Patent Publication Number: US-2012044339-A1

Title: Opto-fluidic microscope system with evaluation chambers

Description:
This application claims the benefit of provisional patent application No. 61/439,684, filed Feb. 4, 2011 and provisional patent No. 61/375,227, filed Aug. 19, 2010, which are hereby incorporated by reference herein in their entireties. 
    
    
     BACKGROUND 
     This relates generally to systems such as opto-fluidic microscope systems, and, more particularly, to using such systems to image and evaluate fluid samples containing cells and other specimens. 
     Opto-fluidic microscopes have been developed that can be used to generate images of cells and other biological specimens. The cells are suspended in a fluid. The fluid flows over a set of image sensor pixels in a channel. The image sensor pixels may be associated with an image sensor pixel array that is masked using a metal layer with a pattern of small holes. In a typical arrangement, the holes and corresponding image sensor pixels are arranged in a diagonal line that crosses the channel. As cells flow through the channel, image data from the pixels may be acquired and processed to form high-resolution images of the cells. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a diagram of an illustrative system for imaging and evaluating cells and other biological specimens in accordance with an embodiment of the present invention. 
         FIG. 2  is a cross-sectional side view of a portion of an image sensor pixel array of the type that may be used in a fluid channel in a system of the type shown in  FIG. 1  in accordance with an embodiment of the present invention. 
         FIG. 3  is a top view of an illustrative fluid channel having image pixels arranged in a line to form an imager in accordance with an embodiment of the present invention. 
         FIG. 4  is a cross-sectional diagram showing how image sensor pixels may be used to form a light sensor associated with a chamber in accordance with an embodiment of the present invention. 
         FIG. 5  is a top view of an illustrative system having multiple channels and multiple chambers in accordance with an embodiment of the present invention. 
         FIG. 6  is a cross-sectional end view of an illustrative chamber having an entrance port for receiving a sample in accordance with an embodiment of the present invention. 
         FIG. 7  is a cross-sectional end view of an illustrative chamber having a heater and a flow control electrode in accordance with an embodiment of the present invention. 
         FIG. 8  is a cross-sectional end view of an illustrative chamber having a light source and a reactant in accordance with an embodiment of the present invention. 
         FIG. 9  is a top view of an illustrative chamber showing how the chamber may be provided with regions having different reactant coatings or other individualized properties in accordance with an embodiment of the present invention. 
         FIG. 10  is a top view of an illustrative chamber showing how a reactant may be supplied from an ancillary chamber in accordance with an embodiment of the present invention. 
         FIG. 11  is a top view of an illustrative system in which a channel has multiple braches with multiple respective evaluation regions and in which controllable gate structures are used to control fluid flow within the system in accordance with an embodiment of the present invention. 
         FIG. 12  is a cross-sectional side view of a system in which a reactant is located at the beginning of a channel and in which an evaluation chamber is located at the end of the channel in accordance with an embodiment of the present invention. 
         FIG. 13  is a flow chart of illustrative steps involved in using a system with fluid channels and evaluation chambers to evaluate samples in accordance with an embodiment of the present invention. 
     
    
    
     DETAILED DESCRIPTION 
     A system of the type that may be used to image and otherwise evaluate cells and other samples such as biological specimens is shown in  FIG. 1 . As shown in  FIG. 1 , system  10  may include opto-fluidic microscope  12 . Microscope  12  may include an image sensor integrated circuit such as image sensor integrated circuit  34 . Image sensor integrated circuit  34  may be formed from a semiconductor substrate material such as silicon and may contain numerous image sensor pixels  36 . Complementary metal-oxide-semiconductor (CMOS) technology or other image sensor integrated circuit technologies may be used in forming image sensor pixels  36  and integrated circuit  34 . 
     Image sensor pixels  36  may form part of an array of image sensor pixels on image sensor integrated circuit  34  (e.g., a rectangular array). Some of the pixels may be actively used for gathering light. Other pixels may be inactive or may be omitted from the array during fabrication. In arrays in which fabricated pixels are to remain inactive, the inactive pixels may be covered with metal or other opaque materials, may be depowered, or may otherwise be inactivated. There may be any suitable number of pixels fabricated in integrated circuit  34  (e.g., tens, hundreds, thousands, millions, etc.). The number of active pixels in integrated circuit  34  may be tens, hundreds, thousands, or more). 
