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Precise evaluation of breast tumor malignancy based on tissue calcifications has important practical value in the disease diagnosis, as well as the understanding of tumor development. Traditional X-ray mammography provides the overall morphologies of the calcifications but lacks intrinsic chemical information. In contrast, spontaneous Raman spectroscopy offers detailed chemical analysis but lacks the spatial profiles. Here, we applied hyperspectral stimulated Raman scattering (SRS) microscopy to extract both the chemical and morphological features of the microcalcifications, based on the spectral and spatial domain analysis. A total of 211 calcification sites from 23 patients were imaged with SRS, and the results were analyzed with a support vector machine (SVM) based classification algorithm. With optimized combinations of chemical and geometrical features of microcalcifications, we were able to reach a precision of 98.21% and recall of 100.00% for classifying benign and malignant cases, significantly improved from the pure spectroscopy or imaging based methods. Our findings may provide a rapid means to accurately evaluate breast tumor malignancy based on fresh tissue biopsies.

Van der Waals heterostructures of monolayer crystals are promising platforms for novel electronic, photonic and spintronic devices [1] - [2] . Fine control of the stacking rotation angle, i.e. the twist angle θ, allows to tailor their electronic and optical properties. In twisted bilayer graphene (tBLG), interlayer coupling leads to a complex subband electronic structure and van Hove singularities (vHs) with θ-dependent energy position [3] .

Coherent anti-Stokes Raman scattering (CARS) can be used to excite vibrational bonds with chemical selectivity, high spatial and spectral resolution, and high sensitivity, which has many applications in biomedical research. The common way to realize CARS imaging is illuminating sample with two synchronized ultra-short pulses. Recent development of various fiber laser schemes based on nonlinear optical effect provides compact laser source for CARS imaging. However, the nonlinear conversion in optical fiber may inevitably introduce temporal or spectral noise to the newly generated pulses. In this paper, we have proposed a polarization-maintaining (PM) passive-synchronized picosecond fiber laser system that generates dual-color picosecond pulses for CARS. An Er-doped fiber laser and a Yb-doped fiber laser were passively synchronized by cross phase modulation based on master-slave injection scheme. In the experiment, the wavelength of one branch was fixed at 791 nm, which was generated by second harmonic generation of Er-doped fiber laser. The wavelength of the other branch can be continuously tuned from 1017-1047 nm, which was obtained by adding an active spectral broadening module and an optical bandpass filter after Yb-doped fiber laser. As a result, the laser source allows to probe vibrational bonds with frequencies difference between 2809 cm-1 and 3091 cm-1 . Finally, the achieved tunable synchronized pulses enabled us to microscopically image mouse ear samples. The compact optical fiber laser proposed with PM fiber design, stable synchronization and large wavelength tunability would become a promising laser source for CARS imaging in clinical use.

Photochromic probes with reversible fluorescence have revolutionized the fields of single molecule spectroscopy and super-resolution microscopy, but lack sufficient chemical specificity. In contrast, Raman probes with stimulated Raman scattering (SRS) microscopy provides superb chemical resolution for super-multiplexed imaging, but are relatively inert. Here we report vibrational photochromism by engineering alkyne tagged diarylethene to realize photo-switchable SRS imaging. The narrow Raman peak of the alkyne group shifts reversibly upon photoisomerization of the conjugated diarylethene when irradiated by ultraviolet (UV) or visible light, yielding “on” or “off” SRS images taken at the photoactive Raman frequency. We demonstrated photo-rewritable patterning and encryption on thin films, painting/erasing of cells with labelled alkyne-diarylethene, as well as pulse-chase experiments of mitochondria diffusion in living cells. The design principle provides potentials for super-resolution microscopy, optical memories and switches with vibrational specificity., Probes with reversible fluorescence are useful in super-resolution microscopy, but lack sufficient chemical specificity. Here, the authors engineer alkyne tagged diarylethene to realize photo-switchable stimulated Raman scattering probes with high chemical resolution, for applications in living cells.

