Figure S1 Hyperspectral Imaging. (a) Schematic representation of the hyperspectral imaging system used in this work.

Figure S2 Optical effects induced by the skull. (a) Narrowband fluorescence image of a brain half covered by a piece of skull (left) and a piece of skull isolated (right). (b) Comparison of autofluorescence spectra of the isolated brain and skull. (c) Transmittance of the skull in the 900–1600 nm wavelength range according to previously published data [22]. (d) Comparison between the autofluorescence provided by the isolated brain (black), the skull mounted on the brain (red) and the simulation using the data of [22] for the values of transmittance.

Figure S3 In vivo hyperspectral analysis of brain under a cranial window.

Figure S4 Residual spectra for reported NIR-II liver autofluorescence and the data obtained in this work. (a) Residual spectra was calculated for both reported and experimentally obtained curves after applying the Savitzky–Golay smoothing method (Polynomial order 5, 9 points of window). (b) original spectra from which the noise was estimated [23].

Figure S5 Ex vivo ratiometric images of different tissues and organs. Figure S III: Ratiometric images of the different tissues. (a) Ratiometric image as obtained by calculating the ratio between emitted intensities at 1080/1125 nm. (b) Ratiometric image as obtained by calculating the ratio between emitted intensities at 1020/990 nm. (c) Intensity ratio between 1080 and 1125 nm. (d) Intensity ratio between 1020 and 990 nm.

Figure S6 Optical frontal view of the brain of a C57BL/6J mouse. The olfactory bulbs have been removed to expose the frontal area in which meningeal melanocytes enriched in neuromelanin can be observed by naked-eye examination (gray square).

Table S1 Recent advances in NIR-II fluorophores for transcranial brain imaging. An updated list of some of the most relevant NIR-II nanoprobes and their in vivo applications. (Wt = wild type, MCAO = Middle cerebral artery occlusion).