     Image sensor integrated circuit  34  may be covered with a transparent layer of material such as glass layer  28  or other covering layers. Layer  28  may, if desired, be colored or covered with filter coatings (e.g., coatings of one or more different colors to filter light). Image sensor pixels  36  may be covered with color filter layer  37 . Color filter layer  37  may be color filtering material formed individually on image sensor pixels  36  or applied as a flat planar coating covering the lower surface channel  16 . Color filter layer  37  may include with red filters, portions with blue color filters, portions having green color filers, portions having tiled color filters (e.g., tiled Bayer pattern filters, etc.). If desired, color filter layer  37  may include infrared-blocking filters, ultraviolet light blocking filters, visible-light-blocking-and-infrared-passing filters, etc. Structures such as standoffs  40  (e.g., polymer standoffs) may be used to elevate the lower surface of glass layer  28  from the upper surface of image sensor integrated circuit  34 . This forms one or more channels such as channels  16 . Channels  16  may have lateral dimensions (dimensions parallel to dimensions x and z in the example of  FIG. 1 ) of a millimeter or less (as an example). The length of each channel (the dimension of channel  16  along dimension y in the example of 
       FIG. 1 ) may be 1-10 mm, less than 10 mm, more than 10 mm, or other suitable length. Standoff structures  40  may be patterned to form sidewalls for channels such as channel  16 . 
     During operation, fluid flows through channel  16  as illustrated by arrows  20 . A fluid source such as source  14  may be used to introduce fluid into channel  16  through entrance port  24 . Fluid may, for example, be dispensed from a pipette, from a drop on top of port  24 , from a fluid-filled reservoir, from tubing that is coupled to an external pump, etc. Fluid may exit channel  16  through exit port  26  and may, if desired, be collected in reservoir  18 . Reservoirs (sometimes referred to as chambers) may also be formed within portions of channel  16 . 
     The rate at which fluid flows through channel  16  may be controlled using fluid flow rate control structures. Examples of fluid flow rate control structures that may be used in system  10  include pumps, electrodes, microelectromechanical systems (MEMS) devices, etc. If desired, structures such as these (e.g., MEMs structures or patterns of electrodes) may be used to form fluid flow control gates (i.e., structures that selectively block fluid flow or allow fluid to pass and/or that route fluid flow in particular directions). In the example of  FIG. 1 , channel  16  has been provided with electrodes such as electrodes  38 . By controlling the voltage applied across electrodes such as electrodes  38 , the flow rate of fluids in channel  16  such as ionic fluids may be controlled by control circuitry  42 . 
     Fluid  20  may contain cells such as cell  22  or other biological elements or particles. As cells such as cells  22  pass by sensor pixels  36 , image data may be acquired. In effect, the cell is “scanned” across the pattern of sensor pixels  36  in channel  16  in much the same way that a printed image is scanned in a fax machine. Control circuitry  42  (which may be implemented as external circuitry or as circuitry that is embedded within image sensor integrated circuit  34 ) may be used to process the image data that is acquired using sensor pixels  36 . Because the size of each image sensor pixel  36  is typically small (e.g., on the order of 0.5-5.6 microns or less in width), precise image data may be acquired. This allows high-resolution images of cells such as cell  22  to be produced. A typical cell may have dimensions on the order of 1-10 microns (as an example). Images of other samples (e.g., other biological specimens) may also be acquired in this way. Arrangements in which cells are imaged are sometimes described herein as an example. 
     During imaging operations, control circuit  42  (e.g., on-chip and/or off-chip control circuitry) may be used to control the operation of light source  32 . Light source  32  may be based on one or more lamps, light-emitting diodes, lasers, or other sources of light. Light source  32  may be a white light source or may contain one or more light-generating elements  32 - 1 ,  32 - 2 ,  32 - 3  . . .  32 -N that emit different colors of light. For example, light-source  32  may contain multiple light-emitting diodes of different colors or may contain white-light light-emitting diodes or other white light sources that are provided with different respective colored filters. Light source  32  may be configured to emit laser light of a desired frequency or combination of frequencies. If desired, layer  28  and layer  37  may be implemented using colored transparent material in one or more regions that serve as one or more color filters. In response to control signals from control circuitry  42 , light source  32  may produce light  30  of a desired color and intensity. Light  30  may pass through glass layer  28  to illuminate the sample in channel  16 . 
