Abstract:
A system and method are provided to observe and count particles in polydisperse solutions with dark field microscopy while distinguishing among particles of different sizes and accurately counting particles. A calibration mask, calibration light source, and multiple wavelengths of light are used. Opaque calibration marks on the transparent calibration mask define a region of interest. Multiple beams of various wavelengths are combined into a beam or a light sheet and the perpendicular component of scattered light from the specimen particles is then split into separate wavelengths and detected by separate sensors attuned to each wavelength. By calibrating the region of interest and measuring rotational and translational differences between images captured by differing sensors, the images may be aligned exactly and merged, enabling: i) removal of duplicate particles which yields more accurate particle counts, ii) more accurate estimation of the examined volume, and iii) accurate particle concentration measurements.

Description:
RELATED APPLICATIONS 
       [0001]    This application is also related to U.S. Provisional Patent Application No. 62/007,312, filed on Jun. 3, 2014, titled “Nanoparticle Analyzer,” the disclosure of which is herein incorporated by reference in its entirety, and U.S. patent application Ser. No. 14/730,138, filed on Jun. 3, 2015, titled “Nanoparticle Analyzer,” the disclosure of which is herein incorporated by reference in its entirety. 
     
    
     TECHNICAL FIELD 
       [0002]    The present invention relates to a system for detecting electromagnetic radiation, and more particularly to the observation of nanoparticles in liquid samples using a microscope equipped with digital video cameras/sensors. 
       BACKGROUND 
       [0003]    Nanoparticles are ubiquitous and by far the most abundant particle-like entities in natural environments on Earth and are widespread across many applications associated with human activities. There are many types of naturally occurring nanoparticles and man-made (engineered) nanoparticles. Nanoparticles occur in air, aquatic environments, rain water, drinking water, bio-fluids, pharmaceuticals, drug delivery and therapeutic products, and a broad range of many industrial products. Nanoparticles usually occur within polydisperse assemblages which are characterized by co-occurrence of differently-sized particles 
         [0004]    Given the widespread usage of nanoparticles, the ability to control and accurately characterize their properties may be useful to many applications. Conventional methods for measuring nanoparticle properties may be inaccurate for polydisperse samples of mixed nanoparticle sizes, which are common in many applications. Some of these conventional approaches make measurements on an ensemble of a large number of nanoparticles within a sample. Because the light scattered from all nanoparticles is measured simultaneously, it may be difficult to resolve the nanoparticles into their constituent sizes when there is a range of particle sizes. Other approaches fail to account for the large differences in the intensity of scattered light produced by differently-sized nanoparticles across the range of nanoparticle sizes. In these approaches, the low scattering signals from small nanoparticles may be undetected, or the high scattering signals from larger nanoparticles can obscure the signals from smaller nanoparticles. As a result of these deficiencies, the concentration of nanoparticles of any given size, and hence the entire size distribution, can be subject to unknown error. 
         [0005]    These methods of detecting nanoparticles are commonly referred as dark field microscopy. The instrument to perform such an analysis typically comprises a small cell (for example a cuvette) that enables illumination of a liquid with a precisely-defined, narrow light sheet and observation of scattered light from the nanoparticles, usually at a 90-degree angle relative to the light sheet plane. In other words, the direction of observation is perpendicular to the direction of the plane of illumination. Different sizes of particles can be visualized via the camera capturing light scattered by particles, with images having different sizes and intensities (various brightness of pixels) depending on the size of the particles. But as noted above, recording images of light scattered by particles of mixed sizes coexisting in a solution remains somewhat problematic due to the huge difference in the amount of light scattered by particles of different sizes. Specifically, the intensity of scattered light depends very strongly on particle size, changing by many orders of magnitude between 10 nm and 1000 nm diameter nanoparticles, for instance. This problem is also encountered in other areas of photography and videography and is commonly called High Dynamic Range (HDR) imaging. What has been needed is an improved system and method that overcomes the problems that tend to be inherent in dark field microscopy systems. 
