Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy.

Lipid metabolism is a fundamental physiological process necessary for cellular and organism health. Dysregulation of lipid metabolism often elicits obesity and many associated diseases including cardiovascular disorders, type II diabetes, and cancer. To advance the current understanding of lipid metabolic regulation, quantitative methods to precisely measure in vivo lipid storage levels in time and space have become increasingly important and useful. Traditional approaches to analyze lipid storage are semi-quantitative for microscopic assessment or lacking spatio-temporal information for biochemical measurement. Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology that enables rapid and quantitative detection of lipids in live cells with a subcellular resolution. As the contrast is exploited from intrinsic molecular vibrations, SRS microscopy also permits four-dimensional tracking of lipids in live animals. In the last decade, SRS microscopy has been widely used for small molecule imaging in biomedical research and overcome the major limitations of conventional fluorescent staining and lipid extraction methods. In the laboratory, we have combined SRS microscopy with the genetic and biochemical tools available to the powerful model organism, Caenorhabditis elegans, to investigate the distribution and heterogeneity of lipid droplets across different cells and tissues and ultimately to discover novel conserved signaling pathways that modulate lipid metabolism. Here, we present the working principles and the detailed setup of the SRS microscope and provide methods for its use in quantifying lipid storage at distinct developmental timepoints of wild-type and insulin signaling deficient mutant C. elegans.

Raman image-activated cell sorting.

The advent of image-activated cell sorting and imaging-based cell picking has advanced our knowledge and exploitation of biological systems in the last decade. Unfortunately, they generally rely on fluorescent labeling for cellular phenotyping, an indirect measure of the molecular landscape in the cell, which has critical limitations. Here we demonstrate Raman image-activated cell sorting by directly probing chemically specific intracellular molecular vibrations via ultrafast multicolor stimulated Raman scattering (SRS) microscopy for cellular phenotyping. Specifically, the technology enables real-time SRS-image-based sorting of single live cells with a throughput of up to ~100 events per second without the need for fluorescent labeling. To show the broad utility of the technology, we show its applicability to diverse cell types and sizes. The technology is highly versatile and holds promise for numerous applications that are previously difficult or undesirable with fluorescence-based technologies.

Label-free chemical imaging flow cytometry by high-speed multicolor stimulated Raman scattering.

Combining the strength of flow cytometry with fluorescence imaging and digital image analysis, imaging flow cytometry is a powerful tool in diverse fields including cancer biology, immunology, drug discovery, microbiology, and metabolic engineering. It enables measurements and statistical analyses of chemical, structural, and morphological phenotypes of numerous living cells to provide systematic insights into biological processes. However, its utility is constrained by its requirement of fluorescent labeling for phenotyping. Here we present label-free chemical imaging flow cytometry to overcome the issue. It builds on a pulse pair-resolved wavelength-switchable Stokes laser for the fastest-to-date multicolor stimulated Raman scattering (SRS) microscopy of fast-flowing cells on a 3D acoustic focusing microfluidic chip, enabling an unprecedented throughput of up to ∼140 cells/s. To show its broad utility, we use the SRS imaging flow cytometry with the aid of deep learning to study the metabolic heterogeneity of microalgal cells and perform marker-free cancer detection in blood.

Laser power density dependent energy transfer between Tm3+ and Tb3+: tunable upconversion emissions in NaYF4:Tm3+,Tb3+,Yb3+ microcrystals.

Energy transfer between Tm3+ and Tb3+ dependent on the power density of pump laser was investigated in NaYF4: Tb3+,Tm3+,Yb3+ microcrystals. Under the excitation of a 976-nm near-infrared laser at various power densities, Tb3+-Tm3+-Yb3+ doped samples exhibited intense visible emissions with tunable color between green and blue. The ratio of blue and green emission were determined by energy transfer between Tm3+ and Tb3+. When the power density of pump laser was low, the energy transfer process from Tm3+ (3F4) to Tb3+ (7F0) occurred efficiently. Upconversion processes in Tm3+ were inhibited, only visible emissions from Tb3+ with green color were observed. When the power density increased, energy transfer from the 3F4 (Tm3+) to 7F0 level (Tb3+) was restrained and population on high energy levels of Tm3+ was increased. Contribution of upconversion emissions from Tm3+ gradually became dominant. The emission color was tuned from green to blue with increasing the power density. Energy transfer processes between low-lying levels of activators, such as Tm3+ will greatly reduce the population on certain levels for further high-order upconversion processes. The Tb3+-Tm3+-Yb3+ doped phosphors are promising materials for detecting the condition of power density of the invisible near-infrared laser.

