Super-multiplex vibrational imaging

Abstract

The ability to directly visualize a large number of distinct molecular species inside cells is increasingly essential for understanding complex systems and processes. Even though existing methods have been used successfully to explore structural-functional relationships in nervous systems, profile RNA in situ, reveal tumor microenvironment heterogeneity or study dynamic macromolecular assembly1–4, it remains challenging to image many species with high selectivity and sensitivity under biological conditions. For instance, fluorescence microscopy faces a “color barrier” due to the intrinsically broad (~1500 cm−1) and featureless nature of fluorescence spectra5 that limits the number of resolvable colors to 2 to 5 (or 7-9 if using complicated instrumentation and analysis)6–8. Spontaneous Raman microscopy probes vibrational transitions with much narrower resonances (peak width ~10 cm−1) and thus doesn’t suffer this problem, but its feeble signals make many demanding bio-imaging applications impossible. And while surface-enhanced Raman scattering offers remarkable sensitivity and multiplicity, it cannot be readily used to quantitatively image specific molecular targets inside live cells9. Here we show that carrying out stimulated Raman scattering under electronic pre-resonance conditions (epr-SRS) enables imaging with exquisite vibrational selectivity and sensitivity (down to 250 nM with 1-ms) in living cells. We also create a palette of triple-bond-conjugated near-infrared dyes that each display a single epr-SRS peak in the cell-silent spectral window, and that with available fluorescent probes give 24 resolvable colors with potential for further expansion. Proof-of-principle experiments on neuronal co-cultures and brain tissues reveal cell-type dependent heterogeneities in DNA and protein metabolism under physiological and pathological conditions, underscoring the potential of this super-multiplex optical imaging approach for untangling intricate interactions in complex biological systems.

Applications of vibrational tags in biological imaging by Raman microscopy

“…And as the chemical development of vibrational tags progresses for better tags with higher specificity, sensitivity and multiplex capability 70 , while the instrumental 71 and algorithmic development advances for fast acquisition, hyperspectral modality and accurate classifiers, we can readily anticipate even broader applications of the techniques, especially when small molecule metabolism is under investigation. For example, metabolic alterations are usually observed in cancer cells and might be a key to cancer proliferation and metastasis.…”

Metal‐Dielectric Nanocavity for Real‐Time Tracing Molecular Events with Temperature Feedback

“…Despite mK temperature sensitivity of the luminescent techniques at the nanoscale (Figure ) and μK for other quantum techniques, they are associated with a number of challenges: a limited temperature range for thermometry; perturbation of the molecule of interest by the organic label; the possibility of performing only a limited number of temperature measurement cycles; slow acquisition time and/or requirement for post‐processing; modification of the molecule structure during self‐luminescence; as well as a need an additional instrumentation to extract not only the local temperature, but also information about the event (e.g., with Forster Resonant Energy Transport, FRET technique) . Indeed, most of the luminescence based methods are not suitable to monitoring the complex processes at the nanometer scale as compared to the Raman techniques (iii)The intensity of the RS signal usually scales as the fourth power of the enhancement of electric field inside the nanocavity and is typically 10 6 –10 12 times larger than the RS intensity from the molecule in free space.…”

Electronic Preresonance Stimulated Raman Scattering Microscopy

“…56 First, they directly explored the rigorous resonance SRS detection (Figure 2d). Because the Stokes laser wavelength (λ Stokes ) is fixed at 1064 nm in the setup, the pump laser wavelength (λ pump ) for detecting typical electronically coupled vibrational modes (e.g., the total-symmetric vibration of conjugated double bonds, at ∼1600–1660 cm –1 ) is around 906 nm.…”