         [0006]    In U.S. Published Patent Application No. 2015/0346076 A1 to Stramski et al., published Dec. 3, 2015 (“Stramski”), the entirety of which is incorporated herein by reference, these problems were addressed by using several light sources and a single color camera recording simultaneously several different colors of scattered light by the Bayer pattern of pixels corresponding to the three additive primary colors conventionally used in photography. In the Stramski approach, final images were obtained from a single recording device and hence images of the same colloidal volume at different colors were recorded in the same area of the recording device or sensor, thereby resulting in pixel numbering relative to a single point of origin, usually being one of the corners of a sensor in the camera. This made processing images in different colors possible because positions of observed particles were given in the same system of coordinates. However, when multiple sensors are used, the images may become unaligned relative to each other and Stramski did not address alignment. 
         [0007]    What is needed, therefore, is an improved system that overcomes or avoids the alignment problems presented by using multiple detectors that cannot easily be aligned to the exact same point. 
       SUMMARY 
       [0008]    The apparatus, systems, and methods described herein elegantly provide the benefits of simultaneous multi-spectral analysis in the Stramski system with a novel optical configuration that also provides other improvements and benefits such as automated calibration of the volume of investigation as will be apparent to persons of skill in the art. Provided in various example embodiments are apparatus, systems, and methods using a plurality of light sources and a plurality of greyscale cameras to simultaneously record different intensities of light scattered by various particles at different wavebands. Using the apparatus, systems, and methods herein one may accurately and precisely select the same region of interest (ROI) on all cameras, thus allowing for simultaneous recording of light scattered from exactly the same volume of illuminated sample with numbering of pixel coordinates related by known values in both directions, which is important when counting nanoparticles in order to determine their concentration in a colloid. 
         [0009]    Accordingly, provided in various example embodiments are apparatus, systems, and methods for detecting electromagnetic radiation of multiple wavelengths ( 10 ), examples of which may comprise: a first light source ( 15 ) constructed to emit a first beam of electromagnetic radiation at substantially a first wavelength ( 20 ); a second light source ( 25 ) constructed to emit a second beam of electromagnetic radiation at substantially a second wavelength ( 30 ); a combining structure ( 35 ) that merges the first and second beams into a combined beam ( 40 ), wherein the combined beam is directed at a specimen chamber ( 50 ), the chamber is constructed to allow a portion of the combined beam ( 40 ) to scatter the electromagnetic radiation in a direction perpendicular ( 55 ) to the combined beam ( 40 ). The specimen chamber ( 50 ) may further comprise a calibration mask ( 95 ), the mask including at least two calibration marks ( 100 ), with a calibration light source ( 110 ) constructed to emit a calibration beam ( 112 ) of electromagnetic radiation comprising the first and second wavelengths, wherein the calibration light source ( 100 ) directed at the calibration mask ( 95 ) emits its beam ( 112 ) parallel to the scattered portion of the combined beam ( 55 ). Then, the scattered portion of the combined beam ( 55 ) and the calibration beam ( 112 ) may be directed to a decombining (splitting) structure ( 65 ) that separates the electromagnetic radiation into the first wavelength radiation ( 70 ) and the second wavelength radiation ( 80 ). The separated first wavelength radiation ( 70 ) is directed to a first sensor ( 75 ) biased to detect electromagnetic radiation at substantially the first wavelength; the separated second wavelength radiation ( 80 ) is detected by a second sensor ( 85 ) biased to detect electromagnetic radiation at substantially the second wavelength; a processor ( 87 ) is connected to the first ( 75 ) and second ( 85 ) sensors, the processor ( 87 ) being configured to perform the following steps: when the calibration light source ( 110 ) is actuated, obtaining a first calibration image ( 115 ) from the first sensor ( 75 ) and a second calibration image ( 120 ) from the second sensor ( 85 ); detecting the calibration marks ( 100 ) from the calibration mask ( 95 ) in the first calibration image and in the calibration second image; determining a calibration correction ( 125 ,  130 ) based of the location of the calibration marks ( 100 ) in the first calibration image and the location of the calibration marks ( 100 ) in the second calibration image; when the first ( 15 ) and second ( 25 ) light source are actuated, obtaining a first detection image from the first sensor ( 75 ) and a second detection image from the second sensor ( 85 ); adjusting the detection images relative to each other based on the calibration correction ( 125 ,  130 ). 