Experimental observation of multiple dispersive waves emitted by multiple mid-infrared solitons in a birefringence tellurite microstuctured optical fiber.

We experimentally demonstrate multiple dispersive waves (DWs) emitted by multiple mid-infrared solitons in a birefringence tellurite microstuctured optical fiber (BTMOF). To the best of our knowledge, this is the first demonstration of multiple DWs in the non-silica fibers. By using a pulse of ~80 MHz and ~200 fs emitted from an optical parametric oscillator (OPO) as the pump source, DWs and solitons are investigated on the fast and slow axes of the BTMOF at the pump wavelength of ~1800 nm. With the average pump power increasing from ~200 to 450 mW, the center wavelength of the 1st DW decreases from ~956 to 890 nm, the 2nd DW from ~1039 to 997 nm, the 3rd DW from ~1101 to 1080 nm, and the 4th DW from ~1160 to 1150 nm. Meanwhile, obvious multiple soliton self-frequency shifts (SSFSs) are observed in the mid-infrared region. Furthermore, DWs and solitons at the pump wavelength of ~1400 and 2000 nm are investigated at the average pump power of ~350 mW.

Widely tunable third-harmonic generation in a tellurite microstructured optical fiber.

We designed and fabricated a tellurite (76.5TeO(2)-6Bi(2)O(3)-11.5Li(2)O-6ZnO, mol. %) microstructured optical fiber (TMOF) with four air holes for widely tunable third-harmonic generation (THG). The loss of the TMOF is ~0.2 dB/m at 1550 nm. Widely tunable THG from ~567 to 902 nm is obtained when the TMOF is pumped by an optical parametric oscillator with the pump wavelength changing from ~1700 to 2700 nm. The mechanism of THG in this work is further investigated through the third-harmonic signal pattern, which is due to the high nonlinearity of the TMOF and the high pump power, not from the phase-matching process between the fundamental mode and the high-order TH mode.

Broadband cascaded four-wave mixing and supercontinuum generation in a tellurite microstructured optical fiber pumped at 2 µm.

We demonstrate the broadband cascaded four-wave mixing (FWM) and supercontinuum (SC) generation in a tellurite MOF which is made from 76.5TeO(2)-6ZnO-11.5Li(2)O-6Bi(2)O(3) (TZLB, mol%) glass. By using a 2-µm picosecond laser with the center wavelength of ~1958 nm as the pump source, the broadband FWM with the frequency separation of ~1.1 THz is obtained. The bandwidth of the frequency comb spans a range of ~630 nm from ~1620 to 2250 nm at the average pump power of ~125 mW. With the average pump power increasing to ~800 mW, the broadband mid-infrared SC generation with the spectrum from ~900 to 3900 nm is observed. Changing the pump source to a femtosecond laser (optical parametric oscillator, OPO) with the center wavelength of ~2000 nm, solitons and dispersive waves (DWs) are obtained.

Mid-infrared supercontinuum generation in a novel AsSe2-As2S5 hybrid microstructured optical fiber.

novel AsSe(2)-As(2)S(5) hybrid MOF (HMOF) is designed and fabricated by the rod-in-tube drawing technique. The core is made from AsSe2 glass and the cladding is made from As(2)S(5) glass. The loss is ~1.2 dB/m at ~3000 nm. Zero dispersion wavelength (ZDW) of the HMOF is ~3380 nm. Supercontinuum (SC) generation in a 2 cm-long HMOF is investigated with the pump wavelengths of ~3062, 3241 and 3389 nm from a tunable optical parametric oscillator (OPO) system. Broadband midinfrared (MIR) SC generation with the spectrum from ~1256 to 5400 nm is obtained with the peak power of ~1337 kW at the wavelength of ~3389 nm.

Dispersion characterization of two orthogonal modes in a birefringence tellurite microstructured optical fiber.

