Spectroscopic analysis of the sum-frequency response of the carbon-hydrogen stretching modes in collagen type I.

We studied the origin of the vibrational signatures in the sum-frequency generation (SFG) spectrum of fibrillar collagen type I in the carbon-hydrogen stretching regime. For this purpose, we developed an all-reflective, laser-scanning SFG microscope with minimum chromatic aberrations and excellent retention of the polarization state of the incident beams. We performed detailed SFG measurements of aligned collagen fibers obtained from rat tail tendon, enabling the characterization of the magnitude and polarization-orientation dependence of individual tensor elements Xijk2 of collagen's nonlinear susceptibility.

Volumetric Temperature Mapping Using Light-Sheet Microscopy and Upconversion Fluorescence from Micro- and Nano-Rare Earth Composites.

We present a combination of light-sheet excitation and two-dimensional fluorescence intensity ratio (FIR) measurements as a simple and promising technique for three-dimensional temperature mapping. The feasibility of this approach is demonstrated with samples fabricated with sodium yttrium fluoride nanoparticles co-doped with rare-earth ytterbium and erbium ions (NaYF:Yb/Er) incorporated into polydimethylsiloxane (PDMS) as a host material. In addition, we also evaluate the technique using lipid-coated NaYF:Yb/Er nanoparticles immersed in agar.

Two-photon absorption spectrometers for near infrared.

We present a Silicon-based Charge-Coupled Device (Si-CCD) sensor applied as a cost-effective spectrometer for femtosecond pulse characterization in the Near Infrared region in two different configurations: two-Fourier and Czerny-Turner setups. To test the spectrometer's performance, a femtosecond Optical Parametric Oscillator with a tuning range between 1100 and 1700 nm and a femtosecond Erbium-Doped Fiber Amplifier at 1582 nm were employed. The nonlinear spectrometer operation is based on the Two-Photon Absorption effect generated in the Si-CCD sensor.

Multicolor light-sheet microscopy for a large field of view imaging: A comparative study between Bessel and Gaussian light-sheets configurations.

Light-sheet fluorescence microscopy (LSFM) is useful for developmental biology studies, which require a simultaneous visualization of dynamic microstructures over large fields of views (FOVs). A comparative study between multicolor Bessel and Gaussian-based LSFM systems is presented. Discussing the chromatic implications to achieve colocalized and large FOVs when both optical arrays are implemented under the same excitation objective is the purpose of this work.

Second harmonic generation signal from type I collagen fibers grown .

We present a study of the optical second-order nonlinearity of type I collagen fibers grown via second harmonic generation (SHG) experiments and analyze the observed polarization-resolved SHG signal using previously reported SHG analytical expressions obtained for anisotropic tissue. Our results indicate that the effective second-order nonlinearity measured in the grown fibers is one order of magnitude lower than that of native collagen fibers. This is attributed to the formation of loose and dispersive fibrillar networks of thinner collagen fibrils that constitute the reassembled collagen fibers.

Multicolor fluorescence microscopy using static light sheets and a single-channel detection.

We present a multicolor fluorescence microscope system, under a selective plane illumination microscopy (SPIM) configuration, using three continuous wave-lasers and a single-channel-detection camera. The laser intensities are modulated with three time-delayed pulse trains that operate synchronously at one third of the camera frame rate, allowing a sequential excitation and an image acquisition of up to three different biomarkers. The feasibility of this imaging acquisition mode is demonstrated by acquiring single-plane multicolor images of living hyphae of Neurospora crassa.

Light sheet microscopy and SrAl O nanoparticles codoped with Eu /Dy ions for cancer cell tagging.

Light sheet optical microscopy on strontium aluminate nanoparticles (SrAl O NPs)1 codoped with Eu and Dy was used for cancer cell tagging and tracking. The nanoparticles were synthesized by urea-assisted combustion with optimized percentage values of the 2 codoping rare-earth ions for cell viability and for lower cytotoxic effects. The optical properties of these materials showed an excitation wide range of wavelengths (λ = 254-460 nm), a broad emission band (λ = 475-575 nm) with the maximum centered wavelength at 525 nm and a half lifetime within the seconds regime.

Rapid spontaneous Raman light sheet microscopy using cw-lasers and tunable filters.

We perform rapid spontaneous Raman 2D imaging in light-sheet microscopy using continuous wave lasers and interferometric tunable filters. By angularly tuning the filter, the cut-on/off edge transitions are scanned along the excited Stokes wavelengths. This allows obtaining cumulative intensity profiles of the scanned vibrational bands, which are recorded on image stacks; resembling a spectral version of the knife-edge technique to measure intensity profiles.

Image formation by linear and nonlinear digital scanned light-sheet fluorescence microscopy with Gaussian and Bessel beam profiles.

We present the implementation of a combined digital scanned light-sheet microscope (DSLM) able to work in the linear and nonlinear regimes under either Gaussian or Bessel beam excitation schemes. A complete characterization of the setup is performed and a comparison of the performance of each DSLM imaging modality is presented using in vivoCaenorhabditis elegans samples. We found that the use of Bessel beam nonlinear excitation results in better image contrast over a wider field of view.

Second-order nonlinear response of composites containing aligned elongated silver nanoparticles.

We present second-harmonic generation (SHG) measurements and simulations from a silica matrix containing randomly distributed but aligned elongated silver nanoparticles (NPs). The composites were produced by a double ion-implantation process of silver nanoparticles followed by an irradiation with Si ions. It is demonstrated that one can model the experimental results by considering the sub-micrometric composite layer as a nonlinear media containing rod NPs for which the hyperpolarizability tensor is cylindrically symmetric along the NP long axis.

Differential coherent anti-Stokes Raman scattering microscopy with linearly chirped femtosecond laser pulses.

We demonstrate a technique for differential coherent anti-Stokes Raman scattering (CARS) microscopy employing linearly chirped femtosecond laser pulses. By replicating the exciting pump-Stokes pulse pairs to create a pulse train at twice the laser repetition rate, and controlling the instantaneous frequency difference of each pair by glass dispersion, we can adjust the Raman frequency probed by each pair in an intrinsically stable and cost-effective way. The resulting CARS intensities are detected by a single photomultiplier as sum and difference using phase-sensitive frequency filtering.

Quantitative analysis of structural disorder in intervertebral disks using second harmonic generation imaging: comparison with morphometric analysis.

A novel signal processing algorithm for quantifying structural disorder in biological tissue using second harmonic generation (SHG) imaging is described. Both the magnitude and the pattern of disorder in collagenous tissues can be determined with this method. Mathematical models are used to determine the range of disordered states over which the algorithm can be used, because highly disordered biological samples do not generate second harmonic signals.

Sum frequency vibrational spectroscopy: the molecular origins of the optical second-order nonlinearity of collagen.

The molecular origins of second-order nonlinear effects in type I collagen fibrils have been identified with sum-frequency generation vibrational spectroscopy. The dominant contributing molecular groups are: 1), the methylene groups associated with a Fermi resonance between the fundamental symmetric stretch and the bending overtone of methylene; and 2), the carbonyl and peptide groups associated with the amide I band. The noncentrosymmetrically aligned methylene groups are characterized by a distinctive tilt relative to the axis perpendicular to the main axis of the collagen fiber, a conformation producing a strong achiral contribution to the second-order nonlinear effect.