         [0010]    In various example embodiments the processor may count the number of particles on the merged image after adjusting the images relative to each other. In various example embodiments the first and second light sources may be lasers. In various example embodiments the calibration mask may comprise a plate ( 105 ) that is transparent to the first and second wavelengths, and the calibration marks ( 100 ) may be opaque to the first ( 70 ) and second ( 80 ) wavelengths. In various example embodiments either one or both of the combining structure ( 35 ) and the decombining structure ( 65 ) may be a dichroic mirror. In various example embodiments the system may further comprise a light sheet former ( 45 ) that forms the combined beam ( 40 ) into a sheet of electromagnetic radiation, and that sheet may be directed at the specimen chamber ( 50 ). In various example embodiments the system may further comprise an imaging objective ( 60 ) that focuses the scattered beam ( 55 ) on the first and second sensors via the decombining structure ( 65 ). In various example embodiments the position of the decombining structure ( 65 ) can be adjusted ( 67 ) to change the direction of the separated second wavelength radiation ( 80 ) relative to the second sensor ( 85 ). In various example embodiments the position of the second sensor can be adjusted ( 90 ). In various example embodiments the processor ( 87 ) may determine the calibration correction ( 125 ,  130 ) by: aligning the calibration marks ( 100 ) from the first image to the second image; determining a rotational shift phi φ ( 125 ) between the first and second images; and determining a translational delta x and delta y ( 130 ) between the first and second images. In various example embodiments the processor ( 87 ) may be connected to the first ( 15 ) and second ( 25 ) light sources and the calibration light source ( 110 ), and the processor ( 87 ) may perform the following steps: actuating the calibration light source ( 110 ) prior to determining the calibration correction ( 125 ,  130 ); and actuating the first ( 15 ) and second ( 25 ) light sources prior to obtaining an image from the first sensor ( 115 ) and second sensor ( 120 ). In various example embodiments the decombining structure ( 65 ) may be connected to an electro-mechanical adjustor ( 67 ) that can change the position of the decombining structure ( 65 ), the adjustor ( 67 ) may be connected to the processor ( 87 ), and the processor ( 87 ) may actuate the adjustor ( 67 ) to determine the calibration correction ( 125 ,  130 ). In various example embodiments the second sensor ( 85 ) may be connected to an electro-mechanical adjustor ( 90 ) that can change the position of the second sensor ( 85 ), the adjustor ( 90 ) may be connected to the processor ( 87 ), and the processor ( 87 ) may actuate the adjustor ( 90 ) to determine the calibration correction ( 125 ,  130 ). 
         [0011]    Additional aspects, alternatives and variations as would be apparent to persons of skill in the art are also disclosed herein and are specifically contemplated as included as part of the invention. The invention is set forth only in the claims as allowed by the patent office in this or related applications, and the following summary descriptions of certain examples are not in any way to limit, define or otherwise establish the scope of legal protection. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0012]    The invention can be better understood with reference to the following figures. The components within the figures are not necessarily to scale, emphasis instead being placed on clearly illustrating example aspects of the invention. In the figures, like reference numerals designate corresponding parts throughout the different views and/or embodiments. It will be understood that certain components and details may not appear in the figures to assist in more clearly describing the invention. 
           [0013]      FIG. 1  illustrates a system for detecting electromagnetic radiation of multiple wavelengths. 
           [0014]      FIG. 2  illustrates a calibration mask. 
           [0015]      FIG. 3A  illustrates a system for detecting electromagnetic radiation of two wavelengths using a calibration mask and a calibration light source. 
           [0016]      FIG. 3B  illustrates a system for detecting electromagnetic radiation of three wavelengths using a calibration mask and a calibration light source. 
           [0017]      FIG. 4A  illustrates the alignment of the calibration marks from two separate images. 
