To perform expansion microscopy, we used secondary probes (FLAP-Y) from IDT with a 5′-acrydite modification and a 3′-Atto565 label. Primary probes (CRM1 or GAPDH), were pre-hybridized with secondary probes as described in the Supplementary Protocol. smFISH experiments were performed with the Stellaris RNA FISH buffers (Biosearch Technologies) according to the provided protocol (https://www.biosearchtech.com/support/resources/stellaris-protocols ), except that the final mounting step with Vectashield was omitted. Expansion was conducted as described (12 (link)) (a more detailed version is available at http://expansionmicroscopy.org/ ), with some modifications as explained next. Cells were grown on 18 mm coverslips to facilitate the smFISH experiments. To cast the gel, coverslips were quickly air-dried after the final washing step of the smFISH protocol. As a mold, individual wells from non-adhesive silicon insulators with an inner diameter of 4.5 mm (Grace bio-labs, Product #664206) were cut, and gently pressed on the coverslip. Gels were poured with 30 μl of the monomer solution with a cross-linker concentration of 0.2%. After 1 h, coverslips were transferred to Nunc 2-well LabTek chambers (Thermo Scientific). Proteinase K treatment was performed for 4 h at 37°C and expansion was performed as described (12 (link)). Expanded samples were embedded in 2% low melting agarose, to avoid drift during acquisition.
Three-dimensional images were acquired on a Nikon Ti Eclipse, with a LED light-source (Lumencor Spectra X light engine), a 60 × 1.4 NA objective and an Orca flash 4.0 LT sCMOS camera. Before expansion 41 z-slices with a spacing of 300 nm were acquired, after expansion 40 slices with a spacing of 600 nm. DAPI images were acquired by excitation with 390 nm at 4% for 100 ms, smFISH images at 560 nm at 40% and 500 ms.
Nuclear area was measured in 2D maximum intensity projections of DAPI images with CellProfiler (11 (link)) after automated segmentation. mRNA detection was performed with FISH-quant (5 (link)) with local-maximum detection after Laplacian of Gaussian filtering. Signal-to-noise ratio (SNR) was calculated for individual cells as the ratio of the mean amplitude of the fitted 3D Gaussian to the standard deviation of the background in a region without cells. In Supplementary Note 3, we provide carefully validated guidelines to select the best mRNA detection method in FISH-quant.
Specimen free gels were cast in 6 mm silicon tubes with varying concentrations of the cross-linker. Gels were expanded as described above for cells, but without the digestion. Diameter of expanded gels were measured, and ratio to unexpanded gels reported as expansion factor.
Three-dimensional images were acquired on a Nikon Ti Eclipse, with a LED light-source (Lumencor Spectra X light engine), a 60 × 1.4 NA objective and an Orca flash 4.0 LT sCMOS camera. Before expansion 41 z-slices with a spacing of 300 nm were acquired, after expansion 40 slices with a spacing of 600 nm. DAPI images were acquired by excitation with 390 nm at 4% for 100 ms, smFISH images at 560 nm at 40% and 500 ms.
Nuclear area was measured in 2D maximum intensity projections of DAPI images with CellProfiler (11 (link)) after automated segmentation. mRNA detection was performed with FISH-quant (5 (link)) with local-maximum detection after Laplacian of Gaussian filtering. Signal-to-noise ratio (SNR) was calculated for individual cells as the ratio of the mean amplitude of the fitted 3D Gaussian to the standard deviation of the background in a region without cells. In Supplementary Note 3, we provide carefully validated guidelines to select the best mRNA detection method in FISH-quant.
Specimen free gels were cast in 6 mm silicon tubes with varying concentrations of the cross-linker. Gels were expanded as described above for cells, but without the digestion. Diameter of expanded gels were measured, and ratio to unexpanded gels reported as expansion factor.
