Method and system for imaging a cell sample

• US 10,176,565 B2
• Filed: 05/23/2014
• Issued: 01/08/2019
• Est. Priority Date: 05/23/2013
• Status: Active Grant

• Current Assignee: S.D. Sight Diagnostics Ltd
• Original Assignee: S.D. Sight Diagnostics Ltd
• Inventors: Greenfield, Yuval, Bilu, Yonatan, Pollak, Joseph Joel, Yorav-Raphael, Noam
• Primary Examiner(s): Czekaj, Dave
• Assistant Examiner(s): Brumfield, Shanika M
• Application Number:
  US14/285,672
• Publication Number:
  US 20140347459A1
• Time in Patent Office: 1,691 Days
• Field of Search: 348 79
• CPC Class Codes: G02B 21/16 adapted for ultraviolet illumination ; Fluorescence microscopes G02B21/0076 takes precedence
• CPC Class Codes: G02B 21/244 using image analysis techniques
• CPC Class Codes: G02B 21/361 Optical details, e.g. image relay to the camera or image sensor G02B21/364 takes precedence; illumination details G02B21/06 and subgroups
• CPC Class Codes: G02B 21/367 providing an output produced by processing a plurality of individual source images, e.g. image tiling, montage, composite images, depth sectioning, image comparison
• CPC Class Codes: G02B 7/38 measured at different points on the optical axis , e.g. focussing on two or more planes and comparing image data
• CPC Class Codes: G06T 2207/10056 Microscopic image
• CPC Class Codes: G06T 2207/30024 Cell structures in vitro; Tissue sections in vitro
• CPC Class Codes: G06T 7/0012 Biomedical image inspection
• CPC Class Codes: G06T 7/507 from shading G06T7/586 takes precedence

Abstract

The present disclosure provides a method of determining a reference depth level within a cell sample. The method comprises obtaining data representative of a series of images captured by performing a depth scan of the cell sample using a digital microscope, the series of images being associated with a series of depth levels of the cell sample; processing said data for detecting at least one depth level corresponding to a drop in image contrast; and identifying the detected depth level as the reference depth level.

Claims

1. A method for use with a cell sample, the method comprising:

obtaining data representative of a series of images captured by performing a depth scan of the cell sample using a microscope, the series of images being associated with a series of depth levels of the cell sample;

identifying one of the depth levels as being an optimum focal plane for imaging one or more entities within the sample using the microscope, by;

detecting a plurality of depth levels as corresponding to drops in image contrast;

identifying one of the plurality of detected depth levels as corresponding to a deepest drop in image contrast; and

identifying the depth level that corresponds to the deepest drop in image contrast as the depth level that is the optimum focal plane;

and

imaging the cell sample using the microscope, by focusing the microscope at an investigative depth level that is based on the identified depth level.

2. The method according to claim 1, wherein identifying one of the depth levels as the optimum focal plane comprises identifying that the identified depth level is such that image contrast at the identified depth level is lower than image contrast associated with a depth level immediately preceding the identified depth level in the series of depth levels and lower than image contrast associated with a depth level immediately following the identified depth level in the series of depth levels.

3. The method according to claim 1, further comprising obtaining data representative of an additional series of images associated with an additional series of depth levels, wherein a scanning depth interval of the additional series of images is wider than a scanning depth interval of the series of images.

4. The method of claim 3, wherein the obtaining of the data representative of the additional series of images is performed in response to the deepness of the drop in image contrast at the identified depth level being below a predetermined deepness threshold.

5. The method according to claim 1, wherein detecting the plurality of depth levels as corresponding to drops in image contrast comprises computing, for each image, a contrast related value that enables derivation of an image contrast for each image.

6. The method according to claim 1, wherein detecting the plurality of depth levels as corresponding to drops in image contrast comprises calculating image contrast as a function of depth level using a contrast function that increases with image contrast, and detecting one or more wells in a contrast curve representing the image contrast as the function of depth level.

7. The method according to claim 6, wherein identifying one of the depth levels as corresponding to the deepest drop in image contrast comprises detecting that the depth level corresponds to a bottom of one of the wells in the contrast curve.

8. The method according to claim 6, wherein identifying one of the depth levels as corresponding to the deepest drop in image contrast comprises determining a deepness of the well corresponding to that depth level by:

determining right and left boundary depth levels at which the contrast function becomes inferior to the well bottom contrast;

determining right and left highest contrast values reached by the contrast function between the well bottom depth level and respectively the right and left boundary depth levels; and

calculating a minimum of;

a difference between the right highest contrast value and the well bottom contrast; and

a difference between the left highest contrast value and the well bottom contrast.

9. The method according to claim 1, wherein detecting the plurality of depth levels as corresponding to drops in image contrast comprises calculating image contrast as a function of depth level using a contrast function that decreases with image contrast and detecting one or more roofs of a contrast curve representing the image contrast as the function of depth level.

10. The method according to claim 9, wherein identifying one of the depth levels as corresponding to the deepest in image contrast comprises detecting that the depth level corresponds to a top of one of the roofs of the contrast curve.

