High-content stimulated Raman histology of human breast cancer.

Histological examination is crucial for cancer diagnosis, however, the labor-intensive sample preparation involved in the histology impedes the speed of diagnosis. Recently developed two-color stimulated Raman histology could bypass the complex tissue processing to generates result close to hematoxylin and eosin staining, which is one of the golden standards in cancer histology. Yet, the underlying chemical features are not revealed in two-color stimulated Raman histology, compromising the effectiveness of prognostic stratification. Here, we present a high-content stimulated Raman histology (HC-SRH) platform that provides both morphological and chemical information for cancer diagnosis based on un-stained breast tissues. Methods: By utilizing both hyperspectral SRS imaging in the C-H vibration window and sparsity-penalized unmixing of overlapped spectral profiles, HC-SRH enabled high-content chemical mapping of saturated lipids, unsaturated lipids, cellular protein, extracellular matrix (ECM), and water. Spectral selective sampling was further implemented to boost the speed of HC-SRH. To show the potential for clinical use, HC-SRH using a compact fiber laser-based stimulated Raman microscope was demonstrated. Harnessing the wide and rapid tuning capability of the fiber laser, both C-H and fingerprint vibration windows were accessed. Results: HC-SRH successfully mapped unsaturated lipids, cellular protein, extracellular matrix, saturated lipid, and water in breast tissue. With these five chemical maps, HC-SRH provided distinct contrast for tissue components including duct, stroma, fat cell, necrosis, and vessel. With selective spectral sampling, the speed of HC-SRH was improved by one order of magnitude. The fiber-laser-based HC-SRH produced the same image quality in the C-H window as the state-of-the-art solid laser. In the fingerprint window, nucleic acid and solid-state ester contrast was demonstrated. Conclusions: HC-SRH provides both morphological and chemical information of tissue in a label-free manner. The chemical information detected is beyond the reach of traditional hematoxylin and eosin staining and heralds the potential of HC-SRH for biomarker discovery.

Millimeter-deep micron-resolution vibrational imaging by shortwave infrared photothermal microscopy.

Deep-tissue chemical imaging plays a vital role in biological and medical applications. Here, we present a shortwave infrared photothermal (SWIP) microscope for millimeter-deep vibrational imaging with sub-micron lateral resolution and nanoparticle detection sensitivity. By pumping the overtone transition of carbon-hydrogen bonds and probing the subsequent photothermal lens with shortwave infrared light, SWIP can obtain chemical contrast from polymer particles located millimeter-deep in a highly scattering phantom. By fast digitization of the optically probed signal, the amplitude of the photothermal signal is shown to be 63 times larger than that of the photoacoustic signal, thus enabling highly sensitive detection of nanoscale objects. SWIP can resolve the intracellular lipids across an intact tumor spheroid and the layered structure in millimeter-thick liver, skin, brain, and breast tissues. Together, SWIP microscopy fills a gap in vibrational imaging with sub-cellular resolution and millimeter-level penetration, which heralds broad potential for life science and clinical applications.

Stimulated Raman photothermal microscopy toward ultrasensitive chemical imaging.

Stimulated Raman scattering (SRS) microscopy has shown enormous potential in revealing molecular structures, dynamics, and couplings in complex systems. However, the sensitivity of SRS is fundamentally limited to the millimolar level due to shot noise and the small modulation depth. To overcome this barrier, we revisit SRS from the perspective of energy deposition. The SRS process pumps molecules to their vibrationally excited states. The subsequent relaxation heats up the surroundings and induces refractive index changes. By probing the refractive index changes with a laser beam, we introduce stimulated Raman photothermal (SRP) microscopy, where a >500-fold boost of modulation depth is achieved. The versatile applications of SRP microscopy on viral particles, cells, and tissues are demonstrated. SRP microscopy opens a way to perform vibrational spectroscopic imaging with ultrahigh sensitivity.

Stimulated Raman Photothermal Microscopy towards Ultrasensitive Chemical Imaging.

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.

A high-sensitivity high-resolution intravascular photoacoustic catheter through mode cleaning in a graded-index fiber.

Intravascular photoacoustic imaging has been developed to evaluate the possibility of plaque rupture in atherosclerosis by high spatial resolution imaging of lipid. However, the detection sensitivity and spatial resolution are compromised by the poor focusing caused by a multimode fiber. In this work, we report an intravascular photoacoustic catheter with mode self-cleaning in a graded-index fiber to improve the beam quality and the sensitivity for lipid detection. Compared with the higher-order modes in a step-index multimode fiber, the lower-order modes generated by the self-cleaning effect in the graded-index fiber greatly enhanced the photoacoustic spatial resolution and detection sensitivity. The dominant ringing artifact caused by laser absorption of the ultrasound transducer was further reduced by using stripe suppression. A lipid plaque mimicking phantom was imaged for evaluation. Lipid particles with a small diameter of 75.7 µm were clearly observed.

Nanosecond-resolution photothermal dynamic imaging via MHZ digitization and match filtering.

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.

Multiwindow SRS Imaging Using a Rapid Widely Tunable Fiber Laser.

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.

Volumetric chemical imaging in vivo by a remote-focusing stimulated Raman scattering microscope.

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.

Identification of hub methylated-CpG sites and associated genes in oral squamous cell carcinoma.

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.

Preparation and influencing factors of sirolimus liposome by supercritical fluid.

We prepared the sirolimus liposome by rapid expansion of supercritical solution (RESS) technology of supercritical fluid, and studied the effects of temperature, pressure, and equilibrium time on the average particle size and envelop rate of liposome. The conditions of the minimal average particle size of liposome were 328K of temperature (35MPa), 35MPa of pressure (333K), and 50 minutes of equilibrium (343K) time, respectively. The conditions of the maximal envelop rate of liposome were 333K of temperature (30MPa), 35MPa of pressure (343K), and 50 minutes of equilibrium (323K) time, respectively.