Multiwindow SRS Imaging Using a Rapid Widely Tunable Fiber Laser

Abstract

Spectroscopic stimulated Raman scattering (SRS) imaging has become a useful tool finding a broad range of applications. Yet, wider adoption is hindered by the bulky and environmentally sensitive solid-state optical parametric oscillator (OPO) in a current SRS microscope. Moreover, chemically informative multiwindow SRS imaging across C−H, C−D, and fingerprint Raman regions is challenging due to the slow wavelength tuning speed of the solid-state OPO. In this work, we present a multiwindow SRS imaging system based on a compact and robust fiber laser with rapid and wide tuning capability. To address the relative intensity noise intrinsic to a fiber laser, we implemented autobalanced detection, which enhances the signal-to-noise ratio of stimulated Raman loss imaging by 23 times. We demonstrate high-quality SRS metabolic imaging of fungi, cancer cells, and Caenorhabditis elegans across the C−H, C−D, and fingerprint Raman windows. Our results showcase the potential of the compact multiwindow SRS system for a broad range of applications.

Label-Free Imaging of Lipid Droplets in Prostate Cells Using Stimulated Raman Scattering Microscopy and Multivariate Analysis

“…Furthermore, the significant SRS signal detected at 3015 and 2965 cm –1 is indicative of unsaturated lipids (TAGs) and cholesterol esters (CEs), respectively . The average SRS spectra of cellular LDs suggest that TAGs are a major component of LDs in prostate cells due to the spectral similarity to a previous report that identified TAGs in ovarian cancer cells . We assessed the ratio 3015/2965 cm –1 (TAGs/CEs) for the three prostate cell lines (Figure B).…”

“…This result is also in agreement with a recent report that segmented protein and lipid rich regions in ovarian cancer cells using least absolute shrinkage and selection operator (LASSO) spectral unmixing. 34 It is interesting to note that the nucleoli cluster differently to the rest of the nucleus in each of the three cell lines, and may be a reflection of the different levels of DNA, RNA, and protein content across the nucleus as a whole. Areas corresponding to the non-cell background were clustered in (iv).…”

“…The clustering of the nucleus and cytoplasm regions in (i) suggest that protein signal is the likely discriminator for this cluster because cellular DNA is largely confined to the nucleus. This result is also in agreement with a recent report that segmented protein and lipid rich regions in ovarian cancer cells using least absolute shrinkage and selection operator (LASSO) spectral unmixing . It is interesting to note that the nucleoli cluster differently to the rest of the nucleus in each of the three cell lines, and may be a reflection of the different levels of DNA, RNA, and protein content across the nucleus as a whole.…”

“… 37 The average SRS spectra of cellular LDs suggest that TAGs are a major component of LDs in prostate cells due to the spectral similarity to a previous report that identified TAGs in ovarian cancer cells. 34 We assessed the ratio 3015/2965 cm –1 (TAGs/CEs) for the three prostate cell lines ( Figure 4 B). These data indicated that in the LNCaP and PNT2 cells, the LDs contain large quantities of TAGs with 3015/2965 cm –1 ratios >0.75, whereas in the case of PC3 cells, the ratio 3015/2965 cm –1 is ∼0.6, which indicated greater levels of CEs.…”

Visualization of a Limonene Synthesis Metabolon Inside Living Bacteria by Hyperspectral SRS Microscopy

“…By using three standard reference spectra: pure limonene, the wild-type strain, and the background (of each hSRS image stack), the potential Raman signal contribution from the precursors was forced to go to the cell body channel. To discriminate limonene and three precursors (IPP, DMAPP, and GPP), a broadband coherent Raman imaging system could be applied (50,51). By collecting a wider Raman spectral window, more metabolites could be quantified simultaneously, providing direct evidence of metabolite channeling (52).…”