Extended Data Fig. 2: The instrument and chip design.

(a) The ‘black-box’ prototype with major components including the camera, the imaging optics, the motion stages equipped with sequencing modules, as well as the liquid delivery. (b) Inside the ‘black-box’. Two sequencing modules sit on the moving stages. Above one of the sequencing module is the microscopic objective lens mounted on a piezo focusing adjustment stage. The manifolds are used for connection between the flow cell and the fluidic system (the selector and solenoid valves are shown in the photo). (c) The graphical user interface of the sequencer operating software (written in LabVIEW). (d) The schematic of the structure and fabrication of the flow-cell chip. (e) The fully assembled flow-cell chip. (f) The flow-cell chip and its holder. (g) The fluid flowing process in each reaction period that contains two reaction cycles. (h) The bleaching test to confirm the effectiveness of sealing of the microreactors. Top: Field-of-view (FOV) where fluorophores were sealed inside the microwells by FC-40. Middle: Using the pinhole to selectively photo-bleach part of the FOV, the fluorescent intensity is lower in the exposed area. Bottom: After several minutes, the margin of the exposed area remains sharp and clear, indicating the sealing is successful so that non-bleached fluorophores in the unexposed area cannot diffuse into the bleached area. (i) When sealing fails, the margin of exposed area is blurred, and the intensity of the exposed area recovers as a result of fluorophore diffusion. (j) A typical example of light stains (blue) and dark stains (red). (k) A typical example of bright-field image with tetris-like positioning markers.