Patent Publication Number: US-2010118130-A1

Title: Digital imaging system

Description:
CROSS-REFERENCE TO RELATED APPLICATION(S) 
     This application claims benefit of priority from U.S. Provisional Patent Application No. 61/113,322, filed Nov. 11, 2008, which application is incorporated by reference herein in its entirety. 
    
    
     FIELD OF INVENTION 
     The present invention relates to a digital imaging system for image analysis of samples particularly in high throughput research. 
     BACKGROUND OF THE INVENTION 
     High throughput research (sometimes also referred to as combinatorial) has become a wide practice nowadays especially in pharmaceutical and chemical industry to speed up the chemical discovery process and formulation research respectively. One aspect that is of interest in such research is the nature of samples as determined by visual or other optical inspection. The ability to view and analyze such mixtures in a high throughput context can be important. 
     WO 2004/053468 describes a combinatorial technique for systematically studying variations in heterogeneous mixtures such as oil-in-water and water-in-oil type of emulsions using automated and high throughput techniques. The system comprises a vial receptacle, an image capturing device, a light source directed at the first location and programmable processor operatively coupled to the image capturing device to detect a behavior in a captured image of a sample for analyzing a plurality of samples containing a dispersion of one or more incompletely miscible components in a continuous fluid phase. The device is available as the digital imaging feature of Symyx Technologies&#39; Core Module tool. 
     SUMMARY OF THE INVENTION 
     The inventors have discovered that the imaging quality of the Symyx tool is not always adequate to detect important features of the samples being investigated. They have now discovered an improved data image system. 
     Thus, in one aspect, the invention is a digital imaging device for analysis of one or more samples. The device comprises: an image capturing device comprising a lens to capture the image of the one or more samples which are located in an image capturing chamber, a nest receptacle to hold the one or more samples during imaging, and at least one illuminating source to illuminate the one or more samples. The digital image analysis system further comprises one or more features selected from the following: (a) at least one side of an inner surface of the image capturing chamber is a light absorbing non-reflective surface; (b) at least a first portion of the lens of the image capturing device is covered and a second portion exposed to the interior surface of the image capturing chamber to capture image; (c) the nest receptacle is provided with a spacer having through holes for the spacer to fit in vial nest pins to provide elevation of the samples in the vial nest receptacle; (d) the illuminating source is placed exterior to a housing containing the image capturing chamber which housing is opaque except for at least one aperture wherein the illuminating source is placed at an angle to illuminate the image capturing chamber through the aperture; (e) the illuminating source is placed exterior to a housing containing the image capturing chamber which housing is opaque except for at least one aperture wherein the aperture is fitted with a diffuser for diffusing the illuminating source, and (f) the light is directed at the sample at an angle of about 70 to 110 degrees relative to the lens and a reflective material is placed on the opposite side of the sample from the illuminating source and the reflective material is covered entirely by the sample. 
     A method of analyzing digital image(s) of plurality of samples using the digital image analysis system as described above was implemented by receiving the one or more samples in the image capturing chamber and placing the sample receptacles in a field view of the image capturing device and on the vial nest receptacle, illuminating the sample receptacles with at least one illuminating source and capturing the image. The image is processed digitally using computer interface to provide data on the samples. The digital image of the sample is analyzed using the digital image analysis system. Multiple sample mixtures such as liquid and emulsion samples can be prepared at one time, and these samples can be screened for desired properties in an automated high throughput manner. 
     Although the details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below, other features, objects and advantages of the invention will be apparent from the description and drawings and from the claims. 
    
    
     
       DESCRIPTIONS OF THE DRAWINGS 
         FIG. 1  illustrates a screening system according to a prior art. 
         FIG. 2  illustrates inside view of an image capturing chamber and components of an image capturing device of a digital imaging system according to prior art. 
         FIG. 3  illustrates a view inside an image capturing chamber of a digital imaging system, according to one embodiment of the present invention. 