We have proposed and implemented a polarization-maintaining passively synchronized fiber laser system, which could deliver tunable dual-color picosecond pulses by including a frequency-doubling module and a spectral broadening module. Specifically, the output from the involved Er-doped fiber laser were used to generate second-harmonic pulses at 790 nm with a quadratic nonlinear crystal. In parallel, the amplified pulses from the synchronized Yb-doped fiber laser were launched into a 150-m single mode fiber, which resulted in not only substantial spectral bandwidth broadening from 0.1 to 20.1 nm, but also a significant Raman-induced signal around 1080 nm. Consequently, narrow spectra from 1018-1051 nm and 1070-1095 nm could be continuously tuned via a tunable bandpass filter, corresponding to Raman bonds from 2835-3143 cm−1 and 3312-3525 cm−1. Finally, the achieved tunable synchronized pulses enabled us to microscopically examine mouse ear samples based on coherent anti-Stokes Raman and second harmonic generation imaging. Therefore, our tunable passively-synchronized fiber laser system would be promising to provide a simple and compact laser source for subsequent coherent Raman microscopy.

As the graphene is transforming from a laboratory niche material into a mass-produced industrial commodity, maintaining high quality standard across different chemical vapor deposition (CVD) systems becomes the first priority. In this paper, we investigated the carbon diffusion behavior involving two kinds of annealing gases, which reduced the nucleation density in different rate. Compared to the time-consuming and hazardous high-pressure H 2 annealing, O 2 annealing represents a more efficient route to reduce the nucleation density down to ∼10 nuclei cm −2 in half an hour. We also proposed a well-defined carbon depletion and refilling mechanism, with which we can precisely gauge the minute carbon concentration inside Cu (less than 10 ppm), and control the nucleation density in a reproducible manner. Moreover, we find that O 2 displays an impressive carbon depletion activity, ∼6 order more efficient than H 2 . The graphene single crystals' quality was confirmed with Raman spectroscopy and field effect transistor (FET) devices.

Hexagonal boron nitride (h-BN) is an important member of two-dimensional (2D) materials with a large direct bandgap, and has attracted growing interest in ultraviolet optoelectronics and nanoelectronics. Compared with graphene and graphite, h-BN has weak Raman effect because of the far off-resonance excitation; hence, it is difficult to exploit Raman spectroscopy to characterize important properties of 2D h-BN, such as thickness, doping, and strain effects. Here, we applied stimulated Raman scattering (SRS) to enhance the sensitivity of the E2g Raman mode of h-BN. We showed that SRS microscopy achieves rapid high resolution imaging of h-BN with a pixel dwell time 4 orders of magnitude smaller than conventional spontaneous Raman microscopy. Moreover, the near-perfect linear dependence of signal intensity on h-BN thickness and isotropic polarization dependence allow convenient determination of the flake thickness with SRS imaging. Our results indicated that SRS microscopy provides a promising tool for high-speed quantification of h-BN and holds the potential for vibrational imaging of 2D materials.

The lignification triggered by biotic or abiotic stresses hardens fruits and vegetables and eventually influences their consumer appeal. Extensive prior efforts have been made to unveil the underlying mechanism of flesh lignification, primarily focused on its physicochemical and molecular biological properties. Nevertheless, most of these studies used destroyed and homogenized bulk tissues as analytes; as a result, potentially valuable spatial information was lost. In this study, the deposition of lignin in loquat flesh during lignification was visualized from the tissue level to the single-cell level by combining the advantages of stimulated Raman scattering (SRS) and spontaneous Raman microscopy using label-free in situ molecular imaging. SRS has the advantages of being fast and providing large-area chemical imaging to reveal the spatial heterogeneity of lignin and cell wall polysaccharide distribution in loquat flesh. After 2 days of storage at 0 °C, increased lignins were observed by large-area SRS imaging. In addition, microscopic SRS images of the flesh cells indicated that the increased lignins were trapped in the cell corner (CC) and middle lamella (ML). Furthermore, the compositional and structural features of lignified cells (LCs), CC and ML of loquat flesh were investigated by spontaneous Raman microscopy, and the results showed that the LCs were a combination of lignin, cellulose, and hemicellulose, whereas CC and ML showed only deposited lignin and pectin without cross-linked cellulose and hemicellulose. This result further suggests that the lignins in the CC and ML regions of loquats were later synthesized alone during postharvest storage. This innovative combination of SRS and spontaneous Raman microscopy allows the label-free macroscale and fine chemical imaging of plant cell walls and will enhance our fundamental understanding of the structures and functions of the plant cell wall.