           [0018]      FIG. 4B  illustrates the alignment of the calibration marks from the merged image of  FIG. 4A  and a third calibration image. 
           [0019]      FIG. 5  is a flow chart detailing a method of determining a calibration correction in a system for detecting electromagnetic radiation of multiple wavelengths. 
           [0020]      FIG. 6  is a flow chart detailing a method of applying a calibration correction in a system for detecting electromagnetic radiation of multiple wavelengths. 
       
    
    
     DETAILED DESCRIPTION 
       [0021]    Reference is made herein to some specific examples of the present invention, including any best modes contemplated by the inventor for carrying out the invention. Examples of these specific embodiments are illustrated in the accompanying figures. While the invention is described in conjunction with these specific embodiments, it will be understood that it is not intended to limit the invention to the described or illustrated embodiments. To the contrary, it is intended to cover alternatives, modifications, and equivalents as may be included within the spirit and scope of the invention as defined by the appended claims. 
         [0022]    In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. Particular example embodiments of the present invention may be implemented without some or all of these specific details. In other instances, process operations well known to persons of skill in the art have not been described in detail in order not to obscure unnecessarily the present invention. Various techniques and mechanisms of the present invention will sometimes be described in singular form for clarity. However, it should be noted that some embodiments include multiple iterations of a technique or multiple mechanisms unless noted otherwise. Similarly, various steps of the methods shown and described herein are not necessarily performed in the order indicated, or performed at all in certain embodiments. Accordingly, some implementations of the methods discussed herein may include more or fewer steps than those shown or described. Further, the techniques and mechanisms of the present invention will sometimes describe a connection, relationship or communication between two or more entities. It should be noted that a connection or relationship between entities does not necessarily mean a direct, unimpeded connection, as a variety of other entities or processes may reside or occur between any two entities. Consequently, an indicated connection does not necessarily mean a direct, unimpeded connection unless otherwise noted. 
         [0023]    The following list of example features corresponds with  FIGS. 1-4  and is provided for ease of reference, where like reference numerals designate corresponding features throughout the specification and figures: 
         [0024]    A system for detecting electromagnetic radiation of multiple wavelengths  10 . 
         [0025]    An alternate system for detecting electromagnetic ration of multiple wavelength  10 A. 
         [0026]    First light source at a first wave length  15 . 
         [0027]    First beam of electromagnetic radiation at substantially a first wavelength  20 . 
         [0028]    Second light source at a second wavelength  25 . 
         [0029]    Second beam of electromagnetic radiation at substantially a second wavelength  30 . 
         [0030]    A third light source at a third wave length  32 . 
         [0031]    Third beam of electromagnetic radiation at substantially a third wavelength  34 . 
         [0032]    Combining structure/dichroic mirror  35 . 
         [0033]    A second combining structure/dichroic mirror  37 . 
         [0034]    Combined beam  40 . 
         [0035]    Light sheet former  45 . 
         [0036]    Specimen chamber/cuvette  50 . 
         [0037]    A portion of the third beam that scatters  55 . 
         [0038]    Imaging objective  60 . 
         [0039]    Decombining structure/dichroic mirror  65 . 
         [0040]    Rotation by an electromechanical adjustor  67 . 
         [0041]    Separated first wavelength radiation  70 . 
         [0042]    First sensor biased to detect electromagnetic radiation at substantially the first wave length  75 . 
         [0043]    Separated second wavelength radiation  80 . 
         [0044]    Second sensor biased to detect electromagnetic radiation at substantially the second wave length  85 . 
         [0045]    Separated third wavelength radiation  86 . 
         [0046]    Third sensor biased to detect electromagnetic radiation at substantially the third wave length  87 . 
         [0047]    Second decombining structure/dichroic mirror  88 . 
         [0048]    Processor  87 . 
         [0049]    Movement of sensor by electromechanical adjustor  90 . 
         [0050]    Calibration mask  95 . 
         [0051]    Calibration marks  100 . 
         [0052]    Transparent portion of calibration mask  105 . 
         [0053]    Calibration light source  110 . 
