EP-4010755-B1 - OPTICAL IMAGING PERFORMANCE TEST SYSTEM AND METHOD

EP4010755B1EP 4010755 B1EP4010755 B1EP 4010755B1EP-4010755-B1

Inventors

RIDGEWAY, William K.
DYKE, ALAN
CHEN, PENGYUAN
KURZAVA, Rebecca Di Ricco

Dates

Publication Date: 20260506
Application Date: 20200807

Claims (15)

A method of testing imaging performance of an optical system (104), the method comprising: positioning a test target (116) at an object plane of the optical system; operating the optical system to illuminate the test target and generate an image beam; operating a focusing stage of the optical system to acquire a plurality of images of the test target from the image beam corresponding to a plurality of values of defocus; calculating from each image a plurality of Edge Spread Functions at a plurality of locations within the test target; characterized by constructing a plurality of Point Spread Function models from the respective Edge Spread Functions; and based on the Point Spread Function models, calculating a plurality of imaging performance values corresponding to the plurality of locations, wherein the imaging performance values are based on a metric selected from the group consisting of: Ensquared Energy; Encircled Energy; and Strehl ratio.
The method of claim 1, comprising at least one of: (a) wherein the plurality of locations comprises a plurality of field coordinates (x, y ) in the object plane within the test target; (b) wherein the plurality of locations comprises a plurality of focal positions (z) along an optical axis passing through the test target, and operating the optical system comprises acquiring a plurality of images of the test target at different focal positions (z).
The method of claim 1, comprising producing one or more maps of imaging performance based on a combination of the imaging performance values.
The method of claim 3, comprising comparing two or more of the maps to provide a measure of relative alignment of a focal plane of each imaging device or channel relative to the object plane.
The method of claim 3, wherein the one or more maps correspond to different imaging channels, and the different imaging channels correspond to different imaging devices of the optical system operated to acquire the image, or different wavelengths of the image acquired, or both different imaging devices and different colors; optionally comprising comparing two or more of the maps to provide a measure of relative alignment of each imaging device or channel relative to each other; and both of the foregoing.
The method of claim 3, comprising, after producing the one or more maps, adjusting a position of one or more optical components of the optical system, or replacing the one or more optical components, based on information provided by the one or more maps; wherein, optionally, the one or more optical components are selected from the group consisting of: one or more of the imaging devices; an objective of the optical system; one or more tube lenses of the optical system; one or more mirrors or dichroic mirrors; and a combination of two or more of the foregoing.
The method of claim 6, wherein the one or more maps are one or more initial maps, and further comprising, after adjusting or replacing the one or more optical components, acquiring a new image of the test target, calculating a plurality of new imaging performance values, and producing one or more new maps of imaging performance; optionally comprising comparing the one or more new maps to the one or more initial maps to determine position adjustments to be made to the one or more optical components for optimizing imaging performance.
The method of claim 6, comprising at least one of: (a) wherein the adjusting or replacing finds an optimum pair of conjugate image planes in the optical system; (b) wherein the adjusting or replacing improves an attribute selected from the group consisting of: focus matching of the imaging devices; imaging device tilt; flattening of field curvature; reduction of astigmatism; reduction of wavelength-dependent focus shift; and a combination of two or more of the foregoing.
The method of claim 1, comprising calculating one or more global scores of imaging performance based on a combination of the imaging performance values; optionally comprising at least one of: (a) modifying the one or more global scores to penalize or reward heterogeneity of the imaging performance values over a range of field coordinates (x, y) in the object plane within the test target, or through a range of focal positions (z) of the object plane, or both of the foregoing; (b) modifying the one or more global scores to penalize or reward similarity of different imaging channels as a function of field coordinates (x, y) in the object plane within the test target, or focal position (z) of the object plane, or both of the foregoing, wherein the different imaging channels correspond to different imaging devices of the optical system operated to acquire the image, or different wavelengths of the image acquired, or both different imaging devices and different colors.
The method of claim 1, comprising, after calculating the imaging performance values, adjusting a position of one or more optical components of the optical system based on information provided by the imaging performance values.
The method of claim 10, wherein the imaging performance values are initial imaging performance values, and further comprising, after adjusting the one or more optical components, acquiring a new image of the test target, and calculating a plurality of new imaging performance values; optionally comprising comparing the new imaging performance values to the initial imaging performance values to determine position adjustments to be made to the one or more optical components for optimizing imaging performance.
