Portable broadband photoacoustic spectroscopy for trace gas detection by quantum cascade laser arrays.

We report a portable broadband photoacoustic spectroscopic system for trace gas detection using distributed feedback quantum cascade laser arrays. By sequentially firing 128 lasers, our system acquires a photoacoustic spectrum covering 565cm-1 (935-1500cm-1) with a normalized-noise-equivalent-absorption coefficient of 2.5×10-9cm-1WHz-1/2. The firing sequence that determines when and which laser to activate is programmable, which enables frequency-multiplexing excitation. For demonstration, 12 lasers are modulated simultaneously at distinct frequencies, and a photoacoustic spectrum is acquired within 13 ms. The compactness (28cm×17cm×13cm, 3.5 kg) and low power consumption enable convenient installation for on-site monitoring.

Imaging of demineralized enamel in intact tooth by epidetected stimulated Raman scattering microscopy.

Stimulated Raman scattering microscopy (SRS) was deployed to quantify enamel demineralization in intact teeth. The surfaces of 15 bovine-enamel blocks were divided into four equal-areas, and chemically demineralized for 0, 8, 16, or 24 h, respectively. SRS images (spectral coverage from ∼850 to 1150 cm - 1) were obtained at 10-µm increments up to 90 µm from the surface to the dentin-enamel junction. SRS intensities of phosphate (peak: 959 cm - 1), carbonate (1070 cm - 1), and water (3250 cm - 1) were measured. The phosphate peak height was divided by the carbonate peak height to calculate the SRS-P/C-ratio, which was normalized relative to 90 µm (SRS-P/C-ratio-normalized). The water intensity against depth decay curve was fitted with exponential decay. A decay constant (SRS-water-content) was obtained. Knoop-hardness values were obtained before (SMHS) and after demineralization (SMHD). Surface microhardness-change (SMH-change) [ ( SMHD - SMHS ) / SMHS] was calculated. Depth and integrated mineral loss (ΔZ) were determined by transverse microradiography. Comparisons were made using repeated-measures of analysis of variance. For SRS-P/C-ratio-normalized, at 0-µm (surface), sound (0-h demineralization) was significantly higher than 8-h demineralization and 24-h demineralization; 16-h demineralization was significantly higher than 24-h demineralization. For SRS-water-content, 24-h demineralization was significantly higher than all other demineralization-groups; 8-h demineralization and 16-h demineralization were significantly higher than 0-h demineralization. SRS-water-content presented moderate-to-strong correlation with SMH-change and weak-to-moderate correlation with depth. These results collectively demonstrate the potential of using SRS microscopy for in-situ chemical analysis of dental caries.

High-Speed Spectroscopic Transient Absorption Imaging of Defects in Graphene.

Graphene grain boundaries (GBs) and other nanodefects can deteriorate electronic properties. Here, using transient absorption (TA) microscopy we directly visualized GBs by TA intensity increase due to change in density of state. We also observed a faster decay due to defect-accelerated carrier relaxation in the GB area. By line-illumination and parallel detection, we increased the TA intensity imaging speed to 1000 frames per second, which is 6 orders of magnitude faster than Raman microscopy. Combined with a resonant optical delay tuner which scans a 5.3 ps temporal delay within 92 µs, our system enabled spectroscopic TA imaging, at a speed of 50 stacks per second, to probe and characterize graphene nanodefects based on the TA decay rate. Finally, we demonstrate real-time nondestructive characterization of graphene at a rolling speed of 0.3 m/min, which matches the fastest roll-to-roll manufacturing process reported.

Spectroscopic stimulated Raman scattering imaging of highly dynamic specimens through matrix completion.

Spectroscopic stimulated Raman scattering (SRS) imaging generates chemical maps of intrinsic molecules, with no need for prior knowledge. Despite great advances in instrumentation, the acquisition speed for a spectroscopic SRS image stack is fundamentally bounded by the pixel integration time. In this work, we report three-dimensional sparsely sampled spectroscopic SRS imaging that measures ~20% of pixels throughout the stack. In conjunction with related work in low-rank matrix completion (e.g., the Netflix Prize), we develop a regularized non-negative matrix factorization algorithm to decompose the sub-sampled image stack into spectral signatures and concentration maps. This design enables an acquisition speed of 0.8 s per image stack, with 50 frames in the spectral domain and 40,000 pixels in the spatial domain, which is faster than the conventional raster laser-scanning scheme by one order of magnitude. Such speed allows real-time metabolic imaging of living fungi suspended in a growth medium while effectively maintaining the spatial and spectral resolutions. This work is expected to promote broad application of matrix completion in spectroscopic laser-scanning imaging.

High-speed stimulated hyperspectral Raman imaging using rapid acousto-optic delay lines.

