High-Resolution Low-Power Hyperspectral Line-Scan Imaging of Fast Cellular Dynamics Using Azo-Enhanced Raman Scattering Probes

Abstract

Small-molecule Raman probes for cellular imaging have attracted great attention owing to their sharp peaks that are sensitive to environmental changes. The small cross section of molecular Raman scattering limits dynamic cellular Raman imaging to expensive and complex coherent approaches that acquire single-channel images and lose hyperspectral Raman information. We introduce a new method, dynamic azo-enhanced Raman imaging (DAERI), to couple the new class of azoenhanced Raman probes with a high-speed line-scan Raman imaging system. DAERI achieved high-resolution low-power imaging of fast cellular dynamics resolved at ∼270 nm along the confocal direction, 75 μW/μm 2 and 3.5 s/frame. Based on the azo-enhanced Raman probes with characteristic signals 10 2 −10 4 stronger than classic Raman labels, DAERI was not restricted to the cellular Raman-silent region as in prior work and enabled multiplex visualization of organelle motions and interactions. We anticipate DAERI to be a powerful tool for future studies in biophysics and cell biology.

A critical evaluation of compressed line-scan Raman imaging

“…The line-scan Raman imaging system was described in detail in our previous work. 33 Briefly, the system was constructed as follows: a continuous-wave 532 nm laser source (Changchun New Industries Optoelectronics Technology) was coupled into a line-generating system consisting of a Powell lens (5° fan angle) and two cylindrical lenses (LJ1267L1-A; LJ1996L1-A, Thorlabs). The laser beam was relayed using a 35 mm scanning lens combined with a 200 mm tube lens, and focused on the sample plane using a 60×/1.2 NA water immersion objective lens (Plan Apo, Nikon).…”

“…The cosmic spikes in the Raman dataset were removed using a cosmic-ray rejection algorithm, 36 followed by correcting slit curvature distortion caused by out-of-plane diffraction of the plane grating, using projective transformation. 33 Next, the corrected spectra could be efficiently denoised by projecting in a lower dimensional subspace using singular value decomposition (SVD). 12 Following SVD denoising, the background signal was subtracted from the Raman spectra via asymmetric least squares.…”

Recent Advances in Enhancement of Raman Scattering Intensity for Biological Applications

“…Utilizing various existing alkyne-based Raman tags, it is possible to achieve rapid, accurate, and real-time dynamic monitoring of biological macromolecules and various organelles in living cells. In addition to deuterium and alkynes, azo-enhanced Raman tags have also been extensively studied recently, and a series of azo tags with super sensitivity and fast multiplex imaging have been developed. ,− …”

Label-Free Analysis of Organelle Interactions Using Organelle-Specific Phase Contrast Microscopy (OS-PCM)

“…To analyze interactions between mitochondria and lysosomes, we also need to identify and track lysosomes. Due to their small size and similar shapes compared to other vesicles inside the cell, lysosomes are typically studied with fluorescence or Raman labels . Lysosomes also move and change much faster compared to mitochondria, requiring high acquisition speeds.…”

“…Due to their small size and similar shapes compared to other vesicles inside the cell, lysosomes are typically studied with fluorescence or Raman labels. 29 Lysosomes also move and change much faster compared to mitochondria, requiring high acquisition speeds. As described above, in this work, we have simplified the experimental and analytical systems to perform identification of both lysosomes and mitochondria from a single 2D intensity image.…”