Optical scanning systems for in situ genetic analysis

Abstract

Presented herein are systems and methods for performing sequencing, including fluorescence in situ sequencing. In one embodiment, a confocal time delay and integration (TDI) line scan imaging system may include various pinhole and / or slit aperture mechanisms in front of the image sensor. The system may also include structures with focusing strips on a substrate in contact with the tissue sample to be imaged. Alternatively, these strips may be cut into the tissue sample. The system may also include configurations and methods of placing a tissue sample inside a reaction chamber of a flow cell during the assembly of the flow cell and then performing chemistry operations on the tissue sample. The flow cells may use an open container for performing chemistry operations on the tissue sample.

Description

Background technique [0001] In a conventional (ie, widefield) fluorescence microscope, the entire sample is uniformly immersed in the light from the light source. All parts of the sample in the light path are excited simultaneously and the resulting fluorescence is detected by the microscope's photodetector or camera including the large unfocused background part. In contrast, confocal microscopes use point illumination and pinholes in the optically conjugate plane in front of the detector to eliminate out-of-focus signals. Since only the light generated by fluorescence that is very close to the focal plane can be detected, the optical resolution of the image, especially in the depth direction of the sample, is much better than that of widefield microscopy. However, since most of the light from the sample's fluorescence is blocked at the pinhole, this increased resolution comes at the expense of reduced signal intensity—thus, often long exposures are required. [0002] A disa...

AI Technical Summary

Problems solved by technology

However, since most of the light from the sample's fluorescence is blocked at the pinhole, this increased resolution comes at the cost of reduced signal strength—thus, often long exposures

[0002] A disadvantage of some photoluminescence-based scanning instruments (or imaging systems) used in current fluorescence-based sequencing-by-synthesis (SBS) systems is that they have poor confocality (i.e., semi-confocal at best)

These semi-confocal systems have a low signal-to-noise ratio (S / N ratio) and are therefore insufficient to remove out-of-focus features in samples

Additionally, current jitter focus tracking methods cannot maintain focus during imaging

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