An elliptical core tellurite microstructured optical fiber with high birefringence was demonstrated and the chromatic dispersion of the two orthogonal modes in this fiber was experimentally characterized by a white light spectral interferometric technique over a wide spectral range. A series of spectral interferograms as a function of the optical path difference were recorded in the Mach-Zehnder interferometer. The birefringence dependence of the wavelength in the fiber was determined by interferograms. The measured and calculated dispersion matched well within the whole spectrum range under test.

Fabrication and characterization of a hybrid four-hole AsSe2-As2S5 microstructured optical fiber with a large refractive index difference.

A hybrid four-hole AsSe2-As2S5 microstructured optical fiber (MOF) with a large refractive index difference is fabricated by the rod-in-tube drawing technique. The core and the cladding are made from the AsSe2 glass and As2S5 glass, respectively. The propagation loss is ~1.8 dB/m and the nonlinear coefficient is ~2.03 × 10(4) km(-1)W(-1) at 2000 nm. Raman scattering is observed in the normal dispersion regime when the fiber is pumped by a 2 µm mode-locked picosecond fiber laser. Additionally, soliton is generated in the anomalous dispersion regime when the fiber is pumped by an optical parametric oscillator (OPO) at the pump wavelength of ~3000 nm.

Widely tunable second-harmonic generation in a chalcogenide-tellurite hybrid optical fiber.

When a chalcogenide-tellurite hybrid optical fiber with a high refractive index difference Δn=0.24 is pumped by an optical parametric oscillator with a pump wavelength from 1700 to 3000 nm, widely tunable second-harmonic generation (SHG) from 850 to 1502 nm is obtained. The observation of SHG is primarily due to the surface nonlinearity polarization at the core-cladding interface and the second-harmonic signal remains stable at the maximal level throughout the laser pulse irradiation.

Soliton self-frequency shift and third-harmonic generation in a four-hole As2S5 microstructured optical fiber.

Soliton self-frequency shift (SSFS) and third-harmonic generation (THG) are observed in a four-hole As2S5 chalcogenide microstructured optical fiber (MOF). The As2S5 MOF is tapered to offer an ideal environment for SSFS. After tapering, the zero-dispersion wavelength (ZDW) shifts from 2.02 to 1.61 µm, and the rate of SSFS can be enhanced by increasing the energy density of the pulse. By varying the average input power from 220 to 340 mW, SSFS of a soliton central wavelength from 2.206 to 2.600 µm in the mid-infrared is observed in the tapered segment, and THG at 632 nm is observed in the untapered segment.

Tunable third-harmonic generation in a chalcogenide-tellurite hybrid optical fiber with high refractive index difference.

A chalcogenide-tellurite hybrid optical fiber with a step-index structure is fabricated by the rod-in-tube drawing technique. The core is made of 15Ge-3Ga-12Sb-70S (mol. %) glass, and the cladding is made of 78TeO2-5ZnO-12Li2O-5Bi2O3 (mol. %) glass. The refractive index difference Δn=0.24. Tunable third-harmonic generation from 568 to 869 nm is observed when the optical fiber is pumped by an optical parametric oscillator with the pump wavelength changing from 1700 to 2600 nm.

Optical parametric gain and bandwidth in highly nonlinear tellurite hybrid microstructured optical fiber with four zero-dispersion wavelengths.

The parametric amplification gain and bandwidth in highly nonlinear tellurite hybrid microstructured optical fiber (HMOF) are simulated based on four wave mixing process. The fiber core and cladding materials are made of TeO(2)­Li(2)O­WO(3)­MoO(3)­Nb(2)O(5) and TeO(2)­ZnO­Na(2)O­P(2)O(5) glass, respectively. The fiber has four zero-dispersion wavelengths and the chromatic dispersion is flattened near the zerodispersion wavelengths. A broad gain bandwidth as wide as 1200 nm from 1290 to 2490 nm can be realized in the near infrared window by using a tellurite HMOF as short as 25 cm.

Third-harmonic generation in an elliptical-core ZBLAN fluoride fiber.

