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Spectroscopic analysis of the oxygenation state of hemoglobin using coherent anti-Stokes Raman scattering

Hilde A. Rinia, Mischa Bonn, Erik M. Vartiainen, Chris B. Schaffer, and Michiel Müller

Journal of Biomedical Optics (2006)

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A method for noninvasively determining blood oxygenation in individual vessels inside bulk tissue would provide a powerful tool for biomedical research. We explore the potential of coherent anti-Stokes Raman scattering (CARS) spectroscopy to provide this capability. Using the multiplex CARS approach, we measure the vibrational spectrum in hemoglobin solutions as a function of the oxygenation state and observe a clear dependence of the spectral shape on oxygenation. The direct extraction of the Raman line shape from the CARS data using a maximum entropy method phase retrieval algorithm enables quantitative analysis. The CARS spectra associated with intermediate oxygenation saturation levels can be accurately described by a weighted sum of the fully oxygenated and fully deoxygenated spectra. We find that the degree of oxygenation determined from the CARS data agrees well with that determined by optical absorption. As a nonlinear optical technique, CARS inherently provides the 3-D imaging capability and tolerance to scattering necessary for biomedical applications. We discuss the challenges in extending the proof of principle demonstrated to in vivo applications.

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Spectroscopy of third-harmonic generation: evidence for resonances in model compounds and ligated hemoglobin

G. Omar Clay, Andrew C. Millard, Chris B. Schaffer, Juerg Aus-der-Au, Philbert S. Tsai, Jeffrey A. Squier, and David Kleinfeld

Journal of the Optical Society of America (2006)

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We report on third-harmonic generation (THG) of biomolecular solutions at the fluid/glass interface as a means to probe resonant contributions to their nonlinear absorption spectra that could serve as contrast mechanisms for functional imaging. Our source was 100 fs laser pulses whose center wavelength varied from 760 to 1000 nm. We find evidence of a two-photon resonance in the ratio of third-order susceptibilities, Xsample(3w)/Xglass, for aqueous solutions of Rhodamine B, Fura-2, and hemoglobin and a three-photon resonance in Xsample(3w)/Xglass for solutions of bovine serum albumin. Consistent with past work, we find evidence of a one-photon resonance of Xsample(3w)/Xglass for water, while confirming a lack of resonant enhancement for benzene. At physiological concentrations, hemoglobin in different ligand-binding states could be distinguished on the basis of features of its THG spectrum.

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Ultrafast processes for bulk modification of transparent materials

K. Itoh, W. Watanabe, S. Nolte, and C. B. Schaffer

Materials Research Society Bulletin (2006)

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When a femtosecond laser pulse is focused inside a transparent material, the optical intensity in the focal volume can become high enough to induce permanent structural modifications such as a refractive index change or the formation of a small vacancy. Thus, one can micromachine structures inside the bulk of a transparent material in three dimensions. We review the mechanisms of and techniques for bulk modification of transparent materials using femtosecond laser pulses and discuss the fabrication of photonic and other structures in transparent materials, including waveguides, couplers, gratings, diffractive lenses, optical data storage, and microfluidic channels.

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Ablation of cytoskeletal filaments and mitochondria in live cells using a femtosecond laser nanoscissor

Nan Shen, Dabajyoti Datta, Chris B. Schaffer, Philip LeDuc, Donald E.Ingber, Eric Mazur

Mechanics and Chemistry of Biosystems (2005)

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Analysis of cell regulation requires methods for perturbingmolecular processes within living cells with spatial discrimination on the nanometer-scale. We present a technique for ablating molecular structures in living cells using low-repetition rate, low-energy femtosecond laser pulses. By tightly focusing these pulses beneath the cell membrane, we ablate cellular material inside the cell through nonlinear processes. We selectively removed sub-micrometer regions of the cytoskeleton and individual mitochondria without altering neighboring structures or compromising cell viability. This nanoscissor technique enables non-invasive manipulation of the structural machinery of living cells with several-hundred-nanometer resolution. Using this approach, we unequivocally demonstrate that mitochondria are structurally independent functional units, and do not form a continuous network as suggested by some past studies.

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Femtosecond laser-drilled capillary integrated into a microfluidic device

Tyson N. Kim, Kyle Campbell, Alex Groisman, David Kleinfeld, and Chris B. Schaffer

Applied Physics Letters (2005)

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Recent growth in microfluidic technology is, to a large extent, driven by soft lithography, a high-throughput fabrication technique where polymer materials, such as polysdimethyld siloxane sPDMSd, are molded to form microscopic channel networks. Nevertheless, the channel architectures that can be obtained by molding are limited. We address this limitation by using femtosecond laser micromachining to add unmoldable features to the microfluidic devices. We apply laser ablation to drill microcapillaries, with diameters as small as 0.5 mm and aspect ratios as high as 800:1, in the walls of molded PDMS channels. Finally, we use a laser-drilled microcapillary to trap a polystyrene bead by suction and hold it against a shear flow

