Focusing method

Abstract

A method and system are provided for focusing an imaging device on a liquid sample flowing through a field of view of the imaging device. Objects are segmented in the captured frames and used to account for the fact that the sample is flowing. Object velocities are calculated and used in selecting an appropriate focus value. The calculation of a focus measure takes account of the number of objects in captured frames in order to ensure a consistent calculation of the focus measure.

1 - 5 . (canceled) 
     
     
         6 . A method of focusing an imaging device on a liquid sample flowing through a field of view of the imaging device, the method comprising:
 a) stepping a focus mechanism of the imaging device through a plurality of focus values;   b) acquiring at least one frame at each of the plurality of focus values using the imaging device;   c) calculating a focus measure for each of the plurality of focus values from the respective at least one video frame;   d) selecting a plurality of respective focus values substantially at local extrema of the focus measure;   e) for each selected focus value, determining a respective object velocity for one or more objects in a respective plurality of frames;   f) selecting a focus value of the plurality of focus values based on each of the respective object velocities; and   g) focusing the imaging device using the selected focus value.   
     
     
         7 . A method as claimed in  claim 6 , wherein calculating a focus measure for each focus value includes detecting one or more objects in each respective frame; defining an image patch for each of the one or more objects and calculating the focus measure for at least one of the image patches in the respective frame. 
     
     
         8 . A method as claimed in  claim 7 , wherein calculating a focus measure for each focus value includes averaging the focus measure over objects. 
     
     
         9 . A method as claimed in  claim 6 , wherein step g) comprises:
 h) stepping the focus mechanism of the imaging device through a further plurality of focus values comprising at least one focus value above and at least one focus value below the selected focus value;   i) acquiring at least one video frame at each of the further plurality of focus values using the imaging device;   j) calculating a focus measure for each of the further plurality of focus values from the respective at least one video frame;   k) selecting a further focus value based on the focus measures for the further plurality of focus values; and   l) focusing the imaging device using the selected further focus value.   
     
     
         10 . A method as claimed in  claim 9 , wherein step l) comprising:
 m) repeating steps a) to k) to select at least one more further focus value;   n) combining the further focus values to derive a final focus value, and   o) setting the focus mechanism to the final focus value.   
     
     
         11 . A method as claimed in  claim 9 , wherein a step size between the further plurality of focus values is less than a step size between the plurality of focus values. 
     
     
         12 . A method as claimed in  claim 6 , wherein the sample flows in a microfluidic conduit. 
     
     
         13 - 17 . (canceled) 
     
     
         18 . An imaging system comprising:
 an imaging device for imaging a liquid sample flowing through a field of view of the imaging device, wherein the imaging device has a focus mechanism for positioning at least a portion of the imaging device in accordance with a focus value to form a focused image of one or more objects in the field of view; and   a processor configured to   a) cause stepping of the focus mechanism through a plurality of focus values;   b) acquire at least one frame at each of the plurality of focus values from the imaging device;   c) calculate a focus measure for each of the plurality of focus values from the respective at least one video frame;   d) select a plurality of respective focus values substantially at local extrema of the focus measure;   e) for each selected focus value, determining a respective object velocity for one or more objects in a respective plurality of frames;   f) select a focus value of the plurality of focus values based on the respective object velocities; and   g) focus the imaging device using the selected value.   
     
     
         19 . A system as claimed in  claim 18 , wherein calculating a focus measure for each focus value includes detecting one or more objects in each respective frame; defining an image patch for each of the one or more objects and calculating the focus measure for at least one of the image patches in each respective frame. 
     
     
         20 . A system as claimed in  claim 19 , wherein calculating a focus measure for each focus value includes averaging the focus measure over objects. 
     
     
         21 . A system as claimed in  claim 18 , wherein step g) comprises:
 h) stepping the focus mechanism of the imaging device through a further plurality of focus values comprising at least one focus value above and at least one focus value below the selected focus value;   i) acquiring at least one video frame at each of the further plurality of focus values from the imaging device;   j) calculating a focus measure for each of the further plurality of focus values from the respective at least one video frame;   k) selecting a further focus value based on the focus measures for the further plurality of focus values; and   l) focusing the imaging device using the selected further focus value.   
     
     
         22 . A system as claimed in  claim 21 , wherein step l) comprises:
 m) repeating steps a) to k) to select at least one more further focus value;   n) combining the further focus values to derive a final focus value; and   o) setting the focus mechanism to the final focus value.   
     
     
         23 . A system as claimed in  claim 18 , wherein the sample flows in a microfluidic conduit. 
     
     
         24 . A method as claimed in  claim 21  wherein a step size between the further plurality of focus values is less than a step size between the plurality of focus values. 
     
     
         25 - 27 . (canceled)