Method and System for Imaging a Cell Sample

• US 20140347459A1
• Filed: 05/23/2014
• Published: 11/27/2014
• Est. Priority Date: 05/23/2013
• Status: Active Grant

• Current Assignee: S.D. Sight Diagnostics Ltd
• Original Assignee: Parasight Ltd.
• Inventors: Bilu, Yonatan, Pollak, Joseph Joel, Yorav-Raphael, Noam, Greenfield, Yuval
• Granted Patent: US 10,176,565 B2
• Time in Patent Office: Days
• US Class Current: 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 of determining a reference depth level within 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 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.

2. The method according to claim 1, wherein the detected depth level is such that image contrast at the detected depth level is lower than image contrast associated with a depth level immediately preceding the detected depth level in the series of depth levels and lower than image contrast associated with a depth level immediately following the detected depth level in the series of depth levels.

3. The method according to claim 1, further comprising determining a deepness of the drop in image contrast.

4. 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.

5. The method of claim 4, further comprising determining for the series of images a deepness of the drop in image contrast and wherein the obtaining of data representative of an additional series of images is performed when the deepness of the drop is below a predetermined deepness threshold.

6. The method according to claim 1, wherein a plurality of drops in image contrast are detected and further comprising identifying the depth level corresponding to the deepest drop as the reference depth level.

7. The method according to claim 1, wherein processing the series of images comprises computing, for each image, a contrast related value enabling to derive the image contrast of each image.

8. The method according to claim 1, further comprising calculating image contrast using a contrast function increasing with the image contrast and wherein detecting at least one depth level corresponding to a drop in image contrast comprises detecting a well in a contrast curve representing image contrast as a function of the depth level.

9. The method according to claim 8, wherein the detected depth level corresponds to a bottom of the well in the contrast curve.

10. The method according to claim 8, further comprising determining the deepness of a well by:

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

determining a 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 the 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.

11. The method according to claim 1, further comprising calculating image contrast using a contrast function decreasing with the image contrast and wherein detecting at least one depth level corresponding to a drop in image contrast comprises detecting a roof of a contrast curve representing image contrast as a function of the depth level.

12. The method according to claim 11, wherein the detected depth level corresponds to a top of the roof of the contrast curve.

13. The method according to claim 11, further comprising determining the deepness of a roof by:

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

determining a 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 the minimum of;

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

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

14. The method according to claim 1, further comprising obtaining one or more supplemental depths levels associated to supplemental contrast values by interpolating and/or extrapolating the contrast curve and wherein the reference depth level is one of the one or more supplemental depth levels.

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

16. The method according to claim 1, wherein a first scanning depth and a second scanning depth are endpoints of the series of depth levels and the method further comprises verifying that a first distance between the reference depth level and the first scanning depth and/or a second distance between the reference depth level and the second scanning depth are respectively above a first and/or second predetermined threshold.

17. The method according to claim 1, wherein a span of the series of depth levels is of 5 to 1000 micrometers.

18. The method according to claim 1, wherein a span of the series of depth levels is less than 50 micrometers.

19. The method according to claim 1, further comprising determining an estimated reference depth level and wherein the series of depth levels covers the estimated reference depth level.

20. The method according to claim 1, wherein image contrast is calculated by a contrast function decreasing with the image contrast and further comprising transforming the contrast function into an increasing function of the contrast.

21. A method of imaging a cell sample using a microscope, comprising:

determining a reference depth level according to the method of claim 1; and

focusing the microscope at an investigation level based on the determined reference depth level.

22. The method according to claim 21, further comprising capturing one or more fluorescent lighting images of the cell sample.

23. The method according to claim 21, wherein focusing the microscope at an investigation level further comprises shifting a focus plane of the digital microscope from the reference depth level by a predetermined value.

24. The method according to claim 1, wherein the cell sample comprises predominantly red blood cells.

25. The method according to claim 1, wherein the cell sample is essentially a monolayer of cells.

26. The method according to claim 1, wherein the image contrast of an image is calculated from any of the following contrast functions:

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

27. An autofocus computation module for 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 a digital microscope, the series of images being associated with a series of depth levels of the cell sample;

a calculation unit configured for 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; and

an output unit for outputting data indicative of the reference depth level.

28. An autofocus system for a digital microscope comprising:

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 a cell sample for performing a depth scan of the cell sample thereby providing a series of digital images associated with a set of distances between the focus plane and the sample carrier;

the autofocus computation module according to claim 27, wherein the input unit is configured for receiving said series of digital images and the output unit is configured for outputting data indicative of the reference depth level to the autofocus adaptation module.

29. The autofocus system according to claim 28, wherein the autofocus adaptation module is further configured for commanding the digital microscope to set the focus plane at the reference depth level.

30. The autofocus system according to claim 28, wherein the autofocus adaptation module is further configured to set the focus plane at an investigation level corresponding to the reference depth level shifted of a predetermined value.

31. A microscope system comprising:

an imaging module comprising;

an optical unit configured for forming a magnified image of a 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 a sample carrier intended to receive the cell sample; and

the autofocus system according to claim 28, the autofocus system cooperating with the focus variation module and the image sensor unit.