Sequencer focus quality metrics and focus tracking for periodically patterned surfaces

A sequencing instrument, comprising:

• a sample stage configured to support a sample container;
• an objective lens, a photodetector, and a light source configured to operate in combination to image the sample container when present on the sample stage;
• a focus component configured to control positioning of the objective lens relative to the sample container when present on the sample stage;
• and
• a controller configured to perform operations comprising:
• for each image in a stack of images acquired of a sample surface at different z-heights, determining a respective Fourier transform for a sub-region of the respective image, wherein an optical spread point function (PSF) associated with the stack of images does not have a Gaussian distribution profile;
• for each Fourier transform, determining a respective slope of a linear segment fitted to a portion of a respective power spectrum calculated from the Fourier transform, wherein the slope corresponds to a focus quality metric;
• parameterizing a focus model based upon the focus quality metrics, wherein the focus quality metrics are intensity independent;
• and
• controlling operation of the focus component during a sequencing operation based on the focus model.

A method for determining a focus quality metric, comprising:

• acquiring or accessing an image of a flow cell comprising a plurality of sample wells;
• calculating a one-dimensional Fourier transform of one or more rows of pixels within the image, wherein an optical point spread function (PSF) associated with the image does not have a Gaussian distribution profile;
• determining a power spectrum based on the one-dimensional Fourier transform of each respective row of pixels;
• determining a slope of a linear segment fitted to a portion of the power spectrum, wherein the slope corresponds to the focus quality metric, and wherein the focus quality metric is intensity independent;
• parameterizing or calibrating a focus model based on at least the focus quality metric or a score derived from the focus quality metric;
• and
• adjusting a relative position of an objective lens and a sample stage on which the flow cell is positioned based upon the focus model.

A sequencing instrument, comprising:

• a sample stage configured to support a flow cell;
• an objective lens, a photodetector, and a light source configured to operate in combination to image the flow cell when present on the sample stage;
• a focus component configured to control positioning of the objective lens relative to the flow cell when present on the sample stage;
• and
• a controller configured to perform operations comprising:
• for each image in a stack of images acquired of a sample surface at different z-heights, determining a respective one-dimensional Fourier transform for a row of pixels within the respective image, wherein an optical spread point function (PSF) associated with the respective image does not have a Gaussian distribution profile;
• for each one-dimensional Fourier transform, determining a respective slope of a linear segment fitted to a portion of a respective power spectrum calculated from the one-dimensional Fourier transform, wherein the slope corresponds to a focus quality metric;
• parameterizing a focus model based upon the focus quality metrics, wherein the focus quality metrics are intensity independent;
• and
• controlling operation of the focus component during a sequencing operation based on the focus model.