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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. The achieved spectrometer resolution was 0.6 ± 0.1 nm with a threshold peak intensity of 2×106Wcm2. An analysis of the nonlinear response as a function of the wavelength, the response saturation, and the criteria to prevent it are also presented.

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. The light-sheets FOVs, optical sectioning, and resolution are experimentally characterized and discussed. The advantages of using Bessel beams and the main drawbacks of using Gaussian beams for multicolor imaging are highlighted. Multiple Bessel excitation minimizes the FOV's mismatch's effects due to the beams chromatic defocusing and alleviates the aside object blurring obtained with multiple Gaussian beams. It also offers a fair homogeneous axial resolution and optical sectioning over a larger effective FOV. Imaging over perithecia samples of the fungus Sordaria macrospora demonstrates such advantages. This work complements previous comparative studies that discuss only single wavelengths light-sheets excitations.

We perform multicolor fluorescent microscopy using different color Bessel beams for the light-sheet excitation. The advantages of this setup over conventional multicolor light-sheet imaging using Gaussian beams is shown over growing protoperithecia of Sordaria macrospora.

We present a study of the optical second-order nonlinearity of type I collagen fibers grown in vitro 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. This is confirmed by scanning electronic microscopy (SEM) imaging and the polarization dependence of the SHG signal. The measured values of the anisotropy parameter ρ of the reassembled collagen fibers are found to be similar to that obtained for native fibers on the relevant sub-µm scale.

The second order nonlinear optical response of gold nanoprisms arrays is investigated by means of second harmonic generation (SHG) experiments and simulations. The polarization dependence of the nonlinear response exhibits a 6-fold symmetry, attributed to the local field enhancement through the excitation of the surface plasmon resonances in bow-tie nanoantennas forming the arrays. Experiments show that for polarization of the input light producing excitation of the plasmonic resonances in the bow-tie nanoantennas, the SHG signal is enhanced; this despite the fact that the linear absorption spectrum is not dependent on polarization. The results are confirmed by electrodynamic simulations which demonstrate that SHG is also determined by the local field distribution in the nanoarrays. Moreover, the maximum of SHG intensity is observed at slightly off-resonance excitation, as implemented in the experiments, showing a close relation between the polarization dependence and the structure of the material, additionally revealing the importance of the presence of non-normal electric field components as under focused beam and oblique illumination.

Coherent Raman spectroscopy (CRS) and imaging have been used successfully for the label-free visualization and analysis of functional and endogenous biomolecules in complex biological samples. The interest in implementing these methodologies worldwide in research laboratories has increased in recent years; however, many practical challenges are faced in the implementation process. One of them is identifying from scratch different coherent Raman signals when broadband femtosecond sources are utilized. We present a straightforward methodology to identify and analyze the multiple excited resonant SRS/CARS signals using a simple spectrophotometer in this work to expand the understanding of this particular branch of CRS. The coherent Raman experiments are conducted in the so-called temporal slit configuration using a broadband femtosecond laser source to obtain the excitation pump and Stokes beams and high group velocity dispersion glass to temporally stretch the pulses; but in a non-conventional and non-optimum configuration for CRS experiments neither for spectral focusing (single-frequency measurements) nor for mixing of a broadband pulse with a narrow band pulse (multiplexed detection). Despite that, we demonstrate our methodology's feasibility to extract and analyze complex to discern coherent Raman spectra. We use dimethyl sulfoxide (DMSO) and β-phase barium meta-borate crystal (BBO) and discuss molecular vibrations identified in the spectral range from 300 to 800 cm−1 from both kinds of CRS spectra, stimulated Raman scattering (SRS) and coherent anti-Stokes Raman scattering (CARS).

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. This allows visualizing simultaneously the localization and dynamics of different cellular components involved in apical growth in living hyphae. The configuration presented represents a noncommercial, cost-effective alternative microscopy system for the rapid and simultaneous acquisition of multifluorescent images and can be potentially useful for three-dimensional imaging of large biological samples.

We report the synthesis of small zinc oxide nanoparticles (ZnO NPs) based colloidal suspensions and the study of second-harmonic generation from aggregated ZnO NPs deposited on glass substrates. The colloidal suspensions were obtained using the laser ablation of solids in liquids technique, ablating a Zn solid target immersed in acetone as the liquid medium, with ns-laser pulses (1064 nm) of a Nd-YAG laser. The per pulse laser fluence, the laser repetition rate frequency and the ablation time were kept constant. The absorption evolution of the obtained suspensions was optically characterized through absorption spectroscopy until stabilization. Raman spectroscopy, SEM and HRTEM were used to provide evidence of the ZnO NPs structure. HRTEM results showed that 5–8 nm spheroids ZnO NPs were obtained. Strong second-harmonic signal is obtained from random ZnO monocrystalline NPs and from aggregated ZnO NPs, suggesting that the high efficiency of the nonlinear process may not depend on the NPs size or aggregation state.

