Freudiger, Christian Wilhelm

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Freudiger, Christian Wilhelm

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Freudiger

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Christian Wilhelm

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Freudiger, Christian Wilhelm

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Video Rate Molecular Imaging In Vivo with Stimulated Raman Scattering
(American Association for the Advancement of Science, 2010) Saar, Brian; Freudiger, Christian Wilhelm; Reichman, Jay; Stanley, C. Michael; Holtom, Gary; Xie, Xiaoliang
Optical imaging in vivo with molecular specificity is important in biomedicine because of its high spatial resolution and sensitivity compared with magnetic resonance imaging. Stimulated Raman scattering (SRS) microscopy allows highly sensitive optical imaging based on vibrational spectroscopy without adding toxic or perturbative labels. However, SRS imaging in living animals and humans has not been feasible because light cannot be collected through thick tissues, and motion-blur arises from slow imaging based on backscattered light. In this work, we enable in vivo SRS imaging by substantially enhancing the collection of the backscattered signal and increasing the imaging speed by three orders of magnitude to video rate. This approach 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.
Multicolor Stimulated Raman Scattering Microscopy with a Rapidly Tunable Optical Parametric Oscillator
(Optical Society of America, 2013) Kong, Lingjie; Ji, Minbiao; Holtom, Gary R.; Fu, Dan; Freudiger, Christian Wilhelm; Xie, Xiaoliang
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.
Stimulated Raman Scattering Microscopy with a Robust Fibre Laser Source
(Nature Publishing Group, 2014) Freudiger, Christian Wilhelm; Yang, Wenlong; Holtom, Gary; Peyghambarian, Nasser; Xie, Xiaoliang; Kieu, Khanh Q.
Stimulated Raman scattering microscopy allows label-free chemical imaging and has enabled exciting applications in biology, material science and medicine. It provides a major advantage in imaging speed over spontaneous Raman scattering and has improved image contrast and spectral fidelity compared to coherent anti-Stokes Raman scattering. Wider adoption of the technique has, however, been hindered by the need for a costly and environmentally sensitive tunable ultrafast dual-wavelength source. We present the development of an optimized all-fibre laser system based on the optical synchronization of two picosecond power amplifiers. To circumvent the high-frequency laser noise intrinsic to amplified fibre lasers, we have further developed a high-speed noise cancellation system based on voltage-subtraction autobalanced detection. We demonstrate uncompromised imaging performance of our fibre-laser-based stimulated Raman scattering microscope with shot-noise-limited sensitivity and an imaging speed up to 1 frame \(s^{−1}\).
Quantitative Chemical Imaging with Multiplex Stimulated Raman Scattering Microscopy
(American Chemical Society, 2012) Fu, Dan; Lu, Fake; Zhang, Xu; Freudiger, Christian Wilhelm; Pernik, Douglas R.; Holtom, Gary; Xie, Xiaoliang
Stimulated Raman scattering (SRS) microscopy is a newly developed label-free chemical imaging technique that overcomes the speed limitation of confocal Raman microscopy while avoiding the nonresonant background problem of coherent anti-Stokes Raman scattering (CARS) microscopy. Previous demonstrations have been limited to single Raman band measurements. We present a novel modulation multiplexing approach that allows real-time detection of multiple species using the fast Fourier transform. We demonstrate the quantitative determination of chemical concentrations in a ternary mixture. Furthermore, two imaging applications are pursued: (1) quantitative determination of oil content as well as pigment and protein concentration in microalgae cultures; and (2) 3D high-resolution imaging of blood, lipids, and protein distribution in ex vivo mouse skin tissue. We believe that quantitative multiplex SRS uniquely combines the advantage of fast label-free imaging with the fingerprinting capability of Raman spectroscopy and enables numerous applications in lipid biology as well as biomedical imaging.
Coherent Nonlinear Optical Imaging: Beyond Fluorescence Microscopy
(Annual Reviews, 2011) Min, Wei; Freudiger, Christian Wilhelm; Lu, Sijia; Xie, Xiaoliang
