Coherent Raman scattering microscopy: capable solution in search of a larger audience.

SIGNIFICANCE: Coherent Raman scattering (CRS) microscopy is an optical imaging technique with capabilities that could benefit a broad range of biomedical research studies. AIM: We reflect on the birth, rapid rise, and inescapable growing pains of the technique and look back on nearly four decades of developments to examine where the CRS imaging approach might be headed in the next decade to come. APPROACH: We provide a brief historical account of CRS microscopy, followed by a discussion of the challenges to disseminate the technique to a larger audience. We then highlight recent progress in expanding the capabilities of the CRS microscope and assess its current appeal as a practical imaging tool. RESULTS: New developments in Raman tagging have improved the specificity and sensitivity of the CRS technique. In addition, technical advances have led to CRS microscopes that can capture hyperspectral data cubes at practical acquisition times. These improvements have broadened the application space of the technique. CONCLUSION: The technical performance of the CRS microscope has improved dramatically since its inception, but these advances have not yet translated into a substantial user base beyond a strong core of enthusiasts. Nonetheless, new developments are poised to move the unique capabilities of the technique into the hands of more users.

Infrared chemical imaging through non-degenerate two-photon absorption in silicon-based cameras.

Chemical imaging based on mid-infrared (MIR) spectroscopic contrast is an important technique with a myriad of applications, including biomedical imaging and environmental monitoring. Current MIR cameras, however, lack performance and are much less affordable than mature Si-based devices, which operate in the visible and near-infrared regions. Here, we demonstrate fast MIR chemical imaging through non-degenerate two-photon absorption (NTA) in a standard Si-based charge-coupled device (CCD). We show that wide-field MIR images can be obtained at 100 ms exposure times using picosecond pulse energies of only a few femtojoules per pixel through NTA directly on the CCD chip. Because this on-chip approach does not rely on phase matching, it is alignment-free and does not necessitate complex postprocessing of the images. We emphasize the utility of this technique through chemically selective MIR imaging of polymers and biological samples, including MIR videos of moving targets, physical processes and live nematodes.

Eicosapentaenoic acid (EPA) activates PPARγ signaling leading to cell cycle exit, lipid accumulation, and autophagy in human meibomian gland epithelial cells (hMGEC).

PURPOSE: The purpose of this study was to access the ability of the natural PPAR agonist, eicosapentaenoic acid (EPA), to activate PPAR gamma (γ) signaling leading to meibocyte differentiation in human meibomian gland epithelial cell (hMGEC). METHODS: HMGEC were exposed to EPA, alone and in combination with the specific PPARγ antagonist, T0070907, to selectively block PPARγ signaling. Expression of PPARγ response genes were evaluated by qPCR. Effect on cell cycle was evaluated using Ki-67 labelling and western blots. During differentiation, autophagy was monitored using the Autophagy Tandem Sensor (ATS) and LysoTracker. Lipid accumulation was characterized by Stimulated Raman Scattering microscopy (SRS) and neutral lipid staining in combination with ER-Tracker, LysoTracker, and ATS. Autophagy was also investigated using western blotting. Seahorse XF analysis was performed to monitor mitochondrial function. RESULTS: EPA specifically upregulated expression of genes related to lipid synthesis and induced cell cycle exit through reduced cyclin D1 expression and increased p21 and p27 expression. EPA also induced accumulation of lipid droplets in a time and dose dependent manner (P < 0.05) by specific PPARγ signaling. Lipid analysis identified both de novo synthesis and extracellular transport of lipid to form lipid droplets that were localized to the ER. PPARγ signaling also induced activation of AMPK-ULK1 signaling and autophagy, while inhibition of autophagy induced mitochondrial crisis with no effect on lipid accumulation. CONCLUSIONS: EPA induces meibocyte differentiation through PPARγ activation that is characterized by cell cycle exit, de novo and transported lipid accumulation in the ER, and autophagy.

Going visible: high-resolution coherent Raman imaging of cells and tissues.

A simple change of light source might prove what is needed for high-resolution label-free mapping of thin biological samples.

High-resolution infrared imaging of biological samples with third-order sum-frequency generation microscopy.

We studied the use of vibrationally resonant, third-order sum-frequency generation (TSFG) for imaging of biological samples. We found that laser-scanning TSFG provides vibrationally sensitive imaging capabilities of lipid droplets and structures in sectioned tissue samples. Although the contrast is based on the infrared-activity of molecular modes, TSFG images exhibit a high lateral resolution of 0.5 µm or better. We observed that the imaging properties of TSFG resemble the imaging properties of coherent anti-Stokes Raman scattering (CARS) microscopy, offering a nonlinear infrared alternative to coherent Raman methods. TSFG microscopy holds promise as a high-resolution imaging technique in the fingerprint region where coherent Raman techniques often provide insufficient sensitivity.

Stimulated Raman Scattering: From Bulk to Nano.

Stimulated Raman scattering (SRS) describes a family of techniques first discovered and developed in the 1960s. Whereas the nascent history of the technique is parallel to that of laser light sources, recent advances have spurred a resurgence in its use and development that has spanned across scientific fields and spatial scales. SRS is a nonlinear technique that probes the same vibrational modes of molecules that are seen in spontaneous Raman scattering. While spontaneous Raman scattering is an incoherent technique, SRS is a coherent process, and this fact provides several advantages over conventional Raman techniques, among which are much stronger signals and the ability to time-resolve the vibrational motions. Technological improvements in pulse generation and detection strategies have allowed SRS to probe increasingly smaller volumes and shorter time scales. This has enabled SRS research to move from its original domain, of probing bulk media, to imaging biological tissues and single cells at the micro scale, and, ultimately, to characterizing samples with subdiffraction resolution at the nanoscale. In this Review, we give an overview of the history of the technique, outline its basic properties, and present historical and current uses at multiple length scales to underline the utility of SRS to the molecular sciences.