         [0054]    Calibration beam  112 . 
         [0055]    First calibration image  115 . 
         [0056]    Second calibration image  120 . 
         [0057]    Rotational shift phi φ  125 . 
         [0058]    Translational shift (or delta) X and delta Y  130 . 
         [0059]    Region of interest (ROI) and merged image  135 . 
         [0060]    Third calibration image  140 . 
         [0061]    Second rotational shift phi φ 2    145 . 
         [0062]    Second translational shift (or delta) X 2  and delta Y 2    150 . 
         [0063]    With reference to  FIG. 1 , in various example embodiments the apparatus, systems, and methods for detecting electromagnetic radiation of multiple wavelengths may comprise a first light source at a first wave length  15  and a second light source at a second wavelength  25 , such as two lasers with different beam colors or wavelengths. Each of these two beams is directed at a combining structure  35 , such as a dichroic mirror, which combines the beams from light sources  15 ,  25  into a single combined beam  40  and directs the combined beam  40  to an optical system such as a light sheet former  45 . The light sheet former  45  may comprise a cylindrical lens with long working distance objective that forms a very narrow sheet of illumination. The light sheet may be directed to a transparent specimen chamber  50  (such as a cuvette) that houses a colloid containing particles, i.e. nanoparticles (not shown). A portion of the combined beam that scatters  55  upon impacting the particles present in the colloid solution contained within the cuvette  50  has the same wavelengths as the illuminating light from the light sheet former  45 , and can typically be observed at 90-degree angle by focusing an imaging objective  60 , such as a microscope equipped with another long working distance objective. The scattered light exiting the imaging objective  60  is split into constituent wavelengths at a decombining structure  65  such as a second dichroic mirror, namely the separated first wavelength radiation  70  and the separated second wavelength radiation  80 , that may independently reach the two sensors  75 ,  85  (such as those disposed within digital grey-scale cameras), attuned to detect electromagnetic radiation at substantially the first and second wave lengths  15 ,  25 , respectively. The system can be easily extended into more wavelengths and more corresponding sensors  75 ,  85  by adding more pairs of appropriate dichroic mirrors  35 ,  65  to combine and split more wavelengths of illuminating light sources  15 ,  25 . Such an example is shown in  FIG. 3B , which illustrates a three wavelength system with a third light source at a third wave length  32 , that produces a third beam of electromagnetic radiation at substantially a third wavelength  34 , and a second combining structure/dichroic mirror  37 . On the detection side of the system  10 A, a second decombining structure/dichroic mirror  88 , separates a third wavelength radiation  86 , such that it can be detected by a third sensor biased to detect electromagnetic radiation at substantially the third wave length  87 . 
         [0064]    The decombining structure  65  may be moveable (for instance, tiltable along an axis perpendicular to the line of sight), as illustrated in  FIG. 3 . The viewing area of the second sensor  85  can be changed in relation to that of the first sensor  75 , for instance via rotation by an electromechanical adjustor  67 . A similar result can be obtained by moving the second sensor  85  perpendicularly to the direction of split imaging light beam  80 , for instance by electromechanical adjustor  90 . However, these methods, being mechanical in nature, might not achieve the accuracy desired. For example, typical digital sensors have pixels that are squares with sides 5 micrometers long. Hence, to get positioning of a given image space point within 1-pixel accuracy, one would need to move second sensor  85  with less than 5 micrometers absolute accuracy in two directions or tilt the mirror by much less than a one-degree angle. This accuracy may be hard to obtain and reliably maintain for extended periods of time. 
