Patent Publication Number: US-2006001954-A1

Title: Crystal detection with scattered-light illumination and autofocus

Description:
BACKGROUND OF THE INVENTION  
      1. Field of the Invention  
      This invention is related in general to equipment and processes for high-throughout screening of biological samples. In particular, the invention consists of an optical microscope for detecting the formation of protein crystals in liquid droplets contained in a multi-well tray that is rapidly scanned through the microscope objective.  
      2. Description of the Related Art  
      In fast-throughput screening of biological samples, individual samples are loaded into separate wells of multi-well trays or plates, where they are treated with reagents (or otherwise processed) and screened for target results. In the case of proteins, they are typically crystallized out of a liquor and analyzed for molecular structure using X-ray diffraction techniques. For fast throughput, the protein solution is loaded as a droplet into each well of such a multi-well tray (typically a 96-well plate) and is processed through an incubation step designed to cause the precipitation of protein crystals. Each well is then analyzed by some means, typically an optical procedure, to identify the presence of crystals, which are then harvested and processed through an X-ray diffraction unit to characterize their molecular structure. This knowledge is then utilized to design drugs with specific therapeutic objectives.  
      The optical procedures utilized in the art to identify the presence of crystals in each well are microscopic techniques wherein the well is illuminated, the liquid droplet is imaged, and the presence of crystals is detected by some automatic process (typically based on numerical analysis of light intensity signals). For example, after a sample is imaged and the image is digitized to provide an optical density value for each image pixel (or other measure of light intensity), the information is used in conventional manner to identify and isolate each crystal within the sample, so that the crystals formed in the well can be counted for screening purposes.  
      It is critical that all imaging steps be carried out in focus for all wells in the trays, which are not constructed to optical standards of precision and may vary significantly in the vertical position of each well. Therefore, an autofocus mechanism is used to adjust the distance between the microscope objective and each sample well as the tray is scanned through a plane in front of the objective (or vice versa). A light source (typically a laser light which may or may not be the same as the illumination source used for imaging purposes) is used to provide the automatic focusing function by detecting the position of a reference point in each well (such as the bottom of the well or the corresponding underside of the tray) and adjusting the vertical position of the well relative to the microscope so that the droplet in the well is in focus. An empirical offset with respect to the reference point is normally used to focus the objective at a predetermined height within the droplet deemed appropriate to provide acceptable images throughout the multi-well tray. While the imaging function of the microscope is usually implemented from the top of the tray, the autofocus mechanism may be implemented from either side of the tray.  
      Two illumination techniques have been used in the art to illuminate the liquor and crystals contained in the wells for microscope imaging purposed, either from the top or the bottom of the multi-well tray (which is normally made of transparent material). The most common approach is bright-field illumination, wherein the sample is illuminated through the microscope objective with substantially well behaved light rays (collimated or in a well-defined cone) at a particular focal plane of the microscope objective. As illustrated in  FIG. 1 , the problem with this approach is the fact that the small amount of liquid in each well  10  of a multi-well plate, as a result of the surface tension of the liquid, forms a droplet  12  with a top curvature that makes the drop equivalent to an optical lens. Therefore, the light impinging on the surface  14  of the liquid droplet  12  at an angle of incidence equal to or greater than the critical angle is reflected internally away from the microscope objective and lost for the purpose of imaging the liquid sample. Since the curvature of the surface  14  of the liquid is greater around the wall of the well, standard bright-field illumination techniques produce images with a relatively dark outer ring that hinders the process of identifying and counting crystals present in the corresponding area of the well. As much as 30% of the droplet is lost as a result of the darker ring imaged by this type of bright-field illumination.  
      Another approach used in the art is dark-field illumination, which is produced by blocking the light beam exiting the sample in an intermediate focal plane such that only light diffracted around the block is seen by the camera. This enhances edges within the field, but much of the light is lost and substantially greater intensities of illumination need to be used. Otherwise, noisy images are produced. As is well understood in the art, dark-field illumination also does not work well when there is a significant separation of the sample from the microscope objective or when there is substantial bending of the beam by objects that are not of interest, such as by the liquid drop, as opposed to the protein crystals within the drop.  