         FIG. 4  shows a view of a top portion of the image capturing chamber according to one embodiment of the present invention having a diffusing lens and illuminating source. 
         FIG. 5  illustrates a view according to one embodiment of the present invention of the lens with the lens partly exposed to the inner surface of the image capturing chamber 
         FIG. 6  shows a view of a light diffusing lens according to one embodiment of the present invention 
         FIG. 7  shows a view of a vial nest receptacle according to one embodiment of the present invention with a vial spacer. 
         FIG. 8A  shows a view of an exemplary digital image of a sample before implementing a light absorbing non-reflective surface. 
         FIG. 8B  shows a view of a digital image of the sample after implementing a light absorbing non-reflective surface. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The system and methods described herein can be operated to employ rapid liquid handling and high throughput screening techniques to prepare and analyze libraries of samples for optically-detected characteristics. “Samples” as used in the present invention are preferably fluid mixtures in a liquid phase, including heterogeneous or homogenous mixtures. The term “heterogeneous mixtures” used in the present invention refers to liquid samples that have composition of two or more substances differing one from another that are not chemically combined with each other and are capable of being separated. In particular the term ‘emulsions’ or ‘liquid emulsions’ used herein refers to a suspension of tiny droplets of one liquid in a second liquid or mixing two liquids that ordinarily do not mix well. The liquid emulsion samples tend to display different optical characteristics of behavior when two immiscible liquids are mixed such as separated components, forming of layers or phases or bands. Although the system and apparatus used herein can be employed to analyze samples that are in liquid phase, they can also be used to prepare and analyze solid and gaseous mixtures and combinations of different phases such as solid suspensions in liquid, gels and colloidal systems and the like. 
     The samples are typically prepared in moveable containers such as vials or cuvettes, which collectively form the library where they are also held for analysis. The images of the samples are captured using the image capturing device and processed digitally using computer aided image processing techniques to provide qualitative and quantitative data about the samples. The digital image analysis of the samples can provide information about the phase separation, band forming or layer formation when two dissimilar liquid mixtures or emulsions are formed. Stages of demixing of the liquid samples can also be identified. One may be able to determine such information as volume fraction, optical density, stability of the mixture and the like. 
     Referring to  FIGS. 1 and 2 , there is shown a prior art screening apparatus along with digital image system according to WO 04/053468 entitled “Image analysis of heterogenous mixtures” to analyze heterogeneous mixtures of samples for rapid and high throughput analysis. A screening apparatus  200  as illustrated in  FIG. 1  comprises an image capturing device  202  such as a digital camera or single-lens reflex (SLR) camera or digital camera or with a color video camera such as MuTech color video camera or a charged-coupled device (CCD) capable of capturing digital images. The image captured by the digital camera will generally consist of sample receptacles containing samples. 
     The screening apparatus  200  includes one or more robotic arms  204 A and  204 B to facilitate high-throughput preparation and screening of the libraries of samples. At least one of the robotic arms  204 A includes a vial gripper  206  to transport vials between a vial rack  207  and a location  208  that is within the field of view of digital camera  202 . Vial gripper  206  is capable of picking, holding and releasing one or more vials when robotic arm  204  is transporting vials between vial rack  207  and location  208 . As shown in  FIG. 1  a back panel  290  can be mounted behind location  208 , such that location  208  is positioned between back panel  290  and image capturing device  202 . The mechanism of vial gripper  206  is driven by motorized or pneumatic methods. Other robotic arms  204 B can be used to dispense components that are used to prepare the samples. For example, robotic arm  204 B can aspirate components from a component rack  209 , dispense the components into vials located in vial rack  207  and the components in the vials can then be mixed by a mixing apparatus  211 . In the prior art, mixing apparatus  211  is located on robotic arm  204 B. 