     A cross-sectional side view of illustrative image sensor pixels  36  is shown in  FIG. 2 . As shown in  FIG. 2 , image sensor pixels  36  on integrated circuit  34  may each include a corresponding photosensitive element such as photodiode  44 . Light guides such as light guide  46  may be used to concentrate incoming image light  50  into respective photodiodes  44 . Photodiodes  44  may each convert incoming light into corresponding electrical charge. Circuitry  48 , which may form part of control circuitry  42  of  FIG. 1 , may be used to convert the charge from photodiodes  44  into analog and/or digital image data. In a typical arrangement, data is acquired in frames. Control circuitry  42  may convert raw digital data from one or more acquired image data frames into images of cells  22 . 
     As shown in  FIG. 3 , pixels  36  in channel  16  may be arranged to form imager  54 . Pixels  36  may be arranged in a diagonal line that extends across the width of channel  16  or may be arranged in other suitable patterns. The use of a diagonal set of image acquisition pixels  36  in channel  16  may help improve resolution (i.e., lateral resolution in dimension x perpendicular to longitudinal axis  52 ), by increasing the number of pixels  36  per unit length in dimension x. The image acquisition pixels  36  in channel  16  (i.e., the imager sensor pixels) are sometimes referred to as forming an image acquisition region, image sensor, or imager. 
     Light source  32  may be adjusted to produce one or more different colors of light during image acquisition operations. Channels  16  in system  10  may be provided with one or more imagers  54 . The different colors of light may be used in gathering image data in different color channels. A different light color may be used in illuminating cells  22  as cells  22  pass respective imagers  54  in channel  16  by moving in direction  58  with the fluid in channel  16 . 
     In some situations, it may be desirable to mix fluid  20  and/or cells  22  with a reactant. Examples of reactants that may be introduced into channel  16  with fluid  20  and cells  22  include diluents (e.g., fluids such as ionic fluids), dyes (e.g., fluorescent dyes) or other chemical compounds, biological agents such as antigens, antibodies (e.g., antibodies with dye), etc. With one suitable arrangement, one or more reactants may be introduced within a portion of channel  16 . The portion of channel  16  that receives the reactant may be, for example, a portion of channel  16  that has been widened or a portion of channel  16  that has the same width as the rest of the channel. Portions of channel  16  (whether widened or having other shapes) that receive reactant or that may be used to introduce sample material into channel  16  are sometimes referred to herein as chambers. 
     A cross-sectional side view of an illustrative system having a chamber that has been provided with reactant is shown in  FIG. 4 . In system  10  of  FIG. 4 , a fluid sample can be introduced into channel  16  on image sensor integrated circuit substrate  34  through entrance port  24  in glass layer  28 . The fluid and associated particles within the fluid such as cell  22  may flow through channel  16  as illustrated by fluid flow arrow  20 . Imager  54  may be used to gather images of cell  22  as cell  22  passes over imager  54 . 
     Part of channel  16  may be used to form chamber  66 . Chamber  66  may be provided with reactant such as reactant  62  and/or components for evaluating samples such as cell  22 . As shown in  FIG. 4 , for example, reactant  62  such as a fluorescent dye or other reactant may be used to cover the lower surface and/or upper surface of chamber  66 . The lower surface of chamber  66  (i.e., the lower surface of channel  16 ) may have a pattern of image sensor pixels  36  that form one or more light sensors (e.g., one or more light meters) such as light sensor  60 . The image pixels that make up light sensor  60  may be used collectively (i.e., in a binned fashion) to improve noise performance and/or may be used individually (or in small groups associated with respective light sensors) to gather location-dependent light readings. Reactant  62  may be formed on or near the image sensor pixels  36  in chamber  66  and/or on the upper surface of channel  16  (as examples). When fluid and cells  22  reach chamber  66 , reactant  62  may react with the fluid and/or cells. For example, dye in layers  62  may dye the cells. 
