Multicolor live-cell chemical imaging by isotopically edited alkyne vibrational palette

Abstract

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 (13)C-based isotopic editing strategy. We first synthesized (13)C isotopologues of EdU, a DNA metabolic reporter, by using the newly developed alkyne cross-metathesis reaction. Consistent with theoretical predictions, the mono-(13)C ((13)C≡(12)C) and bis-(13)C ((13)C≡(13)C) labeled alkyne isotopologues display Raman peaks that are red-shifted and spectrally resolved from the originally unlabeled ((12)C≡(12)C) 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.

Figure 1

Isotopically edited alkyne vibrational tags for chemical imaging by SRS microscopy. (a) Setup of SRS microscope for alkyne vibrational imaging. When the energy difference between the pump and the Stokes photons matches with the alkyne vibration mode, their joint action will greatly accelerate the vibrational excitation of alkyne bonds. As a result of energy exchange between the input photons with the alkynes, the output pump and Stokes beams will experience intensity loss and intensity gain, respectively. Such intensity changes measured by SRS microscope generate concentration-dependent alkyne distributions in 3D. (b) Structures of unlabeled, mono, and bis ¹³C-labeled 5-ethynyl-2′-deoxyuridine (EdU). Mono-¹³C-labeled EdU, 2, is retrosynthetically disconnected using alkyne cross-metathesis chemistry.

Scheme 1. Synthesis of 3 by Sonagashira Coupling

Reagents and conditions: (a) Pd(OAc)₂ (10% mol), PPh₃ (20% mol), CuI (10% mol), Et₃N (3.0 equiv), and TMS¹³C≡¹³CH (1.5 equiv), DMF, RT, 15 h, 72%; (b) K₂CO₃ (5.0 equiv), MeOH/H₂O, RT, o/n, 75%.

Scheme 2. Synthesis of 2 by Alkyne Cross-Metathesis

Reagents and conditions: (a) 9 (100 equiv), 8 (5 equiv), CCl₄, 70 °C, 8 h, 27%, 33% BRSM; (b) K₂CO₃, TBAF, MeOH/H₂O, RT, 7 h, 50%.

Figure 2

Raman spectra of HeLa cells incubated with three isotopically edited EdUs. Spectra are acquired from nucleus region of fixed cells after incubation with either 1, 2, or 3. Amide bond stretchings at 1655 cm^–1 are shown as reference. The spectra are normalized according to the alkyne peak. Inset: enlarged Raman spectra from 2000 to 2170 cm^–1.

Figure 3

Live cell SRS imaging of DNA synthesis in HeLa cells incubated with isotopically edited EdUs. For each sample incubated with either 1, 2, or 3, images are acquired in 5 different Raman channels: 1655 (amide bond), 2000 (off-resonant), 2048 (on-resonant with 3), 2077 (on-resonant with 2), and 2125 cm^–1 (on-resonant with 1) in sequential mode. Images are acquired in 512 × 512 pixels with a pixel dwell time of 40 μs.

Figure 4

Three-color chemical imaging using isotopically edited alkyne tags. (a) Structures and normalized Raman spectra of RNA probe EU (11), DNA probe EdU (1), and fatty acid probe 17-octadecynoic acid (12). (b) Structures and normalized Raman spectra of isotopically edited EU–¹³C₂ (13), EdU–¹³C (2), and 17-ODYA (12). (c) Three-color SRS imaging of nascent RNA, DNA, and fatty acyl derivatives in live HeLa cells by spectral targeting of different isotopically edited alkyne tags. Images are acquired in 5 different Raman channels: 1655 (amide bond), 2000 (off-resonant), 2053 (on-resonant with 13), 2077 (on-resonant with 2), and 2125 cm^–1 (on-resonant with 12) in sequential mode. Images are acquired in 341 × 341 pixels with a pixel dwell time of 40 μs.