Fig. 1.

Overview of the customized RS instrument. (A) An SMRT Cell consists of 150,000 ZMWs, of which ∼75,000 ZMWs can be simultaneously imaged, allowing multiplexed detection of thousands of single molecules in real time across four spectral channels. (B) Simplified optical schematic of the custom RS instrument: continuous excitation is provided by a 532-nm laser and a 642-nm laser, which are separated into ∼75,000 beamlets that illuminate the array of ZMWs on the SMRT Cell that sits on a six-axis stage during data acquisition. The emitted light from the SMRT Cell is collected through the same objective in epifluorescence mode; notch filters in the collection path block transmission of excitation wavelengths. Emitted light is collected on four high-speed complementary metal-oxide-semiconductor (CMOS) cameras. (C) Comparison of workflows for standard sequencing and SMFM experimental mode shows steps for users (square boxes) and instrument (ovals). Modifications made for the SMFM mode reduce time to stage, alignment time, and exposure of components to laser illumination to allow for flexible single-molecule studies with labile reagents. DOE, diffractive optical element.