     As in  FIG. 1 , vial rack  207  is implemented to hold an array of vials  207   a , each vial containing one of the samples to be analyzed. Vial rack  207  can be located within screening apparatus  200  to decrease the time required for robotic arm  204  to move vials into and out of location  208 . The number of vials located within vial rack  207  varies based on the number of samples being analyzed. As mentioned above, robotic arm  204  positions sample vials at location  208  which can be situated at a focal point of the lens of digital camera  202 . 
     One or more light sources  212  are positioned to illuminate vials positioned at location  208 . This illuminating light is then reflected back into digital camera  202  to be captured in the form of a digital image. Two of light sources  212  are mounted on either side of digital camera  202 . In this assembly, light sources  212  provide polarized light to illuminate vials and the samples contained therein. 
     Screening apparatus  200  as in  FIG. 1  can also include a filter  214  such as a polarizing filter, mounted directly in front of the lens of digital camera  202  to eliminate unwanted light from the captured images. 
     The prior art screening apparatus  200  also includes computer system  220  which can be configured to control the operation of robotic arm  204 , light sources  212 , and digital camera  202 . 
     Screening apparatus  200  includes a housing  216  that is configured to exclude light from the surroundings, such that stray or ambient light cannot affect the digital images captured by digital camera  202 . Some or all of the elements described above can be located within housing  216 . For example, the sample being analyzed, the light sources, and the camera optics are preferably located within housing  216 , where they can be isolated from ambient light. By contrast, elements for which light exclusion is not crucial, such as computer system  220 , vial rack  207 , and robotic arm  204  can be, located outside of housing  216 . 
     One example of the prior art invention is presented in  FIG. 2  wherein an inner view of a location  208  is shown. This location includes a chamber which is an image capturing chamber ( 208 ) comprising an image capturing device  202  such as digital camera or video cameras for capturing the image of the samples. The resultant images that are captured by this device are the sample vials containing the liquid emulsions. The image capturing device  202  has the flexibility of receiving one or more accessories such as macro, zoom or other lenses for capturing images at different resolutions as needed or various filters intended to intensify or reduce various aspects of the optical signature. These accessories are normally provided by the supplier or optionally can be obtained separately from the markets. 
     The image capturing chamber  208  has an inner surface  218  and a vial nest receptacle  210  to receive the sample receptacle  207   a  for image analysis. The vial receptacle  210  has four vial pins  222  for the vials to rest in the vial nest receptacle. An image capturing chamber  208  of the prior art as illustrated in  FIG. 2  also has a weighing device  235  such as weighing balance beneath the vial nest receptacle  210  for weighing the samples when placed in the vial nest receptacle  210 . The image capturing chamber  208  has a pneumatically operated door  223  (not shown in  FIG. 2 ) situated on the upper surface of the image capturing chamber to isolate the image capturing chamber from the external light. 
     The screening system  200  is generally used to analyze a library of samples. The term library used in this specification refers to any matrix of sites, having two or more members, with parametric diversity between members arranged in such a way that physical processes such as synthesis, characterization and measurements can be carried out. Each library includes two or more members, each of which may be represented as a region in an arrangement (an array) of one or more regions. It can also include such as any number of members for example two or more preferably, four, ten, twenty, hundreds or even thousands or more members. The vial rack  207  typically holds one or more members of the library for analysis within the screening system  200 . The library design for a library of heterogeneous mixtures of liquid samples can be represented by a pie chart that displays the different components making up the sample. These library designs can be generated using computer implemented graphical design techniques. The output generated from the computer programs may include a list of mappings to be performed in preparing the library and will contain a recipe for each sample vial  207   a  that has to be prepared and analyzed in the digital image analysis system. 