We demonstrate third-harmonic generation (THG) in an elliptical-core ZrF4-BaF2-LaF3-AlF3-NaF (ZBLAN) fluoride fiber, for the first time to our best knowledge. Linearly polarized THG around 523 nm is obtained when pumped by a pulse laser at 1560 nm. The extinction ratios of average power and peak power are ~6.7 and ~6.8 dB, respectively, in a 10 m long fiber. The extinction ratios are improved to ~11.1 and ~12.6 dB, respectively, when the fiber length is cut to 35 cm. Tunable THG from 605 to 740 nm is observed when pumped by an optical parametric oscillator with the pump wavelength changed from 1800 to 2200 nm.

Supercontinuum generation from a multiple-ring-holes tellurite microstructured optical fiber pumped by a 2 µm mode-locked picosecond fiber laser.

Supercontinuum (SC) generation from a highly nonlinear tellurite microstructured optical fiber with multiple rings of holes was demonstrated by pumping with a 2 µm mode-locked picosecond fiber laser. The chromatic dispersion of the fiber was measured with a homemade white-light spectral interferometer in a wide wavelength range and agreed with the theoretical calculation. Although the pumped wavelength was far from the zero dispersion wavelength, with flat dispersion profile of the fiber in the anomalous dispersion, the SC could be expanded from 650 to 2850 nm with launched pulse energy of several hundred picojoules. Simulations of SC generation agreed with the experimental results.

Mid-infrared supercontinuum generation in a suspended-core As2S3 chalcogenide microstructured optical fiber.

We demonstrate the supercontinuum (SC) generation in a suspended-core As(2)S(3) chalcogenide microstructured optical fiber (MOF). The variation of SC is investigated by changing the fiber length, pump peak power and pump wavelength. In the case of long fibers (20 and 40 cm), the SC ranges are discontinuous and stop at the wavelengths shorter than 3500 nm, due to the absorption of fiber. In the case of short fibers (1.3 and 2.4 cm), the SC ranges are continuous and can extend to the wavelengths longer than 4 µm. The SC broadening is observed when the pump peak power increases from 0.24 to 1.32 kW at 2500 nm. The SC range increases with the pump wavelength changing from 2200 to 2600 nm, corresponding to the dispersion of As(2)S(3) MOF from the normal to anomalous region. The SC generation is simulated by the generalized nonlinear Schrödinger equation. The simulation includes the SC difference between 1.3 and 2.4 cm long fiber by 2500 nm pumping, the variation of SC with pump peak power in 2.4 cm long fiber, and the variation of SC with pump wavelength in 1.3 cm long fiber. The simulation agrees well with the experiment.

A simple all-solid tellurite microstructured optical fiber.

A simple all-solid tellurite microstructured optical fiber which has only one layer of high-index rods in the cladding is proposed and fabricated in the paper. The core and the cladding with the low index are made from the TeO(2)-ZnO-Na(2)O-La(2)O(3) glass, and the high-index rods are made from the TeO(2)-Li(2)O-WO(3)-MoO(3)-Nb(2)O(5) glass. The guiding regime in this fiber can be explained by ARROW model. The fiber can support the near- and mid-infrared light transmitting in the core within the transmission bands while the all-solid silica microstructured optical fiber cannot. When the pump light is outside the transmission bands, the light will transmit in six TLWMN rods.

Fabrication and characterization of on-chip optical nonlinear chalcogenide nanofiber devices.

Chalcogenide (As(2)S(3)) nanofibers as narrow as 200 nm in diameter are drawn by the fiber pulling method, are successfully embedded in SU8 polymer, and form on-chip waveguides and high-Q microknot resonators (Q = 3.9 × 10(4)) with smooth cleaved end faces. Resonance tuning of resonators is realized by localized laser irradiation. Strong supercontinuum generation with a bandwidth of 500 nm is achieved in a 7-cm-long on-chip chalcogenide waveguide. Our result provides a method for the development of compact, high-optical-quality, and robust photonic devices.

55-fs pulse generation without wave-breaking from an all-fiber Erbium-doped ring laser.

We demonstrate the direct generation of 55 fs pulses from an all-fiber Erbium-doped ring laser oscillator using the nonlinear polarization rotation mode-locking. The average output power is 56.4 mW but limited by available pump power of 330 mW. The linear chirped pulse duration is 55 fs after recompression using standard single-mode fiber. The pulses show to resist optical wave breaking with a smooth spectrum without any side lobe and cw-breakthrough. This all-fiber laser exhibits relatively high transfer efficiency of 17% and the single pulse energy reaches 1.5 nJ.