         [0065]    Other various example embodiments may be employed that tend to overcome the limitations of mechanical methods. For example, with reference to  FIGS. 2-6 , various example embodiments may use a special calibration mask  95  to precisely position the image from one sensor  75  in relation to the other sensor  85  electronically. The calibration mask  95  may be made from glass or another transparent material with several micro-points or calibration marks  100  etched or painted on its transparent surface  105 , as shown in  FIG. 2 . Using more than two calibration marks may render the final calibration more accurate, and easier to implement. In the non-limiting example shown in  FIG. 2 , the calibration marks  100  are arranged in a rectangular pattern, but a triangular or any other multi-point pattern could be just as effective. The calibration mask  95  may also have a calibration scale  102 . This can be helpful if the sensors are not perpendicular to the incoming beams  70 ,  80 . If this happens, then each sensor  75 ,  85  may have a slightly different number of length units per pixel (usually measured in nm/pixel). By adding two perpendicular calibration scales  102  between calibration marks  100 , the processor  87  can effectively account for this difference and enables calibration of field of view of each sensor  75 ,  85  separately and accurately. In fact, such scales enable calibration of any sensor that is being used in the system, even if they are not same or with an unknown size of a pixel, giving precise calibration of the viewable area on both sensors in units of length instead of pixels. As yet another alternative, the processor  87  may simply be programmed to know that the calibration mask  95  has, for example in  FIG. 2 , four calibration marks  100 , and may further know the relative distances between the marks. In this latter alternative, the mask  95  need not contain the calibration scale, and based on the preprogramed lengths, the processor  87  can may the appropriate adjustments to arrive at an appropriate calibration correction. 
         [0066]    Such a calibration mask  95  may be placed within the specimen chamber  50  and may either be illuminated by the scattered beam  55  formed when the light sheet formed via the combining structure  35  and the light sheet former  45  encounters the particles in the colloid solution contained within the specimen chamber  50 , or by a calibration beam  112  emitted by a calibration light source  110 , as shown in  FIG. 3A . Thus, when an image is captured by the sensor  75  or by the sensor  85 , the four points made on the image from the calibration marks  100  on the transparent plate  105  of the calibration mask  95  define a rectangular area smaller than those areas that are recorded by the sensors and that could be encompassed wholly by both sensors  75 ,  85 . Computer-detected images of points from the calibration plate or mask  95  define the very same area on both sensor images with single or even sub-pixel accuracy. This highly accurate limitation (by design) of the ROI  135  ensures that each sensor will be simultaneously examining exactly the same region of the sample.  FIG. 3B  illustrates a system employing three wavelengths of light. At this point, it may be necessary to adjust one or both calibration images such that the calibration marks  100  detected in each calibration image are in the same scale as each other. This is the scale adjustment and may be performed by using the calibrations scales  102 , and/or by the processor  87  being preprogramed to know the relative distances between the calibration marks  100 . 
         [0067]    By applying simple mathematical expressions relating pixel shifts between two or more images, one can relate the absolute positions of recorded images of light scattered by particles, thus assuring counting and tracking of the same particles on different images. This is the translational/rotational shift. More specifically, the mathematical relationship of pixel shifts between two or more images can be expressed as: 
         [0000]        x′=x  cos φ+ y  sin φ+Δ x   Equation (1)
 
         [0000]        y′=−x  sin φ+ y  cos φ+Δ y   Equation (2)
 
         [0068]    where (x, y) are the pixel numbers (coordinates) in two directions on one sensor  75  and (x′, y′) are the coordinates on the second sensor  85  with a translational shift (or delta) between the two images of (Δx, Δy)  130  and a rotational shift or tilt of angle φ  125 , as shown in  FIG. 4A . Therefore, the calibration correction may include a scale adjustment, a translational shift and a rotational shift. In some embodiments it may be preferable to use more than two calibration marks (such as three calibration marks as shown in  FIGS. 4A and 4B ), which would allow the system and method to more accurately identify and compare the marks between the images. 
         [0069]    The above listed values of shift and tilt between two images ( 115  and  120  in  FIG. 4A ) can be easily derived from geometrical positions of the ROI  135  corners and edges on the different images, which can be precisely determined by the computer detection software that is used for tracking particles on the recorded images.  FIG. 4B  illustrates this same method used to align the calibration marks with the third calibration image  140 , using a second rotational shift phi φ 2    145  and a second translational delta x 2  and delta y 2    150 . The aligned image from  FIG. 4A —i.e., the ROI  135  aligning the first and second calibration images ( 135 ,  145 )—is then aligned to the third calibration image  140 . This same technique can be used to align additional calibration images at different wavelengths. 