     For digital image analysis of the sample vial  207   a , the system obtains  207   a  containing the sample from vial rack  207  by liquid handling arms  204  and transports it into the image capturing chamber  208  through the pneumatically operated top door  223  and places it within the view of the image capturing device  202  to capture the digital image of the sample vial. The image thus obtained is in the form of an array or matrix of pixel values that corresponds to the intensity of the light reaching the lens system  214  of the image capturing device  202 . Once the image of the vial  207   a  is captured, it is returned to the vial rack  207 , the image is fed to the computer  220  for further analysis of the sample  207   a . These digital images are used to generate qualitative and quantitative descriptions of the samples&#39; natures which include parameters such as band heights and band intensities if banding layers are observed. This information can then be used to provide a detailed analysis of sample  207   a.    
     Surprisingly the inventors have improved the data analysis system by using one or more image enhancement methods. Turning now to  FIG. 3 , there is shown an image analysis system  300  ( FIG. 3 ) constructed in accordance with the present invention. According to a first embodiment, the inventors have provided a light absorbing, non-reflective surface on at least a portion of the inner surface and preferably the entire inner surface  316  of the image capturing chamber  303  as shown in  FIG. 3 . The light-absorbing non-reflective surface  316  can be of a surface coated with a matte, dark or light-absorbing finish. Alternatively, the interior may have a removable absorbing surface attached by gluing or by fastening means such as using a hook and pile type fastener to cover the inner surface of the image capturing chamber  303 . The light absorbing non-reflective surface  316  preferably used according to the present invention is a black fabric either woven or non-woven type. Alternatively the light absorbing non-reflective covering  316  can be of black paper, or black card board of any ply thickness, or a black plastic sheet or a metal sheet or a black glass or a wooden board coated with black color. It is also possible to coat the inner sides of the image capturing chamber  316  with suitable non-reflective coating such as paint, or varnish or suitable black colors such as powder coating or electrochemical coating or spray painting. 
     According to a second embodiment of the present invention, the lens  310  of the image capturing device  302  is exposed partially through the light absorbing non-reflective surface  316  allowing very minimum portion of the lens  310  to be exposed inside the image capturing chamber  303  ( FIGS. 3 and 5 ). The light absorbing non-reflective surface  316  has a narrow slit  317  opening as indicated in  FIG. 5  that is covering the lens portion of the image capturing device  307  for minimum portion of the lens  310  to be exposed. The narrow slit opening in the non-reflective surface  316  according to one example such as a fabric cloth has a dimension corresponding to the length of the sample receptacle for capturing a sharp and clear image of the sample vial for further digital analysis. 
     In additional embodiments of exemplary improvements in the digital image analysis system  300  according the present invention the light illuminating source is exterior to a housing which is opaque except for where there is an aperture. The light from the illuminating sources shines into the image capture chamber through the aperture and is further characterized by (1) the illuminating source is placed at an angle to the aperture rather than directly through the aperture and/or (2) the aperture includes a diffuser.  FIG. 3  shows one such specific embodiment where the aperture is in a door to the chamber. However, a skilled worker would recognize that the aperture need not be in the door and the door need not be above the image capturing chamber. Thus the system  300  has the upper surface  323  of the image capturing chamber  303  in the housing  324  (shown in  FIG. 3 ) fitted with a pneumatically operated door  313  and a light diffuser  331  fitting the opening of the door  313  as shown in  FIG. 4  to diffuse light into the image capturing chamber  303 . The light diffuser  331  diffuses light and provides an illuminating source into the image capturing chamber and sample vial. The light diffuser  331  is preferably circular in shape fitting exactly in the opening of the door  313 . The light diffuser  331  as illustrated in  FIG. 6  has a dimension ranging between 75-90 mm in diameter and more preferably 90 mm. The light diffuser  331  can be of glass or plastic or acrylic sheets and the like. Typically the light diffusers used for example are the ones that are used to diffuse white fluorescent light. The two illuminating light sources  212  as illustrated in prior art  FIG. 1  are eliminated according to one aspect of the preferred embodiment of the present invention and provided with at least one light source  307  as illustrated in  FIGS. 3 and 4  to provide illumination into the image capturing chamber  303 . Preferably, the illuminating source  307  is fitted on the top portion near the door opening  313  at an oblique angle to illuminate the image capturing chamber ( FIG. 4 ) The light source  307  can be of any type such as lamps, and more preferably of LED type comprising a  16  element white LED source. Optionally the light source  307  can be light selected from visible light, ultraviolet light and infrared light. 