Stimulated Raman scattering (SRS) microscopy that is capable of both high speed imaging and rapid spectroscopy will be advantageous for detailed chemical analysis of heterogeneous biological specimens. We have developed a system based on spectral focusing SRS technology, with the integration of a rapid scanning optical delay line (RSODL), which allows continuous tuning of SRS spectra by scanning a galvo mirror. We demonstrated SRS spectral measurements of dimethyl sulfoxide solution at low concentrations, and multi-color imaging of rice pollens and HeLa cells with line-by-line delay tuning to reduce motion artifacts, as well as fast acquisition of SRS spectra at specific regions of interest.

Two-color stimulated Raman scattering (SRS) microscopy with label-free mapping of lipid/protein distributions has shown promise in virtual histology. Despite previous demonstrations of SRS in tumor delineation and diagnosis, the speed and efficiency of the current technique requires further improvements for practical use. Here, we integrate parallel dual-phase SRS detection with strip mosaicing, which reduces the imaging time of a whole mouse brain section from 70 to 8 minutes. We further verified our method in imaging fresh human surgical tissues, showing its great potential for rapid SRS histology, especially for large scale, large quantity imaging tasks.

Label-free DNA imaging is highly desirable in biology and medicine to perform live imaging without affecting cell function and to obtain instant histological tissue examination during surgical procedures. Here we show a label-free DNA imaging method with stimulated Raman scattering (SRS) microscopy for visualization of the cell nuclei in live animals and intact fresh human tissues with subcellular resolution. Relying on the distinct Raman spectral features of the carbon-hydrogen bonds in DNA, the distribution of DNA is retrieved from the strong background of proteins and lipids by linear decomposition of SRS images at three optimally selected Raman shifts. Based on changes on DNA condensation in the nucleus, we were able to capture chromosome dynamics during cell division both in vitro and in vivo. We tracked mouse skin cell proliferation, induced by drug treatment, through in vivo counting of the mitotic rate. Furthermore, we demonstrated a label-free histology method for human skin cancer diagnosis that provides comparable results to other conventional tissue staining methods such as H&E. Our approach exhibits higher sensitivity than SRS imaging of DNA in the fingerprint spectral region. Compared with spontaneous Raman imaging of DNA, our approach is three orders of magnitude faster, allowing both chromatin dynamic studies and label-free optical histology in real time.

Stimulated Raman scattering (SRS) microscopy that is capable of both high-speed imaging and rapid spectroscopy will be advantageous for detailed chemical analysis of heterogeneous biological specimens. We have developed a system based on spectral focusing SRS technology with the integration of a rapid scanning optical delay line, which allows continuous tuning of SRS spectra by scanning a galvo mirror. We demonstrate SRS spectral measurements of dimethyl sulfoxide solution at low concentrations and multi-color imaging of rice pollens and HeLa cells with line-by-line delay tuning to reduce motion artifacts, as well as fast acquisition of SRS spectra at specific regions of interest.

Stimulated Raman scattering (SRS) microscopy is an emerging label-free imaging technique. It is a nonlinear version of Raman scattering, with the advantages of high chemical selectivity and rapid imaging. We have applied SRS microcopy for brain tumor detection on both in vivo mouse models and ex vivo human surgical specimens, based on the spectral differences between normal and tumor tissues. We further developed dual-phase parallel imaging technique for real-time two color SRS imaging, which provides a key advance for intraoperative virtual histology.

Probing nanoparticle-cell interactions in vivo plays a key role in the research and development of nanomedicine and nano-biophotonics. However, simultaneously imaging nanoparticles and intracellular biomolecules in a label-free manner has been a challenge, especially for the majority of nanoparticles that do not emit one- or two-photon excited fluorescence. In this work, a strong optical transient absorption (TA) signal is observed for synthesized MoS2 nanosheets that could be used for sensitive and quantitative imaging of their intracellular distributions. Furthermore, a dual-modal microscopy technique is developed to simultaneously image MoS2 with TA microscopy and cellular lipids/proteins with stimulated Raman scattering (SRS) microscopy. This technique is based on precise pulse engineering and dual-phase lock-in detection. Its capability to quantify the uptake dynamics of MoS2 into live HeLa cells is demonstrated and the colocalization of MoS2 nanosheets with intracellular lipid rich organelles is observed. Different sized MoS2 nanosheets are found to have distinct uptake dynamics as well as transient photocarrier dynamics. These results indicate that dual-modal SRS/TA microscopy may provide a general solution for investigating a large variety of nanomaterials that absorb light but do not fluoresce.