The method of claim 1, comprising at least one of: (a) wherein positioning the test target comprises aligning the target relative to a datum shared with one or more optical components of the optical system; (b) wherein operating the optical system comprises utilizing an objective in the image beam, and further comprising adjusting a position of the objective along an axis of the image beam to acquire a plurality of images of the test target at different focal positions (z); (c) wherein operating the optical system comprises utilizing an objective in the image beam, and further comprising adjusting a position of the objective along an axis of the image beam to acquire a plurality of images of the test target at different focal positions (z), wherein the objective has a configuration selected from the group consisting of: the objective is configured for infinite conjugate microscopy; and the objective is configured for finite conjugate microscopy; (d) wherein operating the optical system comprises operating two or more imaging devices to acquire respective images of the test target; (e) wherein operating the optical system comprises operating two or more imaging devices to acquire respective images of the test target, wherein the two or more imaging devices acquire the respective images at two or more different wavelengths; (f) wherein operating the optical system comprises operating two or more imaging devices to acquire respective images of the test target, wherein the two or more imaging devices acquire the respective images at two or more different wavelengths, and further comprising splitting an image beam propagating from the test target into two or more image beam portions, and transmitting the two or more image beam portions to the two or more imaging devices, respectively; (g) wherein operating the optical system comprises operating a filter assembly to filter the image beam at a selected wavelength; (h) wherein operating the optical system comprises utilizing a tube lens in the image beam, and further comprising adjusting a the relative position of one or more lenses or lens groups within the tube lens to acquire a plurality of images of the test target at different positions of the tube lens; (i) wherein the test target comprises a dark material and an array of bright features disposed on the dark material; (j) wherein the test target comprises a dark material and an array of bright features disposed on the dark material , and wherein the bright features are polygonal; (k) wherein the test target comprises a dark material and an array of bright features disposed on the dark material, wherein the bright features are polygonal, and the bright features are tilted such that edges of the bright features are oriented at angles to a pixel array of the optical imaging system that acquires the image.
An optical imaging performance testing system (100), comprising: a target holder (112) configured to hold a test target (116); a light source (120) configured to illuminate the test target; an imaging device (124, 128) configured to acquire images of the test target; an objective (132) positioned in an imaging light path between the test target and the imaging device, wherein a position of at least one of the objective or the target holder is adjustable along the imaging light path; and a controller (108) comprising an electronic processor and a memory, and configured to control the steps of the method of claim 1 of calculating the plurality of Edge Spread Functions, constructing the plurality of Point Spread Function models, and calculating the plurality of imaging performance values.
The system of claim 13, comprising at least one of: (a) wherein the objective has a configuration selected from the group consisting of: the objective is configured for infinite conjugate microscopy; and the objective is configured for finite conjugate microscopy; (b) wherein the imaging device comprises a plurality of imaging devices, and further comprises an image separation mirror configured to split the imaging light path into a plurality of imaging light paths respectively directed to the imaging devices; (c) a filter assembly configured to select a wavelength of an image beam in the imaging light path for propagation to the imaging device; (d) a tube lens positioned in the imaging light path, wherein the relative position of one or more lenses or lens groups within the tube lens is adjustable; (e) the test target, wherein the test target comprises a dark material and an array of bright features disposed on the dark material; (f) the test target, wherein the test target comprises a dark material and an array of bright features disposed on the dark material , and wherein the bright features are polygonal. (g) the test target, wherein the test target comprises a dark material and an array of bright features disposed on the dark material , the bright features are polygonal, and wherein the bright features are tilted such that edges of the bright features are oriented at angles to a pixel array of the optical imaging system that acquires the image.
A non-transitory computer-readable medium, comprising instructions stored thereon, that when executed on a processor, perform the steps of the method of claim 1 of calculating the plurality of Edge Spread Functions, constructing the plurality of Point Spread Function models, and calculating the plurality of imaging performance values.

