Chien-Sheng Liao

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

Vibrational imaging reveals vitamin E distribution on mouse skin in vivo and captures human breast cancer tissues in situ. 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.

Label-free spectroscopic detection of membrane potential using stimulated Raman scattering

Hyperspectral stimulated Raman scattering microscopy is deployed to measure single-membrane vibrational spectrum as a function of membrane potential. Using erythrocyte ghost as a model, quantitative correlation between transmembrane potential and Raman spectral profile was found. Specifically, the ratio between the area under Raman band at ∼2930 cm−1 and that at ∼2850 cm−1 increased by ∼2.6 times when the potential across the erythrocyte ghost membrane varied from +10 mV to −10 mV. Our results show the feasibility of employing stimulated Raman scattering microscopy to probe the membrane potential without labeling.

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.

Stimulated Raman spectroscopic imaging by microsecond delay-line tuning

Stimulated Raman scattering (SRS) microscopy is an emerging platform for vibrational imaging of living systems. We present microsecond-scale SRS spectroscopic imaging by temporally tuning two spectrally focused pulses through a resonant delay line. Our platform is able to acquire an SRS spectrum in 42 μs and form a spectral image of 40,000 pixels within 3.3 s. Spectroscopic identification of single bacteria and fungi in blood and 4-D imaging (x–y–z–λ) of intracellular organelles in live C. elegans are demonstrated.

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.

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.

Stimulated Raman Spectroscopic Imaging by Microsecond Delay-line Tuning

We report microsecond-scale acquisition of simulated Raman spectra by resonant delay-line tuning. Our scheme improved the spectral acquisition speed by 100 times compared to previous works by motorized translational-stage tuning. 4-D images (

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

\mathrm{x}-\mathrm{y}-\mathrm{z}-\lambda

High-Speed Spectroscopic Transient Absorption Imaging of Defects in Graphene

) from highly dynamic organelles in live C. elegans was demonstrated.

Sparsely-sampled hyperspectral stimulated Raman scattering microscopy: a theoretical investigation

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.