         [0070]      FIG. 5  enumerates steps of the method  500  by which the calibration correction (pixel shifting) may be determined accurately. In a first step  505 , the calibration mask  95  may be inserted into the specimen chamber  50 . Then, in step  510 , the calibration light source  110  is activated. Step  512 , wherein the first light source  15  and the second light source  25  are deactivated, can be optionally performed simultaneously with  510 . With the calibration light source  110  on from step  510 , a first image is obtained from the first sensor  75  in step  515 . A second image is obtained from the second sensor  85  in step  520 . Now with these two images captured from the calibration beam  112 , in step  525  the calibration marks  100  in each image is detected. The processor  87 , may optionally make a scale adjustment at step  527  by first determining whether there is a difference in scale between the images. If there is, then the scale adjustment is determined at step  529 . 
         [0071]    In the next step,  530 , the processor  87  aligns the calibration marks  100  from the first image with the calibration marks  100  on the second image by calculating pixel coordinates of each of the points from the calibration marks  100  in each of the images and calculating the translation of these points. Optionally, in place of step  530  or as part of step  530 , the processor  87  may facilitate alignment of the first image  115  with the second image  120  using the corners of the ROI  135  by actuating the rotational electromechanical adjustor  67  on the decombining element  65  to assist in aligning the marks  100 , as in step  535 , or by actuating the translational electromechanical adjustor  90  on the second sensor  85  to assist in aligning the marks  100 , as in step  540 . Because the rotational electromechanical adjustor  67  may be connected to the processor  87  and may in fact be actuated by the processor  87 , the processor  87  may directly know the rotation shift φ needed to shift the first calibration image to align it with the second calibration image, and vice versa. Equivalently, by keeping track of how many pixels or how much distance the translational electromechanical adjustor  90  (also actuated by the processor  87 ) had to move the sensor  85  to align the images, the processor  87  may directly know the translational shift. 
         [0072]    Based on the adjustments required in step  530  to align the marks  100  and thereby the ROI  135  between the two images, the rotational shift φ between the images can be determined (step  545 ), as well as the translational delta x and delta y between the images (step  550 ). By combining the information from steps  545  and  550 , the calibration correction of step  555  may be expressed through equations (1) and (2). This calibration correction may then be used to process images taken by the same sensors  75  and  85 , to identify duplicated particles detected and remove them from the tally, providing an accurate and reproducible estimation of the examined colloid volume. With more accurate particle tallies taken from an accurately defined corresponding colloid volume, the concentration of particles can be easily and effectively determined. Note that the calibration can be applied or performed after the specimen has been imaged—i.e., the calibration correction can be applied to the video and then processed. Also note that if the processor  87  determined that a scale adjust was necessary in steps  527  and  529 , then the images would also be adjusted by the scale adjustment when taking the particle count.  FIG. 6  illustrates the steps of the method  600  applying the calibration correction on images. After there is a specimen placed in the chamber  50 , (step  605 ), then the first and second light sources,  15  and  25  respectively, can be activated (step  610 ). It may optionally be desirable at this point to simultaneously deactivate the calibration light source  110 , as shown in step  615 . A first image is obtained from the first sensor  75  (step  620 ), and a second image is obtained from the second sensor  85  (step  625 ). Then, in step  630 , the first and second images can be adjusted relative to each other based on the calibration correction. Thus the first and second images can be merged ( 635 ) so that duplicate particles may be identified. Then, the number of particles in the specimen can be counted more accurately (step  640 ). 
         [0073]    Although exemplary embodiments and applications of the invention have been described herein including as described above and shown in the included example Figures, there is no intention that the invention be limited to these exemplary embodiments and applications or to the manner in which the exemplary embodiments and applications operate or are described herein. Indeed, many variations and modifications to the exemplary embodiments are possible as would be apparent to a person of ordinary skill in the art. The invention may include any device, structure, method, or functionality, as long as the resulting device, system or method falls within the scope of one of the claims that are allowed by the patent office based on this or any related patent application.