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Morphology of femtosecond laser-induced structural changes in bulk transparent materials

Chris B. Schaffer, Alan O. Jamison, and Eric Mazur

Applied Physics Letters (2004)

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Using optical and electron microscopy, we analyze the energy and focusing angle dependence of structural changes induced in bulk glass by tightly focused femtosecond laser pulses. We observe a transition from small density variations in the material to void formation with increasing laser energy. At energies close to the threshold for producing a structural change, the shape of the structurally changed region is determined by the focal volume of the objective used to focus the femtosecond pulse, while at higher energies, the structural change takes on a conical shape. From these morphological observations, we infer the role of various mechanisms for structural change

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All-Optical Histology Using Ultrashort Laser Pulses

Philbert S. Tsai, Beth Friedman, Agustin I. Ifarraguerri, Beverly D. Thompson, Varda Lev-Ram, Chris B. Schaffer, Qing Xiong, Roger Y. Tsien, Jeffrey A. Squier, and David Kleinfeld

Neuron (2003)

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As a means to automate the three-dimensional histological analysis of brain tissue, we demonstrate the use of femtosecond laser pulses to iteratively cut and image fixed as well as fresh tissue. Cuts are accomplished with 1 to 10 uJ pulses to ablate tissue with micron precision. We show that the permeability, immunoreactivity, and optical clarity of the tissue is retained after pulsed laser cutting. Further, samples from transgenic mice that express fluorescent proteins retained their fluorescence to within microns of the cut surface. Imaging of exogenous or endogenous fluorescent labels down to 100um or more below the cut surface is accomplished with 0.1 to 1 nJ pulses and conventional two-photon laser scanning microscopy. In one example, labeled projection neurons within the full extent of a neocortical column were visualized with micron resolution. In a second example, the microvasculature within a block of neocortex was measured and reconstructed with micron resolution.

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Bulk heating of transparent materials using a high-repetition-rate femtosecond laser

Chris B. Schaffer, Jose F. Garcia, and Eric Mazur

Applied Physics (2003)

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Femtosecond laser pulses can locally induce structural and chemical changes in the bulk of transparent materials, opening the door to the three-dimensional fabrication of optical devices. We review the laser and focusing parameters that have been applied to induce these changes and discuss the different physical mechanisms that play a role in forming them. We then describe a new technique for inducing refractive-index changes in bulk material using a high-repetition-rate femtosecond oscillator. The changes are caused by a localized melting of the material, which results from an accumulation of thermal energy due to nonlinear absorption of the high-repetition-rate train of laser pulses.

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Customization of Poly(dimethylsiloxane) Stamps by Micromachining Using a Femtosecond-Pulsed Laser

Daniel B. Wolfe, Jonathan B. Ashcom, Jeremy C. Hwang, Chris B. Schaffer, Eric Mazur, and George M. Whitesides

Advanced Materials (2003)

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This work describes the use of focused, high-intensity light from a Ti:sapphire laser that generates femtosecond pulses to create topographical structure in a flat surface of poly(dimethylsiloxane) (PDMS), and the use of the PDMS surfaces patterned in surface bas-reliefis the material most widely used for printing and stamping in soft lithography, and a material widely used in microfluidic systems. The bas-relief patterns required in these applications are usually fabricated by casting PDMS against a complementary bas-relief pattern in photoresist, fabricated in turn by photolithography. This process works well, but is not applicable to the preparation of PDMS stamps required for all types of problems; printing on spherical surfaces is an example.

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Microexplosions in tellurite glasses

S.K. Sundaram, Chris B. Schaffer, and Eric Mazur

Applied Physics (2003)

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Femtosecond laser pulses were used to produce localized damage in the bulk and near the surface of baseline, Al2O3-doped and La2O3-doped sodium tellurite glasses. Single or multiple laser pulses were non-linearly absorbed in the focal volume by the glass, leading to permanent changes in the material in the focal volume. These changes were caused by an explosive expansion of the ionized material in the focal volume into the surrounding material, i.e. a microexplosion. The writing of simple structures (periodic array of voxels, as well as lines) was demonstrated. The regions of microexplosion and writing were subsequently characterized using scanning electron microscopy (SEM), energy-dispersive spectrometry (EDS) and atomic force microscopy (AFM). Fingerprints of microexplosions (concentric lines within the region and a concentric ring outside the region), due to the shock wave generated during microexplosions, were evident. In the case of the baseline glass, no chemistry change was observed within the region of the microexplosion. However, Al2O3-doped and La2O3-doped glasses showed depletion of the dopant fromthe edge to the center of the region of the microexplosions, indicating a chemistry gradient within the regions. Interrogation of the bulk- and lasertreated regions using micro-Raman spectroscopy revealed no structural change due to the microexplosions and writing within these glasses

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