Method for obtaining an optically-sectioned image of a sample, and a device suitable for use in such a method

1 . A method for obtaining an optically-sectioned image of a sample based on a plurality of images of the sample, the method comprising constructing an optically-sectioned image by:

obtaining a plurality of signal values from the plurality of images, the plurality of signal values corresponding to the same pixel across the plurality of images;

evaluating a threshold value for the pixel based on the plurality of signal values and a desired degree of sectioning of the optically-sectioned image; and

evaluating a pixel value by integrating a fraction of the plurality of signal values above the threshold value,

wherein the plurality of images comprises optical images of the sample obtained by illuminating the sample with a focused patterned illumination beam at a plurality of lateral positions at a focal plane, wherein each image is obtained when the patterned illumination beam is at a respective one of the lateral positions, such that an intensity of the patterned illumination beam on a portion of the sample at the focal plane varies for each of the plurality of lateral positions.

2 . The method of claim 1 ,

wherein evaluating the threshold value for the pixel comprises:

providing a sectioning factor for determining the desired degree of sectioning of the optically-sectioned images;

wherein the threshold value for the pixel is evaluated as a function of the sectioning factor and a background function evaluated at the pixel; and

wherein the fraction is evaluated by subtracting the threshold value from each of the plurality of the signal values or by dividing each of the plurality of the signal values by the threshold value.

3 . The method of claim 2 ,

wherein the threshold value for the pixel is a multiplication of the sectioning factor and the background function evaluated at the pixel.

4 . The method of claim 2 ,

wherein the background function is a statistical function whose output depends on a skewness of the distribution of input data; and

wherein the background function is evaluated at the pixel by performing the statistical function on the plurality of the signal values.

5 . The method of claim 2 , wherein evaluating the background function at the pixel involves one of:

(1) calculating the median of the plurality of signal values for the pixel;

(2) calculating the median of the plurality of signal values for the pixel with subsequent Gaussian blurring over neighbouring pixels;

(3) calculating the mean of the plurality of signal values for the pixel;

(4) calculating the mean of the plurality of signal values for the pixel with subsequent Gaussian blurring over neighbouring pixels;

(5) calculating the mean of the plurality of signal values and subtracting the standard deviation for each signal value;

(6) determining the Pth percentile signal value for the pixel.

6 . The method of claim 1 , wherein the step of evaluating a pixel value comprises summing the difference between each of the signal values for that pixel and the threshold value, and setting the pixel value to zero if the sum produces a negative number.

7 . The method of claim 1 , the method comprising an imaging step involving:

providing the patterned illumination beam through an imaging lens such that the patterned illumination beam is focused at a focal plane of the imaging lens;

obtaining said plurality of images of the sample,

wherein obtaining comprises providing the patterned illumination beam at the plurality of lateral positions on the focal plane and obtaining each image when the patterned illumination beam is at a respective one of the lateral positions, such that the intensity of the patterned illumination beam on the portion of the sample at the focal plane varies for each of the plurality of lateral positions, and

detecting, using a detector, signals collected via the imaging lens.

8 . The method of claim 7 ,

wherein the patterned illumination beam is focused at the focal plane, and

wherein the patterned illumination beam comprises a periodic pattern with a spatial period defined in at least one direction within the focal plane.

9 . The method of claim 8 ,

wherein the patterned illumination beam comprises a periodic array of line foci focused at the focal plane.

10 . The method of claim 8 ,

wherein the patterned illumination beam comprises an array of focused spots focused at the focal plane.

11 . The method of claim 8 ,

wherein the patterned illumination beam comprises a combination of line focus and focused spots focused at the focal plane.

12 . A device for obtaining an optically-sectioned image of a sample, the device comprising:

an imaging lens;

an illumination source configured to provide a patterned illumination beam through the imaging lens such that the illumination beam is focused at a focal plane of the imaging lens;

a detector configured to detect signals collected from the sample via the imaging lens;

a control unit configured to:

obtain a plurality of images of the sample by providing the patterned illumination beam at a plurality of lateral positions on the focal plane, such that an intensity of the patterned illumination beam on a portion of the sample at the focal plane varies for each of the plurality of lateral positions; and

construct the optically-sectioned image based on the plurality of images by:

obtaining a plurality of signal values from the plurality of images, the plurality of signal values corresponding to the same pixel across the plurality of images;

evaluating a threshold value for the pixel based on the plurality of signal values and a desired degree of sectioning of the optically-sectioned image; and

evaluating a pixel value by integrating a fraction of the plurality of signal values above the threshold value.

13 . The device of claim 12 ,

wherein the control unit is further configured to:

receive a sectioning factor for determining the desired degree of sectioning of the optically-sectioned image;

evaluate the threshold value for the pixel as a function of the sectioning factor and a background function evaluated at the pixel; and

evaluate the fraction by subtracting the threshold value from each of the plurality of the signal values or by dividing each of the plurality of the signal values by the threshold value.

14 . The device of claim 13 ,

wherein the threshold value for the pixel is a multiplication of the sectioning factor and the background function evaluated at the pixel.

15 . The device of claim 13 , further comprising:

a spatial modulator configured to provide the patterned illumination beam focused at the focal plane, and

wherein the patterned illumination beam comprises a periodic pattern with a spatial period defined in at least one direction within the focal plane.

16 . The device of claim 15 ,

wherein the patterned illumination beam comprises a periodic array of line foci and/or an array of focused spots focused at the focal plane.

17 . The device of claim 12 ,

wherein the control unit is further configured to:

evaluate the background function at the pixel by performing a statistical function on the plurality of the signal values,

wherein the statistical function receives a plurality of data values and outputs a value based on a skewness of the plurality of the data values.

18 . The device of claim 17 ,

wherein the statistical function outputs a median of the received plurality of data values such that the background function at the pixel is a median of the plurality of signal values.

19 . The device of claim 12 , wherein the detector is a single pixel detector.

20 . The device of claim 12 , wherein the detector is a multi-pixel detector.