      In order to overcome these problems, scattered light is produced for bright-field illumination by introducing a diffuser between the illumination source and the sample. By placing the diffuser as close as possible to the sample, the efficiency of illumination is substantially retained and the ring effect produced by the curvature of the liquid droplet is virtually eliminated. This is because light passing through the diffuser exits at many angles, so that there is substantial amount of light that does not pass the critical-angle threshold described above. While this approach is ideal for single-well measurements, it is not compatible with high-throughput systems because it does not permit autofocusing (which requires unscattered light for proper functioning). Therefore, a separate source of illumination needs to be used for the autofocus mechanism and it typically cannot be on the same side of the sample, which adds cost and complexity to the system.  
      Therefore, there is still a need for fast-throughput imaging system with autofocus that does not suffer from the problems outlined above. This invention is directed at providing such a system with a single source of illumination (or a double source from the same side of the sample) adapted to implement both the imaging and autofocus functions at a very rapid pace.  
     BRIEF SUMMARY OF THE INVENTION  
      According to one embodiment of the invention, a scatter-shutter element is used in combination with a single illumination source to alternately provide bright-field illumination for autofocus purposes and scatter-light illumination for imaging purposes. The scatter shutter consists of a transparent plate that becomes cloudy and produces scattered light practically instantaneously upon application of a voltage. Accordingly, as the sample tray is being scanned continuously through the microscope objective for data acquisition, the scatter shutter is intermittently deactivated to allow unscattered light to focus on the underside of the tray and produce autofocus signals, and then activated to produce diffuse light to better image the droplet in each well, either with or without the need to change additional optics positions. The timing of each step is synchronized so as to place the droplet in focus and centered within the field of view prior to energizing the scatter shutter and switching to the imaging mode.  
      According to another embodiment of the invention, a strobed arc-lamp source is used for image acquisition and a separate source, potentially a laser, is used for autofocus purposes and is kept on continuously as the sample tray is being scanned. When the scatter shutter is not energized, the laser beam produces the optical signals off the underside of the tray required to adjust the focal position of the well approaching the microscope objective. When the well is in focus and centered in the field, the scatter shutter is energized to produce diffuse light and the high-intensity arc lamp is also energized for a length of time sufficient to image the droplet in the well. In this case the dual sources ensure that no additional optics will need to change positions between focusing mode and imaging mode.  
      In both cases described above, the electronic scatter shutter allows one to alternately produce diffuse light for imaging drops or permit light to pass through for focusing, all with high speed and no moving parts which can induce vibration. Various other aspects and advantages of the invention will become clear from its description in the specification that follows and from the novel features particularly pointed out in the appended claims. Therefore, to the accomplishment of the objectives described above, this invention consists of the features hereinafter illustrated in the drawings, fully described in the detailed description of the preferred embodiment and particularly pointed out in the claims. However, such drawings and description disclose but one of the various ways in which the invention may be practiced.  
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       FIG. 1  is a simplified schematic representation, in elevational view, of a well of a conventional multi-well sample tray.  
       FIG. 2  is a schematic representation of the multi-well tray of  FIG. 1  illustrating droplets of protein solution in each well with various surface heights and degrees of formation of protein crystals.  
       FIG. 3  is a schematic illustration of an optical microscope for the sequential testing of crystal solutions contained in a multi-well tray, wherein a scatter shutter element is used to switch the system&#39;s illumination between bright-field and scattered modes according to the invention.  
       FIG. 4  is picture of a protein-solution droplet in a test well, imaged using conventional bright-field illumination and showing the dark ring produced by the curvature of the top surface of the droplet in the well.  
       FIG. 5  shows the same sample of  FIG. 4  imaged through the scatter shutter of the invention, thereby producing an image of greater clarity within which a crystal can be more clearly identified.  
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENT OF THE INVENTION  
      The inventive aspect of this disclosure lies in the idea of using an intermittent scatter plate in the optical axis of illumination of a microscope used for high-throughput testing of multi-well trays. The intermittent light-scatter function provided by the plate allows the alternate bright-field illumination required for the operation of conventional autofocusing mechanisms and the scatter-light illumination required for uniform imaging of the entire well. In addition, the fact that both modes of illumination may be implemented from the same side of the sample tray provides construction, maintenance and operational advantages over prior-art microscope systems used for rapid sample screening.  