     In the illustrative configuration of  FIG. 4 , upper portion  64  of chamber  16  has been provided with elements  64 - 1 ,  64 - 2 , . . .  64 -N. Elements  64 - 1 ,  64 - 2 , . . .  64 -N may be transparent colored filter elements that are arranged in a tiled fashion over the upper surface of chamber  66 . Each filter element may be used to filter light entering and/or exiting chamber  66 . For example, each filter element may be used to filter a white light illumination source, thereby illuminating the interior of chamber  66  with various different types of colored light. The sample within chamber  66  (e.g., the fluid containing dyed cells or other sample particles) may respond differently to different colors of light. For example, the sample may fluoresce in response to illumination with one color of light but not in response to another. The use of different colors of light to illuminate different portions of the sample with different wavelengths of interest can therefore be useful in analyzing the sample. Filter elements  37 - 1 ,  37 - 2 , . . .  37 -N may also be used to filter light emissions from within chamber  66 . Lower portion  37  of chamber  66  has been provided with elements  37 - 1 ,  37 - 2 , . . .  37 -N. Elements  37 - 1 ,  37 - 2 , . . .  37 -N may be transparent colored filter elements that are arranged in a tiled fashion over the upper surface of chamber  66 . Each filter element may be used to filter light entering light sensor  60 . For example, each filter element may be used to filter a white light illumination source, thereby illuminating the portions of light sensor  60  with various different types of colored light. The sample within chamber  66  (e.g., the fluid containing dyed cells or other sample particles) may respond differently to different colors of light. For example, the sample may fluoresce in response to illumination with one color of light but not in response to another. The collection of different colors of light using light sensor  60  can therefore be useful in analyzing the sample. Reactant  62  may be provided in a uniform coating over a sidewall, over a lower chamber surface, over an upper chamber surface, or in other suitable chamber regions. If desired, reactant  62  may be patterned. For example, some regions of a chamber may be coated with reactant and other regions of the chamber may be left uncoated. Different reactants may be provided in different regions (e.g., in a tiled pattern on the lower or upper surface of the chamber, etc.). Any suitable number of different reactants may be used within one chamber (e.g., one, two, three, four, more than four, etc.). 
     System  10  may have a channel pattern that routes fluid to multiple chambers  66 . Different chambers may be used, for example, to make different types of measurements (e.g., using different reactants, different illumination sources, different colors of illumination, different temperatures, etc.). An illustrative configuration for system  10  that has multiple chambers  66  and channel branches on a single image sensor array substrate  34  is shown in  FIG. 5 . As shown in the illustrative arrangement of  FIG. 5 , system  10  may include a chamber such as chamber  68  that serves as an entrance port for channel structures  16 . Channel structures  16  may include a channel that separates into multiple channel branches (i.e., channel structures  16  may include multiple interconnected channels). In the example of  FIG. 5 , channel structures  16  initially form a single channel at the exit of chamber  68 . This single channel then splits into respective left, center, and right channel branches  16 . 
     Channels  16  may be provided with chambers such as chambers  70 . Chambers  70  may contain reactant  72 . For example, chambers  70  may contain dilutant for diluting the sample flowing through each respective channel  16 . Other reactants may be provided in chambers  70  if desired such as dyes or other chemical compounds, biological agents such as antigens, antibodies (i.e., antibodies with dye), etc. 
     Each channel branch  16  may have one or more imagers  54  for gathering image data on the sample. At the end of each channel branch  16 , the sample may be evaluated using a respective evaluation chamber  66 . Each chamber  66  may, if desired, be provided with different capabilities for evaluating the sample. For example, the chamber associated with the left channel in  FIG. 5  may contain a first reactant, the chamber associated with the central channel in  FIG. 5  may contain a second reactant, and the chamber associated with the right channel in  FIG. 5  may contain a third reactant. The first, second, and third reactants may all be different. Each evaluation chamber may also contain additional reactants, may use different types of illumination, may use different light sensor schemes, and may otherwise have duplicative and/or independent capabilities from the other chambers in system  10 . 
     With a multichannel arrangement of the type shown in  FIG. 5 , a sample may be evaluated using different types of tests in different chambers. In one chamber, for example, a dye or tiled pattern of dyes may be present and light and light sensors may be used to make fluorescence measurements, whereas different types of measurements using different dyes, light colors, illumination intensities, and/or different environmental characteristics such as different temperatures may be made in other chambers. 
       FIGS. 6 ,  7 , and  8  are cross-sectional end views of illustrative types of chambers that may be used in implementing chambers in system  10 . As shown in  FIG. 6 , entrance chamber  68  may contain an entrance port. Samples may be introduced into chamber  68  for distribution to an array of channels  16 . 