     As shown in  FIG. 3 , the light is at an angle relative to the lens  310 , the angle being between about 70 and 110 degrees and preferably 80 to 100 degrees and most preferably approximately a right angle. In addition, it is preferred that a reflective material be located on the opposite side of the sample from the incident light. Thus, in one preferred embodiment, a portion of the vial nest directly opposite from the incident light is painted white or covered with a highly reflective white coating and the rest of the vial nest is black. Preferably the incident light is from above and a white dot is added directly under the sample and is covered by the sample and the rest of the vial nest and spacer is black. This white dot reflects light upward through the sample from below and aids in distinguishing the interface between clear and opaque phases as well as increase the definition of the meniscus. 
     The image obtained prior to implementations of the preferred light absorbing non-reflective surface as illustrated in  FIG. 8A  was not complete and part of the base portion is not visible for thorough analysis of the sample. 
     Now, in a sixth embodiment, the inventors have found that providing a vial spacer  322  gives additional height for the sample vials  302   a  to be clearly visible in the image capturing device  302  as shown in  FIG. 8B . The spacer has four holes  341  ( FIG. 7 ) which can rest on the four vial pins  311  in the vial nest receptacle  310 . The vial spacer  322  has a dimension ranging between 0.4-0.8 mm in diameter, more preferably about 0.6 mm in diameter, and a thickness of about 1-1.5 mm. The vial spacer  322  as illustrated in  FIGS. 7 &amp; 8B  can be of any material such as a metal, plastic, cardboard, wood or any suitable material that provides additional height for the vial in the vial nest receptacle. Materials which reduce thermal transfer to the vial nest are advantageous. The vial spacer  322  can also be of any shape such as circular, square, rectangular, triangular. Specifically the vial spacer is a metal in the form of a disk. 
     The vial spacer  322  preferably has a light absorbing non-reflective surface  326  ( FIG. 7 ). The surface optionally can be of any coating such as a black coating or powder coating or painting to avoid any reflection of the light from the illuminating source  302  inside the chamber. This helps in better image quality that can be captured by the image capturing device. The vial pins  311  also has a surface coated with non-reflective black coating and preferably the vial pins  311  have shrink sleeving or tubing such as with black polypropylene (PP), or polyvinylchloride (PVC) film or a black tubing made of rubber or silicone as illustrated in  FIG. 9   b  to avoid any reflection from the illuminating source. 
     The image capturing chamber  303  optionally has a weighing device  312  such as a weighing balance. The weighing device  312  is placed at the base of the vial nest receptacle  310  where the sample vial  302   a  rests within the vial pins  311  attached to the vial nest receptacle for weighing the sample as shown in  FIG. 3 . 
     The digital image obtained from the image capturing device is generally in the form of a matrix with defined pixel values that correspond to the intensity of light reaching the lens of the image capturing device  302 . The illumination and image capture can be automated through the computer interface using a computer program. Alternatively some or all of the illumination and image capture can be carried out based on manual interaction with a user. For example, the settings of the image capturing device  302  such as aperture, zoom, ISO speed, exposure time, and stored image quality, can be determined and adjusted by the image capture program using the computer interface. These settings can be adjusted to improve the image quality. In the present invention, the computer program is suitably modified to receive data and make adjustments on such items as frame rate, gain and exposure settings to receive better quality images of the sample. The image thus obtained by the improvements set forth in the above description can help in performing more accurate analysis of the sample. 
     Although the invention has been described in detail for the purpose of illustration, it is understood that such detail is solely for that purpose, and variations can be made therein by those skilled in the art without departing from the spirit and scope of the invention which is defined by the following claims.