Label‐Free Live‐Cell Imaging of Nucleic Acids Using Stimulated Raman Scattering Microscopy

Abstract

Imaging of nucleic acids is important for studying cellular processes such as cell division and apoptosis. A noninvasive label-free technique is attractive. Raman spectroscopy provides rich chemical information based on specific vibrational peaks. However, the signal from spontaneous Raman scattering is weak and long integration times are required, which drastically limits the imaging speed when used for microscopy. Coherent Raman scattering techniques, comprising coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) microscopy, overcome this problem by enhancing the signal level by up to five orders of magnitude. CARS microscopy suffers from a nonresonant background signal, which distorts Raman spectra and limits sensitivity. This makes CARS imaging of weak transitions in spectrally congested regions challenging. This is especially the case in the fingerprint region, where nucleic acids show characteristic peaks. The recently developed SRS microscopy is free from these limitations; excitation spectra are identical to those of spontaneous Raman and sensitivity is close to shot-noise limited. Here we demonstrate the use of SRS imaging in the fingerprint region to map the distribution of nucleic acids in addition to proteins and lipids in single salivary gland cells of Drosophila larvae, and in single mammalian cells. This allows the imaging of DNA condensation associated with cell division and opens up possibilities of imaging such processes in vivo.

Label-free DNA imaging in vivo with stimulated Raman scattering microscopy

“…1E). Chromatin structures within the cell nuclei such as nucleoli, heterochromatin, and euchromatin were visualized at the optical diffraction-limited resolution, demonstrating that SRS imaging of DNA in the CH band offers higher sensitivity than that of the fingerprint spectral region (22).…”

“…SRS has been demonstrated to be valuable for DNA imaging in cultured cells based on detection of the phosphate peaks within the fingerprint spectral region (22). However, imaging of DNA in this spectral region is difficult for cells in interphase because of the lower DNA density, especially in live tissue.…”

Chemically specific imaging and in-situ chemical analysis of articular cartilage with stimulated Raman scattering

“…It has been reported that in some cases where nuclei contain many replica choromosones SRS can be used to image the nucleic acid content via the 785cm -1 vibration for pyrimidine bases [34]. Imaging at this raman shift was attempted in the cartilage however we found there was insufficient contrast to use this Raman shift to image the nucleus.…”

“…The images taken at 2840cm -1 gave a highest contrast between the nucleus and the surrounding cytoplasm due to its lower lipid content [34]. It has been reported that in some cases where nuclei contain many replica choromosones SRS can be used to image the nucleic acid content via the 785cm -1 vibration for pyrimidine bases [34].…”

Fluorescent Labeling of Plasmid DNA and mRNA: Gains and Losses of Current Labeling Strategies

“…Interestingly, label-free detection can occur over a long time, since photobleaching, which is an important bottleneck in live-cell imaging, is no issue anymore. Raman scattering is one of those promising techniques that might be used to visualize nucleic acids 176 . Alkyne-bearing nucleotides were for example used to visualize DNA and RNA synthesis in vivo by stimulated Raman spectroscopy 177 .…”