Extended Data Fig. 9: Imaging pipelines used for automated quantification of condensation in vitro, and associated controls.

(a) Top panel: BSA-Alexa-647 (1 µM) in indicated PEG concentration imaged by SDCM. Bottom panel: the FFT was computed for the images presented in the top panel (right-hand panel), and the fraction of the power spectrum in rings of increasing diameter was measured and plotted on a log scale (left-hand panel, see methods). Each pixel-wide column corresponds to a single image to evaluate variability within the sample (10 images per PEG concentration). Note that an increased presence of spots in the image due to condensation results in higher signal in rings of larger diameters. Also note the similarity of the power spectrum of images in the same condition. (b) Imaging pipeline to quantify the fraction of signal that is condensed in in vitro experiments. Raw images were processed for FFT, then a circular mask was applied. The corresponding low pass image (after inverse FFT, iFFT) corresponds to the background and the signal from the non-condensed protein, while the high pass image corresponds to the signal from the condensed protein. The condensation ratio is defined as the fraction of the power spectrum in the high pass filter. (c) Low pass and high pass filtered image using the mask as in (b) but for an image without condensates (BSA-Alexa-647 (1 µM) alone), showing the absence of signal in the high pass image. (d) The state of condensation of BSA-Alexa647 (1 µM) and Ubiquitin-FITC (100 nM) in solutions where the availability of free water is reduced by non-fluorescent PEG (400 mg ml⁻¹) was assessed by spinning disk microscopy. Images correspond to single confocal planes. The “PopRed” LUT was applied after the dynamic range was adjusted between minimum and maximum grey values of each images (note that the dynamic range was not kept identical between images). Note that the effect on osmotic potential of the three PEGs of different length is similar at the 400 mg ml⁻¹ concentration used here (see Extended Data Fig. 1). Note also that the BSA-Alexa647 panel is the same as in Fig. 4a, reproduced here for comparison. On the contrary to BSA, Ubiquitin does not phase separate when the osmotic potential is high. (e) Differential condensation of BSA and GFP as a function of the decreased availability of free water when macromolecule concentration is increased. BSA-Alexa647 (1 µM) or GFP (1 µM) were imaged in indicated PEG solution at 27 °C by SDCM. (f,g) Reversibility of the condensation of BSA as a function of the availability of free water. BSA-Alexa-647 (1 µM) was shifted from 150 mg ml⁻¹ PEG-10kDa to 300 mg ml⁻¹ before dilution back to 150 mg ml⁻¹, all at 27 °C. The state of condensation of BSA was imaged by SDCM (f) and quantified (g; mean ± SEM of condensation ratio) in each step. Statistics: one-way ANOVA followed by Tukey’s post-hoc test (P value indicated, n: number of images per sample). Note that (f) and (g) are the same data as in Extended Data Fig. 6b-c. (h-i) The state of condensation of GFP (100 nM) in PEG in the presence or absence of 100 nM anti GFP nanobody (GBP-Alexa555) was assessed by spinning disk microscopy (h) and quantified (i), see methods. Statistics: t-test (n: number of images analysed). Scale bars: 5 µm.

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