11. The method according to claim 9, wherein identifying one of the depth levels as corresponding to the deepest drop in image contrast comprises determining a deepness of the roof corresponding to that depth level by:

determining right and left boundary depth levels at which the contrast function becomes superior to the rooftop contrast;

determining right and left lowest contrast values reached by the contrast function between the roof top depth level and respectively the right and left boundary depth levels; and

calculating a minimum of;

a difference between the roof top contrast and the right lowest contrast value, and

a difference between the roof top contrast and the left lowest contrast value.

12. The method according to claim 1,

wherein detecting that the plurality of depth levels as corresponding to drops in image contrast comprises:

calculating image contrast as a function of depth level,

generating a contrast curve representing the image contrast as the function of depth level, and

obtaining one or more supplemental depths levels associated to supplemental contrast values by interpolating or extrapolating the contrast curve; and

wherein identifying the depth level that is at the optimal focal plane comprises identifying, as the depth level that is at the optimum focal plane, one of the one or more supplemental depth levels.

13. The method according to claim 1, wherein obtaining data representative of the series of images comprises scanning a scanning depth interval of the cell sample using the microscope.

14. The method according to claim 1, wherein the series of images are associated with a first scanning depth and a second scanning depth that are endpoints of the series of depth levels and the method further comprises verifying at least one of:

a first distance between the optimum focal plane and the first scanning depth being above a first predetermined threshold, and a second distance between the optimum focal plane and the second scanning depth being above a second predetermined threshold.

15. The method according to claim 1, wherein obtaining the data representative of the series of images comprises obtaining data representative of a series of images captured by performing a depth scan of the cell sample using the microscope, the series of images being associated with a series of depth levels of the cell sample, a span of the series of depth levels being 5 to 1000 micrometers.

16. The method according to claim 1, wherein obtaining the data representative of the series of images comprises obtaining data representative of a series of images captured by performing a depth scan of the cell sample using the microscope, the series of images being associated with a series of depth levels of the cell sample, a span of the series of depth levels being less than 50 micrometers.

17. The method according to claim 1, further comprising determining an estimated optimum focal plane and wherein obtaining the data representative of the series of images comprises obtaining data representative of a series of images captured by performing a depth scan of the cell sample using the microscope, the series of images being associated with a series of depth levels that covers the estimated optimum focal plane.

18. The method according to claim 1, wherein focusing the microscope at the investigative depth level further comprises shifting a focus plane of the microscope from the identified depth level by a predetermined value.

19. The method according to claim 1, wherein the cell sample includes predominantly red blood cells, and obtaining data representative of the series of images captured by performing the depth scan of the cell sample comprises obtaining data representative of a series of images captured by performing a depth scan of the cell sample that includes predominantly red blood cells using the microscope.

20. The method according to claim 1, wherein the cell sample is essentially a monolayer of cells, and obtaining data representative of the series of images captured by performing the depth scan of the cell sample comprises obtaining data representative of a series of images captured by performing a depth scan of the cell sample that is essentially a monolayer of cells using the microscope.

21. The method according to claim 1, wherein detecting the plurality of depth levels as corresponding to drops in image contrast comprises calculating image contrast using a contrast function selected from the group consisting of:

variance, standard deviation, and sum of absolute-value of derivatives.

22. The method according to claim 1, wherein identifying one of the depth levels as the optimum focal plane comprises:

determining a variance deepness value associated with the depth level, the variance deepness value being a function of a variance-related value of the depth level, and a maximum of variance-related values associated with depth levels that are within a given range of distances from the depth level, and

identifying the depth level as the depth level that is at the optimum focal plane at least partially based upon the variance deepness value associated with the depth level.

23. The method according to claim 1, wherein identifying one of the depth levels as the optimum focal plane comprises determining image variance values for depth levels within a given range of distances from the depth level.

24. The method according to claim 1, wherein:

obtaining data representative of the series of images captured by performing the depth scan of the cell sample using the microscope comprises obtaining data representative of a series of images captured by performing a depth scan of the cell sample using the microscope, under a first illumination condition;

identifying one of the depth levels as being the optimum focal plane for imaging the one or more entities within the sample using the microscope comprises identifying one of the depth levels as being an optimum focal plane for imaging the one or more entities within the sample using the microscope, under the first illumination condition; and

imaging the cell sample using the microscope comprises imaging the cell sample under a second illumination condition that is different from the first illumination condition, using the microscope, by focusing the microscope at an investigative depth level that is based on the identified depth level.

25. The method according to claim 24, wherein:

obtaining data representative of the series of images captured by performing the depth scan of the cell sample using the microscope comprises obtaining data representative of a series of images captured by performing a depth scan of the cell sample using the microscope, under brightfield illumination conditions;

identifying one of the depth levels as being the optimum focal plane for imaging the one or more entities within the sample using the microscope comprises identifying one of the depth levels as being an optimum focal plane for imaging the one or more entities within the sample using the microscope, under the brightfield illumination conditions; and

imaging the cell sample using the microscope comprises imaging the cell sample under fluorescent lighting conditions, using the microscope, by focusing the microscope at an investigative depth level that is based on the identified depth level.

