Multiplex protein-specific microscopy with ultraviolet surface excitation

Abstract

Immunohistochemical techniques, such as immunofluorescence (IF) staining, enable microscopic imaging of local protein expression within tissue samples. Molecular profiling enabled by IF is critical to understanding pathogenesis and is often involved in complex diagnoses. A recent innovation, known as microscopy with ultraviolet surface excitation (MUSE), uses deep ultraviolet (≈280 nm) illumination to excite labels at the tissue surface, providing equivalent images without fixation, embedding, and sectioning. However, MUSE has not yet been integrated into traditional IF pipelines. This limits its application in more complex diagnoses that rely on protein-specific markers. This paper aims to broaden the applicability of MUSE to multiplex immunohistochemistry using quantum dot nanoparticles. We demonstrate the advantages of quantum dot labels for protein-specific MUSE imaging on both paraffin-embedded and intact tissue, significantly expanding MUSE applicability to protein-specific applications. Furthermore, with recent innovations in three-dimensional ultraviolet fluorescence microscopy, this opens the door to three-dimensional IF imaging with quantum dots using ultraviolet excitation.

Enhanced fluorescence from semiconductor quantum dot-labelled cells excited at 280 nm

“…The observed increase in fluorescence intensity of QD-labelled cells is expected to apply to all sizes of commercial semiconductor QDs since all sizes of QDs have absorption spectra with broadly similar distributions [10]. Therefore, all sizes of QDs are likely to have a higher absorption efficiency at 280nm compared to longer wavelengths.…”

“…More recently, Microscopy with Ultraviolet Surface Excitation (MUSE) [6,7,8,9] has identified that 280 nm light can be applied to excite fluorescence from different conventional fluorescent dyes whilst reporting further advantages of 280 nm excitation in tissue histology such as improved image contrast due to surface-limited excitation. A further application of MUSE has identified the suitability of QDs for excitation at 280 nm and applied this to multiplexed protein-specific imaging [10]. However, this work did not provide a quantitative insight into the benefits of excitation at this lower wavelength.…”

Pocket MUSE: an affordable, versatile and high-performance fluorescence microscope using a smartphone

“…4 ). Although IHC staining was proposed as a potential application in previous MUSE demonstrations, it has only been demonstrated with quantum dots conjugated to antibodies 32 . To our knowledge, this is the first demonstration of MUSE IHC imaging using fluorophore-conjugated antibodies.…”

Pocket MUSE: an affordable, versatile and high performance fluorescence microscope using a smartphone

“…3). Although IHC staining was proposed as a potential application in previous MUSE demonstrations, it has only been demonstrated with quantum dots conjugated to antibodies 27 . To our knowledge, this is the first demonstration of MUSE IHC imaging using conventional fluorophore-conjugated antibodies.…”