Dynamic azo-enhanced Raman imaging of cellular organelle physiology

Raman spectroscopy reflects molecular vibrational fingerprints of highly narrow bands. Thus Raman microscopy is well-known for offering an attractive solution to multiplex cellular imaging. However, despite the multiplex capability, Raman imaging has never gotten close to the dream of observing high-speed cellular dynamics that are truly significant for biological studies, due to the fundamental bottleneck of low strength of Raman scattering signals. Even with highly advanced Raman techniques, such as stimulated Raman scattering (SRS) microscopy, researchers have rarely achieved dynamic imaging or at the cost of high laser powers and complex systems, along with sacrificing the full-spectrum information that is the major advantage of Raman spectroscopy. This manuscript summarized our efforts on the improvement of high quality spontaneous Raman imaging, which were recently published on the high-impact journals. We combined novel azo-enhanced Raman scattering (AERS) probes with a highly efficient and speed-optimized line-scan Raman imaging system. The AERS probes targeted cellular organelles and featured back-ground free Raman signals with strength 4 orders of magnitude larger than the traditional Raman probes, e.g. 5-ethynyl-2'-deoxyuridine (EdU), without resorting to the complex coherent Raman approaches. The dynamic azo-enhanced Raman imaging (DAERI) system was able to obtain Raman images of live cells at the temporal resolution of 3.5 s per frame and the confocal spatial resolution of ~270 nm using a low power density of 75 μW/μm2. Empowered by this performance, we demonstrated DAERI of lysosomal and mitochondrial dynamics within live cells. In particular, DAERI' s preservation of the full-spectrum information utilized the hallmark of spontaneous Raman signals in fingerprint region and allowed us to easily and accurately unmix triple-stained cell images by resolving individual organelles clearly in a single image scan.