The combination of optical tissue transparency with immunofluorescence allows the molecular characterization of biological tissues in 3D. However, adult human organs are particularly challenging to become transparent because of the autofluorescence contributions of aged tissues. To meet this challenge, we optimized SHORT (SWITCH—H₂O₂—antigen Retrieval—TDE), a procedure based on standard histological treatments in combination with a refined clearing procedure to clear and label portions of the human brain. 3D histological characterization with multiple molecules is performed on cleared samples with a combination of multi-colors and multi-rounds labeling. By performing fast 3D imaging of the samples with a custom-made inverted light-sheet fluorescence microscope (LSFM), we reveal fine details of intact human brain slabs at subcellular resolution. Overall, we proposed a scalable and versatile technology that in combination with LSFM allows mapping the cellular and molecular architecture of the human brain, paving the way to reconstruct the entire organ.

Lipid nanoparticles (LNPs) play a crucial role in addressing genetic disorders, and cancer, and combating pandemics such as COVID-19 and its variants. Yet, the ability of LNPs to effectively encapsulate large-size DNA molecules remains elusive. This is a significant limitation, as the successful delivery of large-size DNA holds immense potential for gene therapy. To address this gap, the present study focuses on the design of PEGylated LNPs, incorporating large-sized DNA, departing from traditional RNA and ionizable lipids. The resultant LNPs demonstrate a unique particle morphology. These particles were further engineered with a DNA coating and plasma proteins. This multicomponent bionanoconstruct exhibits enhanced transfection efficiency and safety in controlled laboratory settings and improved immune system evasion in in vivo tests. These findings provide valuable insights for the design and development of bionanoarchitectures for large-size DNA delivery, opening new avenues for transformative gene therapies.

Imaging of nuclear structures within intact eukaryotic nuclei is imperative to understand the effect of chromatin folding on genome function. Recent developments of super-resolution fluorescence microscopy techniques combine high specificity, sensitivity, and less-invasive sample preparation procedures with the sub-diffraction spatial resolution required to image chromatin at the nanoscale. Here, we present a method to enhance the spatial resolution of a stimulated-emission depletion (STED) microscope based only on the modulation of the STED intensity during the acquisition of a STED image. This modulation induces spatially encoded variations of the fluorescence emission that can be visualized in the phasor plot and used to improve and quantify the effective spatial resolution of the STED image. We show that the method can be used to remove direct excitation by the STED beam and perform dual color imaging. We apply this method to the visualization of transcription and replication foci within intact nuclei of eukaryotic cells.

Expansion microscopy is a super‐resolution method that allows expanding uniformly biological samples, by increasing the relative distances among fluorescent molecules labeling specific components. One of the main concerns in this approach regards the isotropic behavior at the nanoscale. The present study aims to determine the robustness of such a technique, quantifying the expansion parameters i.e. scale factor, isotropy, uniformity. Our focus is on the nuclear pore complex (NPC), as well‐known nanoscale component endowed of a preserved and symmetrical structure localized on the nuclear envelope. Here, we show that Nup153 is a good reporter to quantitatively address the isotropy of the expansion process. The quantitative analysis carried out on NPCs, at different spatial scales, allows concluding that expansion microscopy can be used at the nanoscale to measure subcellular features with an accuracy from 10 to 5 nm. Therefore, it is an excellent method for structural studies of macromolecular complexes.

Brain cells are arranged in laminar, nuclear, or columnar structures, spanning a range of scales. Here, we construct a reliable cell census in the frontal lobe of human cerebral cortex at micrometer resolution in a magnetic resonance imaging (MRI)–referenced system using innovative imaging and analysis methodologies. MRI establishes a macroscopic reference coordinate system of laminar and cytoarchitectural boundaries. Cell counting is obtained with a digital stereological approach on the 3D reconstruction at cellular resolution from a custom-made inverted confocal light-sheet fluorescence microscope (LSFM). Mesoscale optical coherence tomography enables the registration of the distorted histological cell typing obtained with LSFM to the MRI-based atlas coordinate system. The outcome is an integrated high-resolution cellular census of Broca’s area in a human postmortem specimen, within a whole-brain reference space atlas.