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1.  Multicolored Stain-free Histopathology with Coherent Raman Imaging 
Conventional histopathology with hematoxylin & eosin (H&E) has been the gold standard for histopathological diagnosis of a wide range of diseases. However, it is not performed in vivo and requires thin tissue sections obtained after tissue biopsy, which carries risk, particularly in the central nervous system. Here we describe the development of an alternative, multicolored way to visualize tissue in real time through the use of coherent Raman imaging (CRI), without the use of dyes. CRI relies on intrinsic chemical contrast based on vibrational properties of molecules and intrinsic optical sectioning by nonlinear excitation. We demonstrate that multi-color images originating from CH2 and CH3 vibrations of lipids and protein, as well as two-photon absorption of hemoglobin, can be obtained with subcellular resolution from fresh tissue. These stain-free histopathological images show resolutions similar to those obtained by conventional techniques, but do not require tissue fixation, sectioning or staining of the tissue analyzed.
doi:10.1038/labinvest.2012.109
PMCID: PMC3622202  PMID: 22906986
Coherent anti-Stokes Raman scattering; CARS; Histology; In vivo microscopy; Stimulated Raman scattering; SRS
2.  Label-Free Biomedical Imaging with High Sensitivity by Stimulated Raman Scattering Microscopy 
Science (New York, N.Y.)  2008;322(5909):1857-1861.
Label-free chemical contrast is highly desirable in biomedical imaging. Spontaneous Raman microscopy provides specific vibrational signatures of chemical bonds, but is often hindered by low sensitivity. Here we report a three-dimensional multiphoton vibrational imaging technique based on stimulated Raman scattering (SRS). The sensitivity of SRS imaging is significantly greater than that of spontaneous Raman microscopy, which is achieved by implementing high-frequency (megahertz) phase-sensitive detection. SRS microscopy has a major advantage over previous coherent Raman techniques in that it offers background-free and readily interpretable chemical contrast. We show a variety of biomedical applications, such as differentiating distributions of omega-3 fatty acids and saturated lipids in living cells, imaging of brain and skin tissues based on intrinsic lipid contrast, and monitoring drug delivery through the epidermis.
doi:10.1126/science.1165758
PMCID: PMC3576036  PMID: 19095943
3.  Video-Rate Molecular Imaging In Vivo with Stimulated Raman Scattering 
Science (New York, N.Y.)  2010;330(6009):1368-1370.
Optical imaging in vivo with molecular specificity is important in biomedicine because of its high spatial resolution and sensitivity compared to MRI. Stimulated Raman scattering (SRS) microscopy allows highly sensitive optical imaging based on vibrational spectroscopy without adding toxic or perturbative labels. However, SRS tissue imaging in living animals and humans has not been feasible because of weak signals from thick tissues and motion blur due to limited acquisition speed. Here we make in vivo SRS imaging possible by significantly enhancing the collection of the backscattered signal and by increasing the imaging speed by three orders of magnitude, to video rate. This allows label-free in vivo imaging of water, lipid and protein in skin and mapping of penetration pathways of topically-applied drugs in mice and humans.
doi:10.1126/science.1197236
PMCID: PMC3462359  PMID: 21127249
4.  Synchronized time-lens source for coherent Raman scattering microscopy 
Optics express  2010;18(23):24019-24024.
We use the time-lens concept to demonstrate a new scheme for synchronization of two pulsed light sources for biological imaging. An all fiber, 1064 nm time-lens source is synchronized to a picosecond solid-state Ti: Sapphire mode-locked laser by using the mode-locked laser pulses as the clock. We demonstrate the application of this synchronized source for CARS and SRS imaging by imaging mouse tissues. Synchronized two wavelength pulsed source is an important technical difficulty for CARS and SRS imaging. The time-lens source demonstrated here may provide an all fiber, user friendly alternative for future SRS imaging.
PMCID: PMC3408908  PMID: 21164749
5.  Optical heterodyne-detected Raman-Induced Kerr Effect (OHD-RIKE) microscopy 
The journal of physical chemistry. B  2011;115(18):5574-5581.
