with N being the average number density of the fluorescent objects, S the structural parameter of the detection volume (S = z0/w0 with w0 and z0 lateral and axial parameters, determined through calibration), and τD the decay time. 2.9 Cryo-Transmission Electron Microscopy (Cryo-TEM) Raman-SERS spectra were recorded in the wavenumber range of 800-2500 cm -1 , the acquisition time was 60 s and the measurements were repeated twice for spectral averaging. To avoid spurious signals, calcium fluoride Raman slides (CaF2, Crystran, Dorset, UK) were used as substrates. To extract the Raman signal of interest, fluorescence and background signals were subtracted from the acquired raw spectra through Vancouver Raman Algorithm, a dedicated software for automatic autofluorescence background subtraction for Raman spectroscopy. Data was further analyzed with Origin software.