      Referring to the drawings, wherein like parts are designated throughout with like numerals and symbols,  FIG. 2  illustrates in schematic cross-sectional view a multi-well plate  20  used to carry out high-throughput screening of protein crystal samples according to the invention. Each well  10  is aligned in a row that is scanned in front of a microscope objective  22  (or vice versa) for the purpose of imaging the droplet  12  contained in each well and identifying the presence of protein crystals  24  that may have formed in the well. As illustrated, the droplets form a lens structure in the wells by virtue of their curved top surfaces  14  and their height in the various wells are not necessarily the same because of the different amounts of liquid originally placed in the wells and the different effects produced by the incubation stage. The images of the wells are processed in conventional manner to carry out the crystal identification and counting functions. The tray  20  is mounted on a stage (not shown) that allows the continuous translation of the tray to successively position each well in the tray under the microscope objective  22 .  
       FIG. 3  illustrates an embodiment of the invention wherein a switching scatter-shutter plate  26  is introduced into the illumination system. The plate is activated intermittently by an electrical signal provided by a computer  28  in synchrony with the operation of an x,y-scanner  30  adapted to position each well  10  in optical alignment with the microscope objective  22 . From the bottom of the tray  20  (opposite to the imaging side), a light source  32  is used to project a beam that is collimated by a lens  34 , passed through a neutral-density filter  36 , and then through a beam splitter  38  toward the scatter shutter  26 . Under deactivated conditions, the scatter shutter  26  is transparent and the light through it is reflected off the underside  40  of the tray  20  and is redirected by the beam splitter and appropriate optics toward a conventional autofocus mechanism  44 . In turn, the signal generated by the autofocus mechanism  44  is used, through the computer  28 , to drive a z-scanner  46  to adjust the distance between the objective microscope  22  and the well  10  that is in in the process of being positioned under it, so that the microscope is focused at the desired level within the droplet  12  in the well.  
      Subsequent to the autofocus adjustment, the scatter shutter  26  is energized, thereby producing scattered light that illuminates the droplet  12  through the transparent bottom of the sample plate  20 . The scattered light illuminates substantially uniformly across the section of the droplet, thereby producing an image wherein differences in intensity signals correspond to structural boundaries, such as produced by the presence of a crystal  24  within the liquid droplet. The light is collected by the microscope objective  22  and viewed by an eyepiece or detected by a sensor or camera  48 . The data so acquired may be digitized in a conversion unit  50  and stored in the computer  28  for processing and/or viewing on a monitor  52 , either on line or after the scan is completed.  
      In one embodiment of the invention, the images are acquired by precisely timing the acquisition of sensor  48  over a small time period so that a clear picture is generated even though the sample tray  20  is in motion with respect to the microscope objective  22  and the sensor  48 . In another embodiment, the fast image acquisition is accomplished by strobing the light source  32 , which could be an arc lamp, laser, or LED. In this case, an additional source  54 , preferably a low-intensity laser, may be used on axis with the source  32  to illuminate the system for autofocusing purposes.  
      Using a solid state liquid crystal display shutter (such as Anteryon&#39;s Model LCP250) and conventional microscope hardware, stage mechanisms, optics, and processing equipment, it was possible to process images continuously at a rate of about 8,000 images per hour. The autofocus was of the continuous laser type and adapted to operate on reflections from the underside of the tray. An offset of 100 micrometers was used into the autofocus in order to focus the objective approximately in the center of the well drops. A HeNe laser source was used for autofocus purposes in combination with a Xenon flash lamp arc lamp for image acquisition. The arc lamp was strobed to provide illumination for about a 50 milliseconds period at about a 3 Hz frequency  
       FIGS. 4 and 5  illustrate typical images of protein solution droplets in one of a multi-well sample tray. The image of  FIG. 4  was acquired using conventional bright-field illumination. It clearly shows the dark ring produced by the curvature of the droplet in the well. As a result to the darker illumination within the ring, it is difficult to discern the presence of the crystal in the liquid.  FIG. 5  shows the same sample imaged through the scatter shutter of the invention. The image illustrates the greater clarity with which the crystal can be seen, which enables automatic analysis and screening of the samples.  
      It is understood that the concept of the invention could be implemented in similar fashion by any means that permitted the alternate illumination in bright-field mode and scattered-field mode. Thus, the idea could conceivably be implemented by a mechanism that allowed rapid switch between modes, or other equivalent means, but at substantially greater cost, complication, and loss of efficiency.  
      Therefore, various changes in the details, steps and components that have been described may be made by those skilled in the art within the principles and scope of the invention herein illustrated and defined in the appended claims. While the invention has been shown and described herein in what is believed to be the most practical and preferred embodiments, it is recognized that departures can be made therefrom within the scope of the invention, which is not to be limited to the details disclosed but is to be accorded the full scope of the claims so as to embrace any and all equivalent apparatus and methods.