Label-free microscopy based on Raman scattering has been increasingly used in biomedical research to image samples that cannot be labeled or stained. Stimulated Raman scattering (SRS) microscopy, allows signal amplification of the weak Raman signal for fast imaging speeds without introducing the non-resonant background and coherent image artifacts that are present in coherent anti-Stokes Raman scattering (CARS) microscopy. Here we present the Raman-induced Kerr effect (RIKE) as a contrast for label-free microscopy. RIKE allows us to measure different elements of the non-linear susceptibility tensor, both the real and imaginary parts by optical heterodyne detection (OHD-RIKE). OHD-RIKE microscopy provides information similar to polarization CARS (P-CARS) and interferometric CARS (I-CARS) microscopy, with a simple modification of the two-beam SRS microscopy setup. We show that while OHD-RIKE micro-spectroscopy can be in principle more sensitive than SRS, it does not supersede SRS microscopy of heterogeneous biological samples, such as mouse skin tissue, because it is complicated by variations of linear birefringence across the sample.
doi:10.1021/jp1113834
PMCID: PMC3412286  PMID: 21504149
6.  Imaging Drug Delivery to Skin with Stimulated Raman Scattering Microscopy 
Molecular pharmaceutics  2011;8(3):969-975.
Efficient drug delivery to the skin is essential for the treatment of major dermatologic diseases, such as eczema, psoriasis and acne. However, many compounds penetrate the skin barrier poorly and require optimized formulations to ensure their bioavailability. Here, stimulated Raman scattering (SRS) microscopy, a recently-developed, label-free chemical imaging tool, is used to acquire high resolution images of multiple chemical components of a topical formulation as it penetrates into mammalian skin. This technique uniquely provides label-free, non-destructive, three-dimensional images with high spatiotemporal resolution. It reveals novel features of (trans)dermal drug delivery in the tissue environment: different rates of drug penetration via hair follicles as compared to the intercellular pathway across the stratum corneum are directly observed, and the precipitation of drug crystals on the skin surface is visualized after the percutaneous penetration of the co-solvent excipient in the formulation. The high speed three-dimensional imaging capability of SRS thus reveals features that cannot be seen with other techniques, providing both kinetic information and mechanistic insight into the (trans)dermal drug delivery process.
doi:10.1021/mp200122w
PMCID: PMC3109166  PMID: 21548600
Skin; topical drug delivery; stimulated Raman scattering microscopy; skin penetration pathways; dermatopharmacokinetics
7.  Coherent Raman scanning fiber endoscopy 
Optics letters  2011;36(13):2396-2398.
Coherent Raman scattering methods allow for label-free imaging of tissue with chemical contrast and high spatial and temporal resolution. However, their imaging depth in scattering tissue is limited to less than 1 mm, requiring the development of endoscopes to obtain images deep inside the body. Here, we describe a coherent Raman endoscope that provides stimulated Raman scattering images at seven frames per second using a miniaturized fiber scanner, a custom-designed objective lens, and an optimized scheme for collection of scattered light from the tissue. We characterize the system and demonstrate chemical selectivity in mouse tissue images.
PMCID: PMC3164497  PMID: 21725423
8.  Synchronized time-lens source for coherent Raman scattering microscopy 
Optics Express  2010;18(23):24019-24024.
We use the time-lens concept to demonstrate a new scheme for synchronization of two pulsed light sources for biological imaging. An all fiber, 1064 nm time-lens source is synchronized to a picosecond solid-state Ti: Sapphire mode-locked laser by using the mode-locked laser pulses as the clock. We demonstrate the application of this synchronized source for CARS and SRS imaging by imaging mouse tissues. Synchronized two wavelength pulsed source is an important technical difficulty for CARS and SRS imaging. The time-lens source demonstrated here may provide an all fiber, user friendly alternative for future SRS imaging.
doi:10.1364/OE.18.024019
PMCID: PMC3408908  PMID: 21164749
(320.7090) Ultrafast lasers; (060.2380) Fiber optics sources; (180.5655) Raman microscopy
9.  High-power picosecond fiber source for coherent Raman microscopy 
Optics letters  2009;34(13):2051-2053.
We report a high-power picosecond fiber pump laser system for coherent Raman microscopy (CRM). The fiber laser system generates 3.5 ps pulses with 6 W average power at 1030 nm. Frequency doubling yields more than 2 W of green light, which can be used to pump an optical parametric oscillator to produce the pump and the Stokes beams for CRM. Detailed performance data on the laser and the various wavelength conversion steps are discussed, together with representative CRM images of fresh animal tissue obtained with the new source.
PMCID: PMC3142585  PMID: 19571996

Results 1-9 (9)