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Bleaching of Ede1-EGFP in endocytic condensates (Figure 2A and B) was performed using a custom-built set-up that focuses a 488-nm laser beam at the sample plane, on the Olympus IX81 widefield microscope described above. The diameter of the bleach spot was approximately 0.5 μm.
Bleaching of unperturbed endocytic sites (Figure 2C) was performed with a 405 nm laser line controlled by the iLas2 targeting system during simultaneous excitation with 488 nm and 561 nm lasers in TIRF mode on the Olympus IX83 microscope described above. The emission light was collected through a Gemini beam splitter (Hamamatsu) equipped with a Di03-R488/561-t1 dichroic, and FF03-525/50-25 and FF01-630/92-25 emission filters (Semrock).

The animals were head-fixed on a custom-designed rotary treadmill apparatus placed under a custom-built optical macroscope for cortex-wide Ca²⁺ and IOS imaging. A high-power blue light source (450-nm LED) was used to excite the GCaMP6s to generate fluorescence, which was collected by a pair of video lenses that yielded a total magnification of ∼1.3× and passed through a band-pass filter (FF03-525/50-25, Semrock). An array of green LEDs (four LEDs with a central wavelength of 530 nm) were used to project homogeneous illumination onto the cortex for IOS imaging. GCaMP6s fluorescence and green-light reflectance images were captured by a scientific camera (sCMOS, Panda 4.2, PCO) in an interleaved manner at a frame rate of 30 Hz and frame size of 512 × 512 pixels. The TTL exposure signal from the camera output was used to sequentially control and synchronise the illumination of the blue and green LEDs. The treadmill apparatus was equipped with a rotary encoder (E40H6-2000-3-V-5, OMRON, Japan) to record the location/speed of behaving mice during cortex-wide optical imaging.

Single molecule fluorescence microscopy and imaging is performed using an inverted epifluorescence microscope (IX71, Olympus) coupled to an electron-multiplying charge coupled device (EMCCD) camera (iXon, Andor Technology). Labeled DNA solutions are illuminated using a 50 mW 488 nm laser (Spectra-Physics, CA, USA) directed through a 2.2 neutral density filter (ThorLabs, NJ, USA), a 488 nm single-edge dichroic mirror (ZT488rdc, Chroma). Fluorescence emission is collected by a 1.45 NA, ×100 oil immersion objective lens (UPlanSApo, Olympus), and a 525 nm single-band bandpass filter (FF03-525/50-25, Semrock) is used in the detection path. Finally, images are acquired by an Andor iXon EMCCD camera (512 × 512 pixels, 16 μm pixel size) under frame transfer mode at a frame rate of 33 Hz (0.030 s⁻¹). Experimental images obtained using fluorescence microscopy are analyzed using an in-house Matlab code based on algorithms reported in the literature^{49 (link),50 (link)}. The Kuhn step size of fluorescently labeled DNA is taken to be 0.132 μm^{27 (link)}, and the contour length of fluorescently labeled λ-DNA is approximately 21.2 μm. Also, the contour length of the full circular topology of fluorescently labeled Fos 45 DNA is approximately 20 μm, with the fully stretched length of the Fos 45 ring polymer equal to L_circ = 10 μm.