Wide-field fluorescence microscopy imaging coupled with AFM (JPK Instruments, Nanowizard3) was performed on an inverted optical microscope (Nikon, Eclipse Ti-U). The AFM head was mounted on top of the optical microscope. Fluorescence from the sample was collected through an objective lens (Nikon, 60×, 0.95 NA) excited by a 445 nm LED light source (Lumencor, Spectra X) through a dichroic excitation filter (Semrock, Di02-R442). Signals were recorded through an emission filter (Semrock, BLP01-458R) with an EMCCD camera (Andor, iXon 897). The exposure time for each image was adjusted to collect appropriate signals. Color images were obtained by combining three images recorded with appropriate emission filters: blue (Semrock, 451/106, FF01-451/106-25), green (Semrock, 550/88, FF01-550/88-25), and red channel (Semrock, 650/100, FF02-650/100-25), which selectively collect the fluorescence from (R)-1NDI donors, FRET pairs ((R)-1NDI -(S)-2PDI), and (S)-2PDI aggregates, respectively (Figure , top), and were then merged in Image J software. Localized fluorescence spectra were recorded using the optical microscope equipped with a piezo-scanner (PI Instruments, P-545.3C8S) and a custom Labview software that allows user-defined spectra to be recorded in synchronization with a CCD spectrograph (Acton SP2356 with Spec-10 400Br/LN-eXcelon CCD detector cooled with liquid nitrogen, from Princeton Instrument). AFM images were recorded in the intermittent contact mode (tapping) under ambient conditions using silicon cantilevers (Nanosensors, PPP-NCHR-20) with a nominal spring constant of 40 N m -1 and a resonance frequency of 330 kHz. Fluorescence polarization images were obtained on the same optical microscope using a fixed polarizer in the excitation light path, which selects the horizontal polarization excitation to the CCD field of view, and a rotating emission polarizer placed in the emission path. Polarized emission images and spectra were collected with the EMCCD camera or the CCD spectrograph, respectively. After correcting the geometric distortions of each image for vertical and horizontal emission polarization, and estimating the G-factor for the blue, green and red channels, the polarization parameter (P) was calculated at each image pixel using the following equation: