Multicolor Live-Cell Chemical Imaging by Isotopically Edited Alkyne Vibrational Palette

Vibrational imaging such as Raman microscopy is a powerful technique for visualizing a variety of molecules in live cells and tissues with chemical contrast. Going beyond the conventional label-free modality, recent advance of coupling alkyne vibrational tags with stimulated Raman scattering microscopy paves the way for imaging a wide spectrum of alkyne-labeled small biomolecules with superb sensitivity, specificity, resolution, biocompatibility, and minimal perturbation. Unfortunately, the currently available alkyne tag only processes a single vibrational “color”, which prohibits multiplex chemical imaging of small molecules in a way that is being routinely practiced in fluorescence microscopy. Herein we develop a three-color vibrational palette of alkyne tags using a ¹³C-based isotopic editing strategy. We first synthesized ¹³C isotopologues of EdU, a DNA metabolic reporter, by using the newly developed alkyne cross-metathesis reaction. Consistent with theoretical predictions, the mono-¹³C (¹³C¹²C) and bis-¹³C (¹³C¹³C) labeled alkyne isotopologues display Raman peaks that are red-shifted and spectrally resolved from the originally unlabeled (¹²C¹²C) alkynyl probe. We further demonstrated three-color chemical imaging of nascent DNA, RNA, and newly uptaken fatty-acid in live mammalian cells with a simultaneous treatment of three different isotopically edited alkynyl metabolic reporters. The alkyne vibrational palette presented here thus opens up multicolor imaging of small biomolecules, enlightening a new dimension of chemical imaging

Polarized Raman Spectroscopy of Oligothiophene Crystals To Determine Unit Cell Orientation

Raman spectra were recorded experimentally and calculated theoretically for bithiophene, terthiophene, and quaterthiophene samples as a function of excitation polarization. Distinct spectral signatures were assigned and correlated to the molecular/unit cell orientation as determined by X-ray diffraction. The ability to predict molecular/unit cell orientation within organic crystals using polarized Raman spectroscopy was evaluated by predicting the unit cell orientation in a simulated terthiophene crystal given a random set of simulated polarized Raman spectra. Polarized Raman spectroscopy offers a promising tool to quickly and economically determine the unit cell orientation in known organic crystals and crystalline thin films. Implications of our methodologies for studying individual molecule conformations are discussed