The quest for ultrahigh detection sensitivity with spectroscopic contrasts other than fluorescence has led to various novel approaches to optical microscopy of biological systems. Coherent nonlinear optical imaging, especially the recently developed nonlinear dissipation microscopy (including stimulated Raman scattering and two-photon absorption) and pump-probe microscopy (including excited-state absorption, stimulated emission, and ground-state depletion), provides new image contrasts for nonfluorescent species. Thanks to the high-frequency modulation transfer scheme, these imaging techniques exhibit superb detection sensitivity. By directly interrogating vibrational and/or electronic energy levels of molecules, they offer high molecular specificity. Here we review the underlying principles and excitation and detection schemes, as well as exemplary biomedical applications of this emerging class of molecular imaging techniques.
Raman Spectroscopy: Stimulated Raman Scattering Microscopy for Label-Free Chemical Imaging
(Optical Society of America, 2009) Freudiger, Christian Wilhelm; Min, Wei; Saar, Brian; Xie, Xiaoliang
Coherent anti-Stokes Raman techniques are increasing the utility of Raman scattering for chemical and biological diagnostics.
Label-Free Biomedical Imaging with High Sensitivity by Stimulated Raman Scattering Microscopy
(American Association for the Advancement of Science, 2008) Freudiger, Christian Wilhelm; Min, Wei; Saar, Brian G.; Lu, Sijia; Holtom, Gary R.; He, Chengwei; Tsai, Jason C.; Kang, Jing; Xie, Xiaoliang
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.
Hyperspectral Imaging with Stimulated Raman Scattering by Chirped Femtosecond Lasers
(American Chemical Society, 2013-03-22) Fu, Dan; Holtom, Gary; Freudiger, Christian Wilhelm; Zhang, Xu; Xie, Xiaoliang
Raman microscopy is a quantitative, label-free, and noninvasive optical imaging technique for studying inhomogeneous systems. However, the feebleness of Raman scattering significantly limits the use of Raman microscopy to low time resolutions and primarily static samples. Recent developments in narrowband stimulated Raman scattering (SRS) microscopy have significantly increased the acquisition speed of Raman based label-free imaging by a few orders of magnitude, at the expense of reduced spectroscopic information. On the basis of a spectral focusing approach, we present a fast SRS hyperspectral imaging system using chirped femtosecond lasers to achieve rapid Raman spectra acquisition while retaining the full speed and image quality of narrowband SRS imaging. We demonstrate that quantitative concentration determination of cholesterol in the presence of interfering chemical species can be achieved with sensitivity down to 4 mM. For imaging purposes, hyperspectral imaging data in the C–H stretching region is obtained within a minute. We show that mammalian cell SRS hyperspectral imaging reveals the spatially inhomogeneous distribution of saturated lipids, unsaturated lipids, cholesterol, and protein. The combination of fast spectroscopy and label-free chemical imaging will enable new applications in studying biological systems and material systems.
Rapid, Label-Free Detection of Brain Tumors with Stimulated Raman Scattering Microscopy
(American Association for the Advancement of Science (AAAS), 2013) Ji, Minbiao; Orringer, Daniel A.; Freudiger, Christian Wilhelm; Ramkissoon, Shakti H.; Liu, Xiaohui; Lau, Darryl; Golby, Alexandra; Norton, Isaiah Hakim; Hayashi, Marika; Agar, Nathalie; Young, Geoffrey; Spino, Cathie; Santagata, Sandro; Camelo-Piragua, Sandra; Ligon, Keith; Sagher, Oren; Xie, Xiaoliang
Surgery is an essential component in the treatment of brain tumors. However, delineating tumor from normal brain remains a major challenge. We describe the use of stimulated Raman scattering (SRS) microscopy for differentiating healthy human and mouse brain tissue from tumor-infiltrated brain based on histoarchitectural and biochemical differences. Unlike traditional histopathology, SRS is a label-free technique that can be rapidly performed in situ. SRS microscopy was able to differentiate tumor from nonneoplastic tissue in an infiltrative human glioblastoma xenograft mouse model based on their different Raman spectra. We further demonstrated a correlation between SRS and hematoxylin and eosin microscopy for detection of glioma infiltration (κ = 0.98). Finally, we applied SRS microscopy in vivo in mice during surgery to reveal tumor margins that were undetectable under standard operative conditions. By providing rapid intraoperative assessment of brain tissue, SRS microscopy may ultimately improve the safety and accuracy of surgeries where tumor boundaries are visually indistinct.
Synchronized Time-Lens Source for Coherent Raman Scattering Microscopy
(Optical Society of America, 2010) Wang, Ke; Freudiger, Christian Wilhelm; Lee, Jennifer H.; Saar, Brian G.; Xie, Xiaoliang; Xu, Chris
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.