Stimulated Raman scattering (SRS) is a powerful, label-free imaging technique that holds significant potential for medical imaging. To allow chemical specificity and minimize spectral distortion in the imaging of live species, a high-speed multiplex SRS imaging platform is needed. By combining a spectral focusing excitation technique with a rapid acousto-optic delay line, we demonstrate a hyperspectral SRS imaging platform capable of measuring a 3-dB spectral window of ∼200 cm-1 within 12.8 µs with a scan rate of 30 KHz. We present hyperspectral images of a mixture of two different microsphere polymers as well as live fungal cells mixed with human blood.

Label-Free Vibrational Spectroscopic Imaging of Neuronal Membrane Potential.

Detecting membrane potentials is critical for understanding how neuronal networks process information. We report a vibrational spectroscopic signature of neuronal membrane potentials identified through hyperspectral stimulated Raman scattering (SRS) imaging of patched primary neurons. High-speed SRS imaging allowed direct visualization of puff-induced depolarization of multiple neurons in mouse brain slices, confirmed by simultaneous calcium imaging. The observed signature, partially dependent on sodium ion influx, is interpreted as ion interactions on the CH3 Fermi resonance peak in proteins. By implementing a dual-SRS balanced detection scheme, we detected single action potentials in electrically stimulated neurons. These results collectively demonstrate the potential of sensing neuronal activities at multiple sites with a label-free vibrational microscope.

Coherent anti-Stokes Raman scattering imaging under ambient light.

We demonstrate an ambient light coherent anti-Stokes Raman scattering microscope that allows CARS imaging to be operated under environmental light for field use. The CARS signal is modulated at megahertz frequency and detected by a photodiode equipped with a lab-built resonant amplifier, then extracted through a lock-in amplifier. The filters in both the spectral domain and the frequency domain effectively blocked the room light contamination of the CARS image. In situ hyperspectral CARS imaging of tumor tissue under ambient light is demonstrated.

In Situ and In Vivo Molecular Analysis by Coherent Raman Scattering Microscopy.

Coherent Raman scattering (CRS) microscopy is a high-speed vibrational imaging platform with the ability to visualize the chemical content of a living specimen by using molecular vibrational fingerprints. We review technical advances and biological applications of CRS microscopy. The basic theory of CRS and the state-of-the-art instrumentation of a CRS microscope are presented. We further summarize and compare the algorithms that are used to separate the Raman signal from the nonresonant background, to denoise a CRS image, and to decompose a hyperspectral CRS image into concentration maps of principal components. Important applications of single-frequency and hyperspectral CRS microscopy are highlighted. Potential directions of CRS microscopy are discussed.

Spectrometer-free vibrational imaging by retrieving stimulated Raman signal from highly scattered photons.

In vivo vibrational spectroscopic imaging is inhibited by relatively slow spectral acquisition on the second scale and low photon collection efficiency for a highly scattering system. Recently developed multiplex coherent anti-Stokes Raman scattering and stimulated Raman scattering techniques have improved the spectral acquisition time down to microsecond scale. These methods using a spectrometer setting are not suitable for turbid systems in which nearly all photons are scattered. We demonstrate vibrational imaging by spatial frequency multiplexing of incident photons and single photodiode detection of a stimulated Raman spectrum within 60 µs. Compared to the spectrometer setting, our method improved the photon collection efficiency by two orders of magnitude for highly scattering specimens. We demonstrated in vivo imaging of vitamin E distribution on mouse skin and in situ imaging of human breast cancerous tissues. The reported work opens new opportunities for spectroscopic imaging in a surgical room and for development of deep-tissue Raman spectroscopy toward molecular level diagnosis.

Fabrication of Sub-25 nm Diameter GaSb Nanopillar Arrays by Nanoscale Self-Mask Effect.

GaSb individual nanowires and nanowire arrays are considered as intriguing candidates for electronic and photonic applications. In this paper, we report a new mask-free method to fabricate large area GaSb nanopillar arrays through reactive ion etching of GaSb substrates facilitated by O2 plasma. We have shown that nanoscale oxide self-masks could form thereby facilitating the formation of GaSb nanopillars. We have achieved GaSb nanowires with diameters less than 25 nm and an aspect ratio of 24. Additionally, GaSb nanopillar arrays with desired heights, diameters, and density can be obtained by choosing the plasma chemistry and/or controlling etching parameters, such as bias power and pressure. The nanopillar arrays prepared also exhibit tunable broadband antireflection properties.

Microsecond Scale Vibrational Spectroscopic Imaging by Multiplex Stimulated Raman Scattering Microscopy.

