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. Herein 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.

Figure 1

(a) Experimental schematic of stimulated Raman loss (SRL) microscope. For SRG, the pump beam is modulated instead of the Stokes beam and an InGaAs photodetector is used instead of a silicon photodetector because of its better reponsivity at 1064 nm. (b) Detection scheme of SRL. Stokes beam is modulated at 10.4 MHz at which the resulting amplitude modulation of the pump beam due to the stimulated Raman loss can be detected. (c) Detection scheme of SRG. Pump beam is modulated at 10.4 MHz at which the resulting amplitude modulation of the Stokes beam due to the stimulated Raman gain can be detected.

Figure 2

Raman spectra of (a) DNA, (b) BSA and (c) Oleic Acid.

Figure 3

(a) Raman spectrum of a Drosophila cell (b)-(g) SRS images of a salivary gland cell from Drosophila melanogaster, via the stimulated Raman loss detection scheme. (b) Lipid specific image taken at 2845 cm⁻¹, (c) Amide I band at 1655 cm⁻¹ (d) nucleic acids at 785 cm⁻¹ and (e) 1090 cm⁻¹. (f) multicolor image generated by combining images (b)-(e). (g) Nucleic acid map recorded at 785 cm⁻¹ via the stimulated Raman gain detection scheme. Scale bar is 20 μm. Each image has a size of 512 × 512 pixels.

Figure 4

(a) Raman spectrum of a HEK-293 cell pellet. (b)-(f) SRS images of HEK-293 cells at (b) 2845 cm⁻¹, primarily lipid (c) 1004cm⁻¹, phenylalanine (d) 785 cm⁻¹, nucleic acid (e) 1090 cm⁻¹, primarily nucleic acid. (f) multicolor overlay of (b),(c),(d). Scale bar is 20 μm.

Figure 5

SRS images of MCF-7 cells at (a) 2845 cm⁻¹, primarily lipid (b) 1655cm⁻¹, primarily protein (c) 785cm⁻¹, nucleic acid, and (d)overlay of (a)-(c). SRS images of a few other MCF-7 cells at (e) 2845 cm⁻¹, (f) 1655cm⁻¹, (g) 785cm⁻¹ and (h)overlay of (e)-(g). Scale bar is 20 μm.