Microscopy with ultraviolet surface excitation for rapid slide-free histology

Abstract

Histologic examination of tissues is central to the diagnosis and management of neoplasms and many other diseases, and is a foundational technique for preclinical and basic research. However, commonly used bright-field microscopy requires prior preparation of micrometre-thick tissue sections mounted on glass slides, a process that can require hours or days, that contributes to cost, and that delays access to critical information. Here, we introduce a simple, non-destructive slidefree technique that within minutes provides high-resolution diagnostic histological images resembling those obtained from conventional haematoxylin-and-eosin-histology. The approach, which we named microscopy with ultraviolet surface excitation (MUSE), can also generate shape and colour-contrast information. MUSE relies on ~280-nm ultraviolet light to restrict the excitation of conventional fluorescent stains to tissue surfaces, and it has no significant effects on downstream molecular assays (including fluorescence in situ hybridization and RNA-seq). MUSE promises to improve the speed and efficiency of patient care in both state-of-the-art and lowresource settings, and to provide opportunities for rapid histology in research. High-quality tissue microscopy is central to the diagnosis and management of neoplasms as well as other diseases. However, the bright-field (transmission) design of clinical Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Moving tissue spectral window to the deep‐ultraviolet via optical clearing

“…The creation of UV windows opens new possibilities for establishment of the novel diagnostics and treatment procedures in the UV range, or possibly to study tissues with UV fluorescence methods . The development of such clinical methods in the future must be made carefully, since it is known that UV radiation induces tissue phototoxicity .…”

Multiscale nonlinear microscopy and widefield white light imaging enables rapid histological imaging of surgical specimen margins

“…However, the ability to image through surgical debris, blood and tissue marking ink is an important advantage of NLM that enables imaging of tissue aspects that have surface contamination typical of breast surgical specimens. In a previous manuscript, we evaluated CFM and found its ability to image subsurface features to be significantly more limited than NLM [28], while MUSE can only image a single plane on the tissue surface and so cannot perform subsurface assessment [32]. OCT provides excellent subsurface imaging but has limited nuclear contrast.…”

“…By reproducing classic histological features, these techniques enable direct histological evaluation of tissue, but without the delay that makes paraffin embedding or cryosectioning prohibitively time-consuming in many surgeries. Furthermore, several groups have demonstrated extraction of exogenous labels following fluorescent imaging, greatly reducing the possible risk of interference with postoperative immunohistochemistry or DNA assays, while retaining the high contrast enabled by molecularly specific labels [32,43]. Of these techniques, NLM is particularly attractive because of its very high imaging speed and ability to image below the tissue surface through blood and surgical debris.…”

“…Because physical sectioning accounts for the vast majority of processing delay for both FFPE and FSA histology, optical sectioning can dramatically reduce processing times and enable intraoperative histological examination in scenarios where FFPE and FSA histology are too time consuming. Various methods have been proposed for imaging breast, prostate and other surgical margins without physical sectioning, including optical coherence tomography (OCT) [18][19][20][21][22], reflectance confocal microscopy (RCM) [23,24], confocal fluorescence microscopy (CFM) [25][26][27][28][29], structured illumination microscopy (SIM) [30], light sheet microscopy [31], microscopy with ultraviolet surface excitation (MUSE) [32,33], and nonlinear microscopy (NLM) [34][35][36][37][38]. Stimulated Raman scattering (SRS) has also been demonstrated for surgical histology [39], but typically has appreciably lower imaging speed or signal to noise ratio when performed without physical sectioning in reflectance mode.…”

Widefield light sheet microscopy using an Airy beam combined with deep-learning super-resolution

“…Several non-destructive depthsectioning fluorescence microscopy techniques, such as confocal microscopy [6] and non-linear microscopy [7] have been pioneering in these areas. Recent advances in high-resolution imaging with structured illumination microscopy (SIM) [8,9] or at ultraviolet excitation wavelengths (MUSE) [10] have revealed novel information on the sub-cellular scale; however, they have often been at the sacrifice of the size of the area probed, expense or speed.…”