Real-time vibrational spectroscopic imaging is desired for monitoring cellular states and cellular processes in a label-free manner. Raman spectroscopic imaging of highly dynamic systems is inhibited by relatively slow spectral acquisition on millisecond to second scale. Here, we report microsecond scale vibrational spectroscopic imaging by lock-in free parallel detection of spectrally dispersed stimulated Raman scattering signal. Using a homebuilt tuned amplifier array, our method enables Raman spectral acquisition, within the window defined by the broadband pulse, at the speed of 32 microseconds and with close to shot-noise limited detection sensitivity. Incorporated with multivariate curve resolution analysis, our platform allows compositional mapping of lipid droplets in single live cells, observation of intracellular retinoid metabolism, discrimination of fat droplets from protein-rich organelles in Caenorhabditis elegans, spectral detection of fast flowing tumor cells, and monitoring drug diffusion through skin tissue in vivo. The reported technique opens new opportunities for compositional analysis of cellular compartment in a microscope setting and high-throughput spectral profiling of single cells in a flow cytometer setting.

Denoising Stimulated Raman Spectroscopic Images by Total Variation Minimization.

High-speed coherent Raman scattering imaging is opening a new avenue to unveiling the cellular machinery by visualizing the spatio-temporal dynamics of target molecules or intracellular organelles. By extracting signals from the laser at MHz modulation frequency, current stimulated Raman scattering (SRS) microscopy has reached shot noise limited detection sensitivity. The laser-based local oscillator in SRS microscopy not only generates high levels of signal, but also delivers a large shot noise which degrades image quality and spectral fidelity. Here, we demonstrate a denoising algorithm that removes the noise in both spatial and spectral domains by total variation minimization. The signal-to-noise ratio of SRS spectroscopic images was improved by up to 57 times for diluted dimethyl sulfoxide solutions and by 15 times for biological tissues. Weak Raman peaks of target molecules originally buried in the noise were unraveled. Coupling the denoising algorithm with multivariate curve resolution allowed discrimination of fat stores from protein-rich organelles in C. elegans. Together, our method significantly improved detection sensitivity without frame averaging, which can be useful for in vivo spectroscopic imaging.

Observation of spontaneous polarization misalignments in periodically poled crystals using second-harmonic generation microscopy.

Periodically poled crystal (PPC) is a key component for nonlinear optical applications. Its poling quality relies largely on successful domain inversion and the alignment of spontaneous polarization (SP) vectors in each domain. Here we report the unexpected observation of bulk second harmonic generation (SHG) in PPC when excitation propagating along its optical axis. Based on its tensorial nature, SHG is highly sensitive to the orientation of SP, and therefore the misalignment of SP in each domain of PPC can be revealed noninvasively by SHG microscopy. This nonlinear imaging modality provides optical sectioning capability with 3D sub-micrometer resolution, so it will be useful for in situ investigation of poling quality in PPC.

Second harmonic generation imaging - a new method for unraveling molecular information of starch.

We present a new method, second harmonic generation (SHG) imaging for the study of starch structure. SHG imaging can provide the structural organization and molecular orientation information of bio-tissues without centrosymmetry. In recent years, SHG has proven its capability in the study of crystallized bio-molecules such as collagen and myosin. Starch, the most important food source and a promising future energy candidate, has, for a decade, been shown to exhibit strong SHG response. By comparing SHG intensity from different starch species, we first identified that the SHG-active molecule is amylopectin, which accounts for the crystallinity in starch granules. With the aid of SHG polarization anisotropy, we extracted the complete χ((2)) tensor of amylopectin, which reflects the underlying molecular details. Through χ((2)) tensor analysis, three-dimensional orientation and packing symmetry of amylopectin are determined. The helical angle of the double-helix in amylopectin is also deduced from the tensor, and the value corresponds well to previous X-ray studies, further verifying amylopectin as SHG source. It is noteworthy that the nm-sized structure of amylopectin inside a starch granule can be determined by this far-field optical method with 1-µm excitation wavelength. Since SHG is a relatively new tool for plant research, a detailed understanding of SHG in starch structure will be useful for future high-resolution imaging and quantitative analyses for food/energy applications.

A diffraction-limited scanning system providing broad spectral range for laser scanning microscopy.

Diversified research interests in scanning laser microscopy nowadays require broadband capability of the optical system. Although an all-mirror-based optical design with a suitable metallic coating is appropriate for broad-spectrum applications from ultraviolet to terahertz, most researchers prefer lens-based scanning systems despite the drawbacks of a limited spectral range, ghost reflection, and chromatic aberration. One of the main concerns is that the geometrical aberration induced by off-axis incidence on spherical mirrors significantly deteriorates image resolution. Here, we demonstrate a novel geometrical design of a spherical-mirror-based scanning system in which off-axis aberrations, both astigmatism and coma, are compensated to reach diffraction-limited performance. We have numerically simulated and experimentally verified that this scanning system meets the Marechal condition and provides high Strehl ratio within a 3 degrees x 3 degrees scanning area. Moreover, we demonstrate second-harmonic-generation imaging from starch with our new design. A greatly improved resolution compared to the conventional mirror-based system is confirmed. This scanning system will be ideal for high-resolution linear/nonlinear laser scanning microscopy, ophthalmoscopic applications, and precision fabrications.