Photothermal microscopy has enabled highly sensitive label-free imaging of absorbers, from metallic nanoparticles to chemical bonds. Photothermal signals are conventionally detected via modulation of excitation beam and demodulation of probe beam using lock-in amplifier. While convenient, the wealth of thermal dynamics is not revealed. Here, we present a lock-in free, mid-infrared photothermal dynamic imaging (PDI) system by MHz digitization and match filtering at harmonics of modulation frequency. Thermal-dynamic information is acquired at nanosecond resolution within single pulse excitation. Our method not only increases the imaging speed by two orders of magnitude but also obtains four-fold enhancement of signal-to-noise ratio over lock-in counterpart, enabling high-throughput metabolism analysis at single-cell level. Moreover, by harnessing the thermal decay difference between water and biomolecules, water background is effectively separated in mid-infrared PDI of living cells. This ability to nondestructively probe chemically specific photothermal dynamics offers a valuable tool to characterize biological and material specimens.

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.

To improve personalized diagnosis and prognosis for oral squamous cell carcinoma (OSCC) by identification of hub methylated-CpG sites and associated genes, weighted gene comethylation network analysis (WGCNA) was performed to examine and identify hub modules and CpG sites correlated with OSCC. Here, WGCNA modeling yielded blue and brown comethylation modules that were significantly associated with OSCC status. Following screening of the differentially expressed genes (DEGs) from gene expression microarrays and differentially methylated-CpG sites (DCGs), integrated multiomics analysis of the DEGs, DCGs, and hub CpG sites from the modules was performed to investigate their correlations. Expression levels of 16 CpG sites-associated genes were negatively correlated with methylation patterns of promoter. Moreover, Kaplan-Meier survival analysis of the hub CpG sites and associated genes was carried out using 2 public databases, MethSurv and GEPIA.Only 5 genes, ACTA1, ACTN2, OSR1, SYNGR1, and ZNF677, had significant overall survival using GEPIA. Hypermethylated-CpG sites ACTN2-cg21376883 and OSR1-cg06509239 were found to be associated with poor survival by MethSurv.Methylation status of specific site and expression levels of associated genes were determined using clinical samples by quantitative methylation-specific PCR and real-time PCR. Pearson's correlation analysis showed that methylation levels of cg06509239 and cg18335068 were negatively related to OSR1 and ZNF677 expression levels, respectively. Our classification schema using multiomics analysis represents a screening framework for identification of hub CpG sites and associated genes. K E Y W O R D SCpG site, methylation, multiomics, oral squamous cell carcinoma, survival analysis, WGCNA | 3175 DAI et Al.

Operable under ambient light and providing chemical selectivity, stimulated Raman scattering (SRS) microscopy opens a new window for imaging molecular events on a human subject, such as filtration of topical drugs through the skin. A typical approach for volumetric SRS imaging is through piezo scanning of an objective lens, which often disturbs the sample and offers a low axial scan rate. To address these challenges, we have developed a deformable mirror-based remote-focusing SRS microscope, which not only enables high-quality volumetric chemical imaging without mechanical scanning of the objective but also corrects the system aberrations simultaneously. Using the remote-focusing SRS microscope, we performed volumetric chemical imaging of living cells and captured in real time the dynamic diffusion of topical chemicals into human sweat pores.

Stimulated Raman scattering (SRS) microscopy has shown enormous potential in revealing molecular structures, dynamics and coupling in a complex system. However, the bond-detection sensitivity of SRS microscopy is fundamentally limited to milli-molar level due to the shot noise and the small modulation depth in either pump or Stokes beam4. Here, to overcome this barrier, we revisit SRS from the perspective of energy deposition. The SRS process pumps molecules to their vibrational excited states. The thereafter relaxation heats up the surrounding and induces a change in refractive index. By probing the refractive index change with a continuous wave beam, we introduce stimulated Raman photothermal (SRP) microscopy, where a >500-fold boost of modulation depth is achieved on dimethyl sulfide with conserved average power. Versatile applications of SRP microscopy on viral particles, cells, and tissues are demonstrated. With much improved signal to noise ratio compared to SRS, SRP microscopy opens a new way to perform vibrational spectroscopic imaging with ultrahigh sensitivity and minimal water absorption.

Photothermal microscopy has enabled highly sensitive label-free imaging of absorbers, from metallic nanoparticles to chemical bonds. Photothermal signals are conventionally detected via modulation of excitation beam and demodulation of probe beam using lock-in amplifier. While convenient, the wealth of thermal dynamic is not revealed. Here, we present a lock-in free, mid-infrared photothermal dynamic imaging (PDI) system by MHz digitization and match filtering at harmonics of modulation frequency. Thermal-dynamic information is acquired at nanosecond resolution within single pulse excitation. Our method not only increases the imaging speed by two orders of magnitude, but also obtains four-fold enhancement of signal-to-noise ratio over lock-in counterpart, enabling high-throughput metabolism analysis at single-cell level. Moreover, by harnessing the thermal decay difference between water and biomolecules, water background is effectively separated in mid-infrared PDI of living cells. This ability to nondestructively probe chemically specific photothermal dynamics offers a valuable tool to characterize biological and material specimens.

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