       FIG. 7  shows how evaluation chamber  66  may be provided with a heater such as heater  74 . Heater  74  may be, for example, a resistive heater that is controlled by control circuitry  42  ( FIG. 1 ). During sample evaluation operations, heater  74  may be turned on and off to cycle the temperature in the interior of the chamber. Voltages may be applied to chambers such as chamber  66  of  FIG. 7  using electrodes such as electrode  38 . By controlling the voltages on electrodes  38  in chambers  66  and/or other channel structures in system  10 , the flow of sample fluids such as ionic fluids may be controlled. 
     As shown in the example of  FIG. 8 , chamber  66  may be provided with a light source such as light source  76  that produces light  78  of one or more different colors (using optional color filters in light source  76  and/or light filters integrated into the upper surface of chamber  66  in a pattern of the type shown in  FIG. 9 ). Reactant  62  may be provided on any of the exposed surfaces of chamber  66 . In the  FIG. 8  example, reactant  62  has been provided on a lower chamber surface (as an example). Image sensor pixels  36  may be used to form one or more image sensors  60 . Image sensor pixels  36  of image sensors  60  may be configured to receive light of various colors (using optional color filters over image sensor pixels  36  or integrated into the lower surface of chamber  66 ). 
     As shown in the illustrative chamber top view of  FIG. 9 , chambers  66  may be provided with upper portions  64  that have a pattern (e.g., a tiled pattern) of different sub-portions such as  16  illustrative subportions  64 - 1 ,  64 - 2 , . . .  64 - 16 . Each subportion may be provided with a different colored filter element, a different reactant coating, etc. 
       FIG. 10  is a top view of an illustrative chamber structure for system  10  in which reactant  62  is introduced into chamber  66  from an ancillary chamber (chamber  82 ). Reactant  62  may be a diluent, a dye or other chemical compound, a biological agent such as an antigen, antibodies (i.e., antibodies with dye), or other substance that reacts with samples that flow through channel  16  into chamber  66  in direction  84 . 
     Any or all of the features of chambers such as chamber  66  of  FIGS. 4 ,  6 ,  7 ,  8 ,  9 , and  10  may be combined to form one or more chambers  66  in system  10 . For example, a chamber may be formed that has electrodes  38 , reactant  62 , light source  76 , heater  74 , image sensor  60 , an ancillary chamber such as chamber  82  of  FIG. 10 , and patterned filters and/or patterned reactant that uses a pattern of the type shown in  FIG. 9 . The chamber layouts of  FIGS. 4 ,  6 ,  7 ,  8 ,  9 , and  10  are merely examples. 
       FIG. 11  shows how system  10  may have a rectilinear pattern of channels  16 . In the arrangement of  FIG. 11 , channels are separated from each other by gate structures such as gate structures  86 A and  86 B. Gate structures  86 A and  86 B may, for example, be formed from MEMs structures, electrode-based structures, or other structures that can selectively permit fluid to flow or block fluid from flowing. Electrodes such as electrodes  38  of  FIG. 1  or other fluid control mechanisms (e.g., MEMs structures, external pumps, etc.) may be used to cause the sample fluid to flow through channel  16 . Gate structures  86 A and  86 B may be used to route the flow of the sample. When closed, gate structure  86 A may prevent fluid from flowing in direction  90 . When gate structure  86 A is open (e.g., when gate structure  86 A is in the open position represented by dashed line  88 ), fluid may flow along path  90 . Gating structures  86 A and  86 B and other fluid flow control structures may be controlled by control circuitry  42 . When it is desired to direct fluid to flow along path  90 , gate structure  86 A may be placed in its open position and gate structure  86 B may be placed in its closed position. When it is desired to route fluid along path  92 , gate structure  86 A may be placed in its closed position and gate structure  86 B may be placed in its open position. 