26. The method according to claim 24, wherein:

obtaining data representative of the series of images captured by performing the depth scan of the cell sample using the microscope comprises obtaining data representative of a series of images captured by performing a depth scan of the cell sample using the microscope, under a first brightfield illumination condition;

identifying one of the depth levels as being the optimum focal plane for imaging the one or more entities within the sample using the microscope comprises identifying one of the depth levels as being an optimum focal plane for imaging the one or more entities within the sample using the microscope, under the first brightfield illumination condition; and

imaging the cell sample using the microscope comprises imaging the cell sample under a second brightfield illumination condition that is different from the first brightfield illumination condition, using the microscope, by focusing the microscope at an investigative depth level that is based on the identified depth level.

27. An autofocus computation module for use with a digital microscope, the autofocus computation module comprising:

an input unit configured for receiving from the digital microscope data representative of a series of images captured by performing a depth scan of the cell sample using the digital microscope, the series of images being associated with a series of depth levels of the cell sample;

a calculation unit configured for identifying one of the depth levels as being an optimum focal plane for imaging one or more entities within the sample using the microscope, by;

detecting a plurality of depth levels as corresponding to drops in image contrast;

identifying one of the plurality of detected depth levels as corresponding to a deepest drop in image contrast; and

identifying the depth level that corresponds to the deepest drop in image contrast as the depth level that is the optimum focal plane;

an autofocus adaptation module configured for commanding the digital microscope to vary a distance between a focus plane of the microscope and a sample carrier intended to receive the cell sample; and

an output unit for outputting data indicative of the identified depth level to the autofocus adaptation module, such that the autofocus adaptation module commands the digital microscope to set the focus plane at an investigative depth level that is based upon the identified depth level.

28. The autofocus computation module according to claim 27, wherein the autofocus adaptation module is configured for commanding the digital microscope to set the focus plane at an investigative depth level that is at the identified depth level.

29. The autofocus computation module according to claim 27, wherein the autofocus adaptation module is configured to set the focus plane at an investigative depth level corresponding to the identified depth level shifted by a predetermined value.

30. The autofocus computation module according to claim 27, further comprising the digital microscope, the digital microscope comprising:

an imaging module comprising;

an optical unit configured for forming a magnified image of the cell sample by conjugating a focus plane and an image plane; and

an image sensor unit positioned in the image plane of the optical unit;

a focus variation module capable of varying a distance between the focus plane and the sample carrier; and

the autofocus adaptation module cooperating with the focus variation module and the image sensor unit.

31. The autofocus computation module according to claim 27, wherein:

the input unit is configured to receive data representative of the series of images captured by performing the depth scan of the cell sample using the microscope by receiving data representative of a series of images captured by performing a depth scan of the cell sample using the microscope, under a first illumination condition;

the calculation unit is configured to identify one of the depth levels as being the optimum focal plane for imaging the one or more entities within the sample using the microscope by identifying one of the depth levels as being an optimum focal plane for imaging the one or more entities within the sample using the microscope, under the first illumination condition; and

the output unit is configured to output data indicative of the identified depth level to the autofocus adaptation module, such that the autofocus adaptation module commands the digital microscope to set the focus plane, for imaging the cell sample under a second illumination condition, at an investigative depth level that is based upon the identified depth level.

32. The autofocus computation module according to claim 31, wherein:

the input unit is configured to receive data representative of the series of images captured by performing the depth scan of the cell sample using the microscope by receiving data representative of a series of images captured by performing a depth scan of the cell sample using the microscope, under brightfield illumination conditions;

the calculation unit is configured to identify one of the depth levels as being the optimum focal plane for imaging the one or more entities within the sample using the microscope by identifying one of the depth levels as being an optimum focal plane for imaging the one or more entities within the sample using the microscope, under the brightfield illumination conditions; and

the output unit is configured to output data indicative of the identified depth level to the autofocus adaptation module, such that the autofocus adaptation module commands the digital microscope to set the focus plane, for imaging the cell sample under fluorescent illumination conditions, at an investigative depth level that is based upon the identified depth level.

33. The autofocus computation module according to claim 31, wherein:

the input unit is configured to receive data representative of the series of images captured by performing the depth scan of the cell sample using the microscope by receiving data representative of a series of images captured by performing a depth scan of the cell sample using the microscope, under a first brightfield illumination condition;

the calculation unit is configured to identify one of the depth levels as being the optimum focal plane for imaging the one or more entities within the sample using the microscope by identifying one of the depth levels as being an optimum focal plane for imaging the one or more entities within the sample using the microscope, under the first brightfield illumination condition; and

the output unit is configured to output data indicative of the identified depth level to the autofocus adaptation module, such that the autofocus adaptation module commands the digital microscope to set the focus plane, for imaging the cell sample under a second brightfield illumination condition that is different from the first brightfield illumination condition, at an investigative depth level that is based upon the identified depth level.