A compact, alignment-free, and inexpensive fiber source for coherent Raman spectroscopy would benefit the field considerably. We present a fiber optical parametric oscillator offering the best performance from a fiber-source to date. Pumping the oscillator with amplified pulses from a 1 μm fiber laser, we achieve widely spaced, narrowband pulses suitable for coherent anti-Stokes Raman scattering microscopy. The nearly transform limited, 2 ps signal pulses are generated through the use of normal dispersion four wave mixing in photonic crystal fiber, and can be tuned from 779-808 nm, limited by the tuning range of the seed laser. The average signal power can reach 180 mW (pulse energies up to 4 nJ). The long-wavelength idler field is resonant in the oscillator, and the use of a narrow bandpass filter in the feedback loop is critical for stable operation, as seen in both simulation and experiment. Due to the self-consistent nature of the oscillator, this source provides lower relative intensity noise on its output pulses than parametric amplifiers based on the same frequency conversion process. We present high quality images of mouse tissues taken with this source that exhibit an outstanding signal to noise ratio at top imaging speeds.

We present a synchronously pumped fiber optical parametric oscillator for coherent anti-Stokes Raman scattering microscopy. Pulses from a 1 μm Yb-doped fiber laser are amplified and frequency converted to 779–808 nm through normal dispersion four-wave mixing in a photonic crystal fiber. The idler frequency is resonant in the oscillator cavity, and we find that bandpass filtering the feedback is essential for a stable, narrow-bandwidth output. Experimental results agree quite well with numerical simulations of the device. Transform-limited 2 ps pulses with energy up to 4 nJ can be generated at the signal wavelength. The average power is 180 mW, and the relative-intensity noise is much lower than that of a similar parametric amplifier. High-quality coherent Raman images of mouse tissues recorded with this source are presented.

Stimulated Raman scattering (SRS) microscopy has opened up a wide range of biochemical imaging applications by probing a particular Raman-active molecule vibrational mode in the specimen. However, the original implementation with picosecond pulse excitation can only realize rapid chemical mapping with a single Raman band. Here we present a novel SRS microscopic technique using a grating-based pulse shaper for excitation and a grating-based spectrograph for detection to achieve simultaneous multicolor SRS imaging with high sensitivity and high acquisition speeds. In particular, we use a linear combination of the measured CH2 and CH3 stretching signals to map the distributions of protein and lipid contents simultaneously.

Label-free histology with chemical contrast has great potential for rapid intraoperative diagnosis. Two-color stimulated Raman scattering (SRS) microscopy has shown success in label-free digital histology with diagnostic results similar to those of hematoxylin and eosin stain. However, achieving real-time two-color SRS imaging has been a challenge. We have precisely engineered the pulse profiles of Stokes beams, and fully utilized in-phase (X) and quadrature (Y) outputs of a phase-sensitive lock-in amplifier to realize simultaneous two-color imaging. Such a method could reach the maximum speed as in single-color SRS, and has proven its robustness and advantages in real-time histology as well as in vivo imaging of live animals, both in transmission and epi modes. Moreover, this method could be conveniently adapted to other pump–probe-based microscopes.

Epi-detected stimulated Raman scattering microscopy using a high NA objective and long-wavelength excitation is presented. The increased NA and reduced scattering loss allow for high resolution imaging with deeper tissue penetration.

Stimulated Raman scattering (SRS) microscopy allows label-free chemical imaging based on vibrational spectroscopy. Narrowband excitation with picosecond lasers creates the highest signal levels and enables imaging speeds up to video-rate, but it sacrifices chemical specificity in samples with overlapping bands compared to broadband (multiplex) excitation. We develop a rapidly tunable picosecond optical parametric oscillator with an electro-optical tunable Lyot filter, and demonstrate multicolor SRS microscopy with synchronized line-by-line wavelength tuning to avoid spectral artifacts due to sample movement. We show sensitive imaging of three different kinds of polymer beads and live HeLa cells with moving intracellular lipid droplets.