Description

RELATED APPLICATIONS TECHNICAL FIELD The present invention generally relates to the testing of the imaging performance of an optical imaging system, such as may be utilized in microscopy to acquire imaging data from a sample under analysis. BACKGROUND In an optical imaging system, including an automated, high-throughput optical imaging system utilized in microscopy, a key metric of the quality of the imaging data obtained is the sharpness of the images obtained. Many applications of microscopic imaging (for example, in DNA sequencing) depend on the ability to produce uniformly sharp images across the field of view of one or more channels or cameras, despite imperfect focus and tilt of the specimen. The optical alignment of the cameras and other optical components of the optical imaging system to each other and to the specimen being imaged can contribute significantly to the image sharpness and thus the quality of imaging data produced. Accordingly, it is desirable to be able to test and evaluate the imaging performance (i.e., quality) of an optical imaging system. For example, it is useful to determine whether the optical imaging system meets or exceeds some predetermined level of minimum quality deemed to be acceptable for a given application of the optical imaging system. For expedient manufacturing and the calculation of manufacturing performance metrics, it is further useful to describe the entire imaging performance using a single precise number. US 2007/0165131 A1 discloses a method for measuring tilt of a sensor die with respect to the optical axis of the lens assembly in a camera module. An image acquired by an optical imaging system may be characterized as a mixture of the real specimen (or object) [S(x, y)] and an instrument response function, which in microscopy is termed a Point Spread Function [PSF(x, y)]. Mathematically, the image I(x, y) is the sum of the convolution of the specimen and the PSF and the noise at each pixel, N(x, y), as follows: Ixy=PSFxy∗Sxy+Nxy An evaluation of imaging performance may involve acquiring an image of a specimen (which may be a test target, i.e., an object provided for the purpose of testing), using the imaging data to measure and quantify the PSF, and compare the results against a predetermined minimum pass criterion. It is desirable to describe the quality of the PSF using a single scalar number. Several metrics are commonly used, such as full width at half maximum (FWHM) or Strehl ratio of the real-space PSF, wavefront error or modulation transfer function (MTF) contrast at a single frequency, and the real-space quantity Encircled Energy or Ensquared Energy. The accuracy and precision of the estimate for the measurement of each quantity is strongly linked to the experimental methods used to calculate them. The advantages and disadvantages of each pertain to the accuracy with which they are estimated, the ease of interpretation, and the ability to catch both poor image resolution and poor image signal to noise ratios (SNRs). The classical formulation of Equation (1) above does not reflect the fact that the PSF changes over the field of the image, which would be the equivalent of saying that an image could be sharp (high quality) in the middle and blurry (poor quality) at the edges. An optical system that is near to diffraction limited and optimized for detection of weak sources will have a compact PSF containing density in an approximately 5 x 5 pixel area, in contrast to a typically much wider image, such as 4096 x 3000 pixel images. The small PSF can vary substantially across the image, necessitating multiple independent measurements of the PSF across the field of view. In addition to changing over field coordinates (x, y), the PSF is strongly affected by the focus of the image in the z direction, and high performance, high numerical aperture (NA) objectives experience a rapid degradation of PSF performance with defocus, which is to say they have a shallow depth of field. Diffraction effects place an upper limit on the depth of field, but poorly corrected or poorly aligned systems will experience even shallower depth of field. The practical limitations of this are significant, as a system of low optical quality might need to be re-focused several times in order to get a sharp image at every point of the field, leading to poor throughput of the system and potential photo-degradation of the sample. Downstream processing of images by image analysis software produces results that can be strongly influenced by image sharpness. Blurry images of high-contrast punctate objects experience a fundamental degradation of signal to noise ratios and reduction of resolution. For example, blurry images of DNA clusters reduce the ability to perform accurate photometry of nucleotide abundance, reduce the number of small distinct clusters that can be observed and introduce crosstalk between neighboring clusters. Image processing algorithms can partially mitigate blurry images by