Method for assessing image focus quality
View Patent ↗A method for determining the quality of focus of a digital image of a biological specimen includes obtaining a digital image of a specimen using a specimen imaging apparatus. A measure of image texture is calculated at two different scales, and the measurements are compared to determine how much high-resolution data the image contains compared to low-resolution data. The texture measurement may, for example, be a Brenner auto-focus score calculated from the means of adjacent pairs of pixels for the high-resolution measurement and from the means of adjacent triples of pixels for the low-resolution measurement. A score indicative of the quality of focus is then established based on a function of the low-resolution and high-resolution measurements. This score may be used by an automated imaging device to verify that image quality is acceptable. The device may adjust the focus and acquire new images to replace any that are deemed unacceptable.
1. A method of focusing on a specimen using an automatic imaging apparatus, the method comprising:
(a) obtaining three digital images of the specimen at different focal heights using the imaging apparatus, the digital image comprising a plurality of pixels;
(b) calculating a Brenner auto-focus score for each of the three digital images;
(c) fitting an exponentiated quadratic function to the (x, y) points formed by the three focal heights and the corresponding image Brenner scores;
(d) moving to a new focal height based on the function of step (c), acquiring a new image, and calculating the Brenner score for the new image, and
(e) repeating step (d) until an ideal focal height is reached.
2. The method of claim 1 , wherein each new image is acquired at the peak of the fit function calculated from the previous data points.
3. The method of claim 2 , wherein the ideal focal height is reached when fit function height, estimated when incorporating the newest data point into the fit, is within a certain threshold of the previous function height estimate.
4. The method of claim 1 , wherein the fit function is calculated using only three data points:
(a) The data point with the highest score found so far;
(b) The data point with the focal height that is closest to point (a), but below it; and
(c) The data point with the focal height that is closest to point (a), but above it.
5. The method of claim 4 , wherein with each step the focal height is adjusted to the height which is as close as possible to the peak of the fit function while remaining at least a certain distance away from all focal heights checked so far.
6. The method of claim 4 , wherein the ideal focal height is reached when the three points used in the fit are all within a certain distance of each other.
7. A method of focusing a specimen imaging apparatus comprising:
(a) obtaining a digital image of a specimen using the specimen imaging apparatus, the digital image comprising a plurality of pixels;
(b) calculating a high-resolution Brenner score by squaring the difference of the means of neighboring pairs of pixels and summing the squared differences over a set of pixels within the digital image;
(c) calculating a low-resolution Brenner score by squaring the difference of the means of neighboring triples of pixels and summing the squared differences over a set of pixels within the digital image;
(d) calculating a ratio comprising the high-resolution Brenner score divided by the low-resolution Brenner score; and
(e) adjusting the focal length of the specimen imaging apparatus based on the ratio.
8. The method of claim 7 , further comprising the step of obtaining a digital image of the specimen at the adjusted focal length and repeating steps (b) through (e).
9. The method of claim 8 , wherein the steps (b) through (e) are repeated a plurality of times with different images at the adjusted focal length.