  110 , a pair of printed circuit boards are provided including an upper printed circuit board  120  and a middle printed circuit board  160 . 
         [0044]    LEDs  130  are surface mounted to the upper printed circuit board  120  with the LEDs  130  oriented to direct light downward from the upper printed circuit board  120 . The upper printed circuit board  120  can be supported laterally adjacent the arm  48  of the housing  40 . Furthermore, standoffs can be provided to space the upper printed circuit board  120  from the middle printed circuit board  160  and other structures within the housing  40 . 
         [0045]    Beneath the LED  130 , preferably a block  150  is provided, such as a solid aluminum block. This block  150  includes a cavity  152  therein sized to receive a band pass filter  140  therein. This cavity  152  is accessed through an upper aperture  154  directly below the LED  130  and a lower aperture  156  at an end of the cavity  152  below the upper aperture  154  and on an opposite surface of the block  150  relative to the upper aperture  154 . 
         [0046]    The band pass filter  140  is preferably a filter of a type which only allows light of a particular narrow band of light frequencies to pass therethrough. In this way, an LED having a broader than desired bandwidth of frequencies can be utilized and still the densitometer  110  can focus on particular frequencies of interest when performing optical density measurements. Preferably, the singular block  150  includes multiple cavities  152 , one cavity  152  for each of the five LEDs  130  shown in this particular embodiment of the densitometer  110 . Five LEDs  130  are surface mounted on the upper printed circuit board  120  directing light down into a separate band pass filter  150  within the separate cavities  152  within the block  150 . If an LED  130  is created or exists which has a narrow bandwidth of appropriate frequency, the band pass filter  140  could be omitted. Similarly, if the densitometer protocols do not require as specific and/or narrow a frequency, the filter  140  could be omitted. The filter  140  could also conceivably be integrated into the LED  130  itself. 
         [0047]    A middle printed circuit board  160  is oriented substantially parallel with and below the upper printed circuit board  120  and below the block  150 . The middle printed circuit board  160  has a hole  165  therein along a light path below the LED  130  and below the band pass filter  140 , and aligned with the upper aperture  154  and lower aperture  156  adjacent the cavity  152  of the block  150 . A photo detector  170  is preferably located adjacent this hole  165  in the middle printed circuit board  160 . The photo detector  170  is adapted to measure intensity of light adjacent the hole  165  in the middle printed circuit board  160 . 
         [0048]    This light intensity is measured before the light has passed through a sample being measured by the densitometer  110 . This photo detector  170  is preferably a portion of a feedback and control loop coupled to a power supply for an associated LED  130 . The LED  130  is preferably configured within a circuit which can adjust power to the LED in a manner causing light intensity emitted by the LED to be increased or decreased. If the photo detector  170  detects light intensity passing through the hole  165  which is greater or less than desired, an appropriate control signal is fed back to the power supply for the associated LED  130  and modified so that the LED  130  will emit light at a desired and controlled intensity. 
         [0049]    This feedback and control loop is important in that LEDs can vary in the amount of light intensity provided, and this variability can change with temperature and age of the LED, as well as other environmental factors. By providing this feedback and control loop associated with the photo detector  170 , such variability in the LED is eliminated for reliable measurements by the densitometer  110 . 
         [0050]    A lens cavity  180  is provided below the hole  165  in the middle printed circuit board  160 . This lens cavity  180  can support a lens above a lens aperture  185  formed in the support  46  of the housing  40 . The lens within the lens cavity  180  preferably sufficiently focuses light from the LED  130  to cause the light to remain substantially as a column until it reaches the detector  190  on the lower printed circuit board  200  associated with the second structure of the densitometer  110  and similar to the lower plate  30  of the densitometer  10  described in detail above. 
         [0051]    The detector  190  preferably is in the form of five detectors  190 , one for each of the LEDs, and with each detector  190  having a variable resister  195  associated therewith for calibrating an operational range of the detectors  190  before their use. In particular, this variable resister  195  is configured so that it can be adjusted so that when no light absorbing objects are placed between the first structure and second structure, such a detector  190  is caused to be at an upper extent of its operational range from the pure light from the LED  130  striking the detector  190  without any absorption thereof. In this way, a maximum signal range can be obtained for each detector  190 . 
         [0052]    When sample supports such as microtiter plates MTP ( FIG. 2 ) are utilized which have an array of wells W therein, the microtiter plate MTP or other sample support can be configured to control positioning of the microtiter plate MTP in an X direction, while the rail  12  and carriage  14  cause the densitometer  10 ,  110  to move in a Y direction. In this way, each LED of the alternate densitometer  110  can be positioned over each well W in a two-dimensional microtiter plate. 
         [0053]    This disclosure is provided to reveal a preferred embodiment of the invention and a best mode for practicing the invention. Having thus described the invention in this way, it should be apparent that various different modifications can be made to the preferred embodiment without departing from the scope and spirit of this invention disclosure. When structures are identified as a means to perform a function, the identification is intended to include all structures which can perform the function specified. When structures of this invention are identified as being coupled together, such language should be interpreted broadly to include the structures being coupled directly together or coupled together through intervening structures. Such coupling could be permanent or temporary and either in a rigid fashion or in a fashion which allows pivoting, sliding or other relative motion while still providing some form of attachment, unless specifically restricted.