Abstract
Innovations in high-resolution optical imaging have allowed visualization of nanoscale biological structures and connections. However, super-resolution fluorescence techniques, including both optics-oriented and sample-expansion based, are limited in quantification and throughput especially in tissues from photobleaching or quenching of the fluorophores, and low-efficiency or non-uniform delivery of the probes. Here, we report a general sample-expansion vibrational imaging strategy, termed VISTA, for scalable label-free high-resolution interrogations of protein-rich biological structures with resolution down to 78 nm. VISTA achieves decent three-dimensional image quality through optimal retention of endogenous proteins, isotropic sample expansion, and deprivation of scattering lipids. Free from probe-labeling associated issues, VISTA offers unbiased and high-throughput tissue investigations. With correlative VISTA and immunofluorescence, we further validated the imaging specificity of VISTA and trained an image-segmentation model for label-free multi-component and volumetric prediction of nucleus, blood vessels, neuronal cells and dendrites in complex mouse brain tissues. VISTA could hence open new avenues for versatile biomedical studies.
Highlights
Innovations in high-resolution optical imaging have allowed visualization of nanoscale biological structures and connections
As a first step to establishing Vibrational Imaging of Swelled Tissues and Analysis (VISTA), we asked whether the sample expansion strategy is compatible with stimulated Raman scattering (SRS) microscopy
Our further correlative imaging identified that all these cell bodies captured by VISTA belong to matured neuronal cells, but not to astrocytes or oligodendrocytes. This is confirmed by the colocalization of VISTA cell bodies with immuno-fluorescencestained NeuN and MAP2; and the lack of cross-localizations to glial fibrillary acidic protein (GFAP, astrocyte cellular maker, Supplementary Fig. 15a–f) and myelin basic proteins (MBP, oligodendrocyte cellular marker, Supplementary Fig. 15g–l). These results suggest that maturated neuronal cells are more protein abundant in the cytosols compared to astrocytes and oligodendrocytes, a result difficult to quantify by other methodologies
Summary
Innovations in high-resolution optical imaging have allowed visualization of nanoscale biological structures and connections. Strategies including excitation saturation, signal suppression with a donut beam or structural illumination have been reported[11,12,13,14,15] They rely on additional specialized optics and the resolution enhancement is only 1.7 times on biological samples[13,14,15]. We report a super-resolution label-free vibrational imaging strategy in cells and tissues that couples the sensitive SRS microscopy with recent sample-treatment innovations. Compared to optics-based Raman imaging, VISTA is easy to implement without any additional instrument and achieves an unprecedented Raman resolution down to 78 nm on biological samples It allows high-resolution imaging deep into the tissues[16], a common limit shared by all instrumentbased super-resolution microscopy. With further implementation of a convolutional neural network (CNN) for image segmentation[17], VISTA could offer specific, multi-component, and volumetric imaging in complex tissues with quality similar to that of fluorescence
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