Ultra-bright Raman dots for multiplexed optical imaging

Abstract

Imaging the spatial distribution of biomolecules is at the core of modern biology. The development of fluorescence techniques has enabled researchers to investigate subcellular structures with nanometer precision. However, multiplexed imaging, i.e. observing complex biological networks and interactions, is mainly limited by the fundamental ‘spectral crowding’ of fluorescent materials. Raman spectroscopy-based methods, on the other hand, have a much greater spectral resolution, but often lack the required sensitivity for practical imaging of biomarkers. Addressing the pressing need for new Raman probes, herein we present a series of Raman-active nanoparticles (Rdots) that exhibit the combined advantages of ultra-brightness and compact sizes (~20 nm). When coupled with the emerging stimulated Raman scattering (SRS) microscopy, these Rdots are brighter than previously reported Raman-active organic probes by two to three orders of magnitude. We further obtain evidence supporting for SRS imaging of Rdots at single particle level. The compact size and ultra-brightness of Rdots allows immunostaining of specific protein targets (including cytoskeleton and low-abundant surface proteins) in mammalian cells and tissue slices with high imaging contrast. These Rdots thus offer a promising tool for a large range of studies on complex biological networks.

Multiplexed live-cell profiling with Raman probes

“…Despite the high potential for multiplexing, the biological application of Raman spectroscopy is greatly limited by its small Raman cross-section, which is typically 10 8~1 0 14 times smaller than that of fluorescent dyes 31 . To amplify the signal, our group has recently developed a simple strategy to prepare ultra-bright Raman dots (Rdots) by non-covalently doping polymer nanoparticles with Raman-active dyes 32 . When coupled with stimulated Raman scattering (SRS) microscopy, these Rdots show a superb imaging performance in immunostaining of intracellular targets.…”

“…In our previous work, Rdots were imaged by narrowband SRS microscope in which only one Raman channel can be imaged at a time 32 . In this work, we aimed to acquire single-cell Raman spectra of all vibrational modes in a cost-effective manner by employing whole-cell spontaneous Raman spectroscopy 38 .…”

Super-multiplex imaging of cellular dynamics and heterogeneity by integrated stimulated Raman and fluorescence microscopy

“…To increase the imaging speed, brighter probes are desired. For example, the Raman signal can be amplified by incorporating alkyne into a polymer backbone ( Tian et al., 2020 ), or loading Carbow into nanoparticles ( Zhao et al., 2021 ). With enhanced signal, it will be possible to conduct super-multiplex imaging in live cells at video rate by our integrated SRS/fluorescence microscopy.…”

A Bioorthogonal Probe for Multiscale Imaging by 19F-MRI and Raman Microscopy: From Whole Body to Single Cells

“… 7 All these probes have been widely used for imaging biological targets in cells and tissues. 8 , 9 While Raman probes paved the way toward super-multiplexed optical imaging 10 and bioorthogonal cell- or organelle-targeted imaging, in most cases they are not directly detectable by clinical imaging modalities (i.e., MRI). An exception is represented by some SERS tags, based on the synthesis of multimetal NPs, which have been proposed as bimodal agents, but are often limited by toxicity and stability issues.…”