Clean microscope coverslips

Clean microscope coverslips (Marienfeld) or Labtek chambers (Nunc) containing cells were placed in an incubator equilibrated at 37 °C located on a DMIRE2 Leica Microscope controlled by HCImageLive software (Leica). Images from a 100X (N.A. 1.3–0.6) oil-immersion objective (HCX PL FLUOTAR, Leica) were recorded with a Hamamatsu ORCA-05G camera. A4 (Leica) and L5 filter cubes (Leica) were used for excitation and emission of Hoechst 33342 and EGFP fluorescence, respectively.

In preparation of the MP measurements, stock solutions of AAVs (ranging from 0.5 × 10¹³ to 5 × 10¹³ Vp/mL) were pre-diluted in PBS (Gibco) to about 0.5 × 10¹⁰ to 2 × 10¹¹ Vp/mL. Clean microscope coverslips (24 mm × 50 mm; Paul Marienfeld GmbH) were acquired by serial rinsing with Milli-Q water and HPLC-grade isopropanol (Fisher Scientific Ltd.). CultureWell gaskets (Grace Biolabs) were placed as container wells for the AAV dilutions. Coverslips were mounted on a Samux mass photometer (Refeyn Ltd.) and 12 μL of PBS buffer was used to set the focus. For each measurement, 3 μL of AAV solution was applied and mixed in the well. Movies were recorded for 60 or 120 s at 100 fps. Contrast-to-mass conversion was done by measuring a thyroglobulin multimer mix (Sigma, T9145). Three contrast rates were aligned with masses of 335, 670, and 1,340 kDa in a calibration curve. MP data were processed using DiscoverMP (Refeyn Ltd.), following export of the data mass histograms, and Gaussian fits were acquired using SciPy and in-house Python scripts.⁴⁵ (link)