Sola se u nir light engine

We imaged RNA FISH samples on an inverted Nikon TI-E microscope equipped with a SOLA SE U-nIR light engine (Lumencor), an ORCA-Flash 4.0 V3 sCMOS camera (Hamamatsu), 20X Plan-Apo λ (Nikon MRD00205), 40X Plan-Fluor (MRH00401) and 60X Plan-Apo λ (MRD01605) objectives, and filter sets for DAPI, Cy3, Alexa Fluor 594, and Atto647N. For barcode ClampFISH and barcode HCR, we first acquired tiled images in a single Z-plane (scan) at 20X or 40X magnification, then, after identifying positions containing cells positive for resistant barcodes, we returned to those positions to acquire a Z-stack at 60X magnification. For subsequent rounds of single-molecule RNA FISH and ERK immunofluorescence, we acquired Z-stacks at 60X magnification. For scans, we used a Nikon Perfect Focus system to maintain focus across the imaging area.

Drug-naive WM989 A6-G3s were transduced with unique barcodes as described above. Cells were plated in 6-well plates at a density of 100,000 cells per well. One plate was fixed in formaldehyde after 24 h using a protocol described above and the second plate was treated with medium containing 1 μM PLX after 24 h. This drug treatment was continued for three weeks which was enough time for resistant colonies to form. This second plate containing resistant populations was fixed in formaldehyde. Wells were imaged on a Nikon TI-E inverted fluorescence microscope equipped with a SOLA SE U-nIR light engine (Lumencor), a Hamamatsu ORCA-Flash 4.0 V3 sCMOS camera, and 10× Plan-Fluor 10×/0.30 (Nikon MRH10101). Images were analysed using the custom Raj Lab image processing software NimbusImage via the CellPose tool^{67 (link)}. Average intensity for each of the cells was calculated also using NimbusImage.

To image RNA FISH and nuclei signal, we used a Nikon TI-E inverted fluorescence microscope equipped with a SOLA SE U-nIR light engine (Lumencor), a Hamamatsu ORCA-Flash 4.0 V3 sCMOS camera, and 4× Plan-Fluor DL 4XF (Nikon MRH20041/MRH20045), 10× Plan-Fluor 10×/0.30 (Nikon MRH10101) and 60× Plan-Apo λ (MRD01605) objectives. We used the following filter sets to acquire different fluorescence channels: 31000v2 (Chroma) for DAPI, 41028 (Chroma) for Atto 488, SP102v1 (Chroma) for Cy3, 17 SP104v2 (Chroma) for Atto 647N, and a custom filter set for Alexa 594. We tuned the exposure times depending on the dyes used (Cy3, Atto 647N, Alexa 594, and DAPI). For large tiled scans, we used a Nikon Perfect Focus system to maintain focus across the imaging area. For imaging RNA FISH signals in tissue sections, we acquired $z$ -stacks (three positions) at 60× magnification, and used maximum intensity projection to visualize the signal. For bright-field imaging of resistant colonies, we used a Nikon Eclipse TS2-FL with an Imagingsource DFK 33UX252 camera and 4× Plan-Fluor 4×/0.13 (Nikon MRH20041) objective. For time-lapse imaging of the emergence of drug-resistant colonies, we used an IncuCyte S3 Live Cell Imaging Analysis System (Sartorius) with a 4× objective on WM989 A6-G3 tagged with an mCherry nuclear reporter (H2B–mCherry).

For imaging we used a Nikon Ti-E inverted microscope controlled by NIS-Elements v5.11.01 on a personal computer running Windows 10 and equipped with an ORCA-Flash4.0 V3 sCMOS camera (Hamamatsu, C13440-20CU), a SOLA SE U-nIR light engine (Lumencor), and a Nikon Perfect Focus System. We used ×100 (1.45 NA) Plan-Apo λ (Nikon, MRD01905), ×60 (1.4 NA) Plan-Apo λ (Nikon, MRD01605), ×20 (0.75 NA) Plan-Apo λ (Nikon, MRD00205) and ×10 (0.45 NA) Plan-Apo λ (Nikon, MRD00105) objectives and filter sets for DAPI, Atto 488, Cy3, Alexa Fluor 594 and Atto 647N (see Supplementary Table 8 for filter sets used; filter sets can also be viewed at https://www.fpbase.org/microscope/455WNQygW6268avMhrTNx8/). All ×60 images were taken using 2 × 2 camera binning, while ×100, ×20 and ×10 images used 1 × 1 binning. Based on the camera’s pixel size of 6.5 μm, the image pixel size for each magnification level is 65 nm (×100, 1 × 1 binning), 216.7 nm (×60, 2 × 2 binning), 325 nm (×20, 1 × 1 binning) and 650 nm (×10, 1 × 1 binning).