Raman and SERS microscopy for molecular imaging of live cells

Abstract

Raman microscopy is a promising technology for visualizing the distribution of molecules in cells. A challenge for live-cell imaging using Raman microscopy has been long imaging times owing to the weak Raman signal. Here we present a protocol for constructing and using a Raman microscope equipped with both a slit-scanning excitation and detection system and a laser steering and nanoparticle-tracking system. Slit scanning allows Raman imaging with high temporal and spatial resolution, whereas the laser beam steering system enables dynamic surface-enhanced Raman imaging using gold nanoparticles. Both features enable mapping of the distributions of molecules in live cells and visualization of cellular transport pathways. Furthermore, its utility can be expanded to small-molecule imaging by using tiny Raman-active tags such as alkyne. For example, DNA synthesis in a cell can be visualized by detecting 5-ethynyl-2'-deoxyuridine (EdU), a deoxyuridine derivative with an alkyne moiety. We describe the optics, hardware and software to construct the Raman microscope, and discuss the conditions and parameters involved in live-cell imaging. The whole system can be built in ∼8 h.

Label‐free live cell imaging by Confocal Raman Microscopy identifies CHO host and producer cell lines

“…2D: 750 (symmetric breathing of aromatic tryptophan), 1130 (C‐C skeletal of acyl backbone in lipids), 1303 (CH 2 twisting of lipids) and 1585 cm −1 (C=C olefinic stretch of proteins, phenylalanine) indicate that there is an enrichment of proteins and lipids in the putative ER. The X‐loadings of the rest of the cell, however, show a quite different pattern of peaks with highest signal coming from 1001 (Phenylalanine), 1445 (CH 2 deformation band and CH 2 bending of aliphatic amino acids) and 1665 cm −1 (amide‐I vibration mode of peptide bonds) 35. Taken together, due to localization, shape and enrichment for proteins and lipids we hypothesize that this subcellular structure corresponds largely to the ER.…”

Trace cancer biomarker quantification using polystyrene-functionalized gold nanorods

“…The characteristic peaks (e.g., 1002 cm −1 ) in the blank spectrum belong to the polystyrene. The ripple-like features (e.g., 700 cm −1 to 900 cm −1 ) come from the etaloning effect of the CCD detector in the spectrometer and are spurious features [29]. The difference spectrum shows a good match to the spectrum of the AcAm powder, which reduces the etaloning and the background signal from the polystyrene.…”

Sphingomyelin distribution in lipid rafts of artificial monolayer membranes visualized by Raman microscopy

“…5A). We used slit-scanning Raman microscopy, which used a line-shaped laser focus for parallel Raman spectroscopic detection (16). Because the imaging speed of the slit-scanning configuration is more than 100 times faster than that of the point-scanning configuration, we could increase the image pixel number to observe fine structure in the sample.…”