JPWO2021081128A5

JPWO2021081128A5 -

Info

Publication number: JPWO2021081128A5
Application number: JP2022523692A
Authority: JP
Publication date: 2023-10-31
Anticipated expiration: 2040-10-21

Claims

A method for improving the performance of a scanner for detecting fluorescence of millions of samples distributed across a flow cell in images collected over multiple cycles, the sample comprising at least some neighbors of the sample. located closer together than the Abbe diffraction limit for optical resolution between the pair,
calibrating the scanner using substantially the entire field of view projected by a lens onto an optical sensor in the scanner;
imaging at least one image tile under the structured illumination at a plurality of angular and phase displacements of structured illumination;
measuring the spacing between intensity peaks and the angle of a projected illumination pattern indicated by the intensity peaks in at least nine subtiles of said image tile, including a near-center subtile; and forming a spacing surface and an angular surface in said measured fitting a spacing and an angle, respectively, wherein both the spacing surface and the angular surface represent corrections for distortion across the subtiles of the image tile. and save the fitting results.
measuring the phase displacement of the structured illumination at the plurality of angular and phase displacements within the subtile, and looking for a coefficient representing the difference between the phase displacement in the near-center subtile and the phase displacement in other subtiles of the image tile; calibrating, including storing an up-table;
processing imaged image tiles covering positions across the flow cell imaged at the plurality of angular and phase displacements of the structured illumination over the plurality of periods;
extrapolating from the measurements of the spacing between intensity peaks, the angle of the projected illumination pattern, and the phase displacement of the structured illumination for the near-center subtile of each imaged image tile; reconfiguring parameters for the near-center subtile and the at least eight additional subtiles using the stored fitting results and coefficients from the stored lookup table;
applying the determined reconstruction parameters to generate improved resolution images for the subtiles over the plurality of periods with resolution better than the Abbe diffraction limit; processing and
ordering the sample over the plurality of periods using the improved resolution image of the subtile.

The method of claim 1, wherein the sample is distributed in millions of nanowells across the flow cell, and at least some adjacent pairs of nanowells are located closer together than the Abbe diffraction limit for optical resolution. .

3. The method of claim 1 or 2, further comprising storing the fitting results of the spacing surface and the angular surface as coefficients of a quadratic surface.

3. The method of claim 1 or 2, further comprising storing the fitting results of the spacing surface and the angular surface as cubic surface coefficients.

3. The method of claim 1 or 2, further comprising storing the fitting results in a look-up table calculated from a quadratic fit of the spacing surface and the angular surface.

remeasuring the spacing between intensity peaks in the subtiles and the angles of the intensity peaks in an intermediate period between the plurality of periods, and refitting the spacing surface and the angular surface; 6. A method according to any one of claims 1 to 5, further comprising: saving the refitting results for use in processing captured image tiles.

the nanowells are arranged in a repeating hexagonal pattern, three diagonals connecting opposite corners of the hexagons in the pattern, and the intensity peaks are oriented substantially perpendicular to the three diagonals. 3. The method of claim 2, further comprising using three structured illumination angles.

8. The method of claim 1, further comprising applying a cropping margin to remove pixels around an edge of the sensor from use in fitting the spacing surface and the angular surface. the method of.

A system comprising one or more processors coupled to a memory , the memory determining the ability of a scanner to detect fluorescence of millions of samples distributed across a flow cell in images collected over a plurality of cycles. loaded with computer instructions for improving said samples, said samples being positioned closer together than the Abbe diffraction limit for optical resolution between at least some adjacent pairs of said samples; When executed on more than one processor,
calibrating the scanner using substantially the entire field of view projected by a lens onto an optical sensor in the scanner;
imaging at least one image tile under the structured illumination at a plurality of angular and phase displacements of structured illumination;
measuring the spacing between intensity peaks and the angle of a projected illumination pattern indicated by the intensity peaks in at least nine subtiles of said image tile, including a near-center subtile; and forming a spacing surface and an angular surface in said measured fitting an optical distortion across the image tile, respectively fitting a spacing and an angle, wherein both the spacing surface and the angular surface represent corrections for distortion across the subtiles of the image tile. calculate and save fitting results;
measuring the phase displacement of the structured illumination at the plurality of angular and phase displacements within the subtile, and looking for a coefficient representing the difference between the phase displacement in the near-center subtile and the phase displacement in other subtiles of the image tile; calibrating, including storing an up-table;
processing imaged image tiles covering positions across the flow cell imaged at the plurality of angular and phase displacements of the structured illumination over the plurality of periods;
extrapolating from the measurements of the spacing between intensity peaks, the angle of the projected illumination pattern, and the phase displacement of the structured illumination for the near-center subtile of each imaged image tile; reconfiguring parameters for the near-center subtile and the at least eight additional subtiles using the stored fitting results and coefficients from the stored lookup table;
applying the determined reconstruction parameters to generate improved resolution images for the subtiles over the plurality of periods with resolution better than the Abbe diffraction limit; processing and
using the improved resolution images of the subtiles to order the sample over the plurality of periods;
A system that implements behaviors that include.

10. The system of claim 9, wherein the sample is distributed in millions of nanowells across the flow cell, and at least some adjacent pairs of nanowells are located closer together than the Abbe diffraction limit for optical resolution. .

5. The computer instructions , when executed on the one or more processors, further implement operations comprising: storing the fitting results of the spacing surface and the angular surface as coefficients of a quadratic surface. 9 or 10.

9. The computer instructions , when executed on the one or more processors, further implement operations comprising storing the fitting results of the spacing surface and the angular surface as coefficients of a cubic surface. or the system described in 10.

The computer instructions , when executed on the one or more processors, further include storing the fitting results in a lookup table computed from a quadratic fit of the spacing surface and the angular surface. A system according to claim 9 or 10, implementing the system.

when the computer instructions are executed on the one or more processors, remeasuring the spacing between intensity peaks in the subtiles and the angle of the intensity peaks at intermediate periods between the plurality of periods; and refitting the spacing surface and the angular surface, and storing the results of the refitting for use in processing subsequent imaged image tiles. The system according to any one of claims 9 to 13, wherein :

the nanowells are arranged in a repeating hexagonal pattern, three diagonals connecting opposite corners of the hexagons in the pattern, and program instructions are further provided, the method comprising: 11. The system of claim 10, further comprising using three structured illumination angles oriented substantially perpendicular to three diagonals.