     Fluid routing structures such as one or more gate structures may be used to cause samples to flow into different chambers  66 . For example, a sample may be introduced into channel  16  of  FIG. 11  in the vicinity of evaluation chamber  66 A. Following evaluation in chamber  66 A, electrodes or other flow control mechanisms may be used to direct the sample to flow past imager  54 A. Gate structures  86 A and  86 B may then be adjusted by control circuitry  42  to direct the sample to flow past imager  54 B into chamber  66 B and/or to flow past imager  54 C into chamber  66 C for evaluation. If desired, different patterns of channels, chambers, and gate structures may be used in evaluating samples. For example, channel  16  may be provided with additional branches, more or fewer chambers  66  may be used, etc. 
       FIG. 12  is a cross-sectional side view of an illustrative arrangement that may be used for system  10  in which reactant  62  is introduced near the beginning of channel  16  (i.e., in a location such as chamber  66 A that is upstream from a channel region such as chamber  102 ). Chamber  102  may contain image sensor pixels  36  for forming an imager  54  and/or one or more sensors  60 . Chamber  102  may also include a heater, a light source such as light source  76  for producing light  78 , color filters, one or more regions of reactant, etc. 
     In general, system  10  may have a channel that contains one or more branches and optional features such as one or more regions that contain reactant, light sensors, imagers, heaters, gating structures and other fluid control structures (e.g., flow rate control structures), illumination devices, etc. 
     Illustrative steps involved in using system  10  to evaluate samples are shown in  FIG. 13 . At step  94 , a sample of fluid such as a fluid containing cells or other particles may be introduced into channel  16  on image sensor array integrated circuit substrate  34 . For example, a sample may be placed in a channel region such as chamber  68  of  FIG. 5 . 
     At step  96 , optional dilutant may be combined with the sample to dilute the sample. For example, one or more dilutant chambers such as chambers  70  of  FIG. 5  may be used to add dilutant to the sample. If desired, other reactants may be added to the sample during the operations of step  96 . For example, dye, antigens, antibodies (e.g., antibodies with dye), or other reactants may be combined with the sample in channel  16  (e.g., using one or more reactant chambers such as chamber  66 A of  FIG. 12 ). 
     During the operations of step  98 , the flow of the sample throughout the branches and other portions of channel  16  may be controlled using flow control structures such as electrodes  38 , using gate structures such as gate structures  86 A and  86 B ( FIG. 11 ), etc. For example, the sample may be routed to different branches of channel  16  and different chambers  66  as described in connection with  FIG. 11 . In a system that contains multiple parallel branches of channel  16  as described in connection with  FIG. 5 , the sample may be routed in parallel to different respective evaluation chambers  66 . 
     At step  100 , chambers  66  may be used to evaluate the sample. For example, reactant in chambers  66  (which may be provided using a tiled pattern of the type shown in  FIG. 9  or in other suitable patterns) may react with the sample. One or more light sources such as light source  76  (and optionally color filters in layer  28 ) may be used to produce illumination for each chamber. The illumination may be provided in the form of white light or one or more different colors of light. Heaters such as heater  74  may be used to adjust the temperature of the sample during evaluation. The amount of light in chambers  66  may be evaluated using sensors  60 . For example, following illumination with a light source, sensors  60  may be used to detect fluorescence signals. A checkerboard pattern or other tiled pattern may be used for color filters, sensors  60 , and/or reactant within each chamber to allow information on the response of the sample to different colors and/or reactants to be measured. The data that is gathered during step  100  may be gathered and processed using control circuitry  42  (as an example). 
     Various embodiments have been described illustrating apparatus for imaging and evaluating samples of fluids containing cells and other materials. An integrated circuit such as an image sensor array integrated circuit may be provided with fluid channels. Sets of image sensor pixels from an image sensor array on the integrated circuit may form imagers in the fluid channels. A sample may be introduced into a channel for imaging by the imagers and for evaluation using other sample evaluation structures. Chambers may be provided for adding dilutant and other reactants such as dyes, antigens, antibodies, chemical compounds, and other materials to the sample fluid. The channel structures on the integrated circuit may have multiple branches. Flow control structures such as electrodes and gate structures such as microelectromechanical systems (MEMs) gate structures may be used to route fluid through various branches in the channel. For example, flow control structures may be used to route a sample to one or more different chambers for evaluation. Chambers in the channel may include reactant for reacting with the sample, a light source for providing illumination for the sample, a heater for heating the sample, and image sensor pixels. The image sensor pixels may be used in forming one or more light sensors in each chamber. 
     The foregoing is merely illustrative of the principles of this invention which can be practiced in other embodiments.