Three color SPIM is performed using three sequential cw-lasers pulsed trains in the excitation synchronized with the acquisition frame rate of a single camera. Multicolor images of living hyphae of Neurospora crassa are acquired.

Light sheet optical microscopy on strontium aluminate nanoparticles (SrAl

We present results for the induction and monitoring of structural modification of a composite consisting of elongated silver nanoparticles films embedded in silica using ultrafast femtosecond laser irradiation and second harmonic generation imaging, respectively. Waveguide-like patterns are written and characterized under a laser scanning nonlinear microscope system by simply changing the laser fluence in the sample; switching in this way between two different physical processes occurring only within the composite film: second harmonic generation and laser induced nanoparticles removal. A study of the nanoparticles damage process as a function of the laser energy, polarization and scan velocity is also presented and discussed. The use of the non-irradiated zone between two written channels is proposed as a potential linear and nonlinear optical waveguide.

We demonstrate a technique for simultaneous detection of coherent anti-Stokes Raman scattering (CARS) at four vibrational frequencies, using simple passive optical elements and without spectrally resolved detection. The technique is based on pump and Stokes femtosecond pulses selectively exciting vibrational resonances through spectral focusing. By replicating the pump and Stokes pair into four pairs, each traveling through appropriate glass elements, we simultaneously excite four different vibrational frequencies. The resulting CARS is a periodic train of intensities detected by a single photomultiplier and frequency analyzed to retrieve its Fourier coefficients. We demonstrate detection of methanol and ethanol mixtures in water and quantitative determination of their concentration owing to the improved chemical selectivity of this quadruplex CARS scheme.

Light sheet optical microscopy on strontium aluminate nanoparticles (SrAl2 O4 NPs)1 codoped with Eu2+ and Dy3+ 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 (λexc = 254-460 nm), a broad emission band (λem = 475-575 nm) with the maximum centered wavelength at 525 nm and a half lifetime within the seconds regime. The feasibility to measure the nanoparticle luminescence under the selective plane illumination configuration was studied by immersing the nanoparticles in 1% Agarose. The potential applicability of the synthesized nanophosphors for cancer cell tagging was demonstrated by using in vitro experiments with human breast adenocarcinoma MCF-7 cells. A single MCF-7 cell observed by the use of light sheet microscopy with UV excitation. The cell has been bio-labeled with FA-SrAl2 04 : Eu2+ , Dy3+ NPs and 4',6-diamidino-2-phenylindole, dihydrochloride for nucleus identification.

Coherent anti-Stokes Raman scattering (CARS) microscopy using linearly chirped femtosecond laser pulses and spectrally and time-integrated detection is discussed both theoretically and experimentally. By matching the linear chirp of Stokes and pump pulses, the spectral resolution is reduced to the Fourier limit of the chirped pulse duration (spectral focusing). We discuss the effect of the time-ordering of Pump and Stokes pulses on the signal strength and selectivity, and give analytical expressions for the relevant quantities. We consider linear chirp obtained by glass elements of known group-velocity dispersion, and analyze the limit of the spectral resolution by higher order glass dispersion. Experiments corroborating the theory are presented, using 100 fs laser pulses, SF57 glass elements, and polystyrene beads as test samples.

We present results for the production and characterization of structural modifications of composites containing elongated silver nanoparticles embedded in silica, using femtosecond laser pulses. Direct writing of patterns in such composites was achieved using a scanning nonlinear optical microscopy system. Writing was achieved by running the laser at high pulse energy, and characterization was performed using second harmonic generation, and running the microscope at lower energies. Results for both the writing and characterization are presented and discussed.

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. A further differentiation of the stack retrieves the Raman spectra at each pixel of the image which inherits the 3D resolution of the host light sheet system. We demonstrate this technique using solvent solutions and composites of polystyrene beads and lipid droplets immersed in agar and by imaging the C–H (2800-3100cm−1) region in a C. elegans worm. The image acquisition time results in 4 orders of magnitude faster than confocal point scanning Raman systems, allowing the possibility of performing fast spontaneous Raman·3D-imaging on biological samples.

We study the induction and characterization of structural modifications of composites containing elongated silver nanoparticles embedded in silica, using laser scanning nonlinear optical microscopy. Both the writing process and characterization are presented and discussed.

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. The second-order macroscopic susceptibility of the composite originates from the coherent summation of the hyperpolarizabilities associated to each NP. We obtain analytical expressions for the p- and s-polarized effective susceptibility tensor as a function of experimental variables, such as the fundamental beam input polarization and sample orientation, and fitting parameters relating the cylindrically shaped hyperpolarizability. In addition, coherent SHG measurements on spherical nanoparticles resulting from the first ion-implantation process are also presented showing an isotropic polar behavior for the total SHG intensity where the p-polarized SHG intensity resulted to be the main contribution.

We demonstrate frequency differential CARS (D-CARS) using femtosecond laser pulses linearly chirped by glass elements of high group-velocity dispersion. By replicating the Pump-Stokes pair into a pulse train at twice the laser repetition rate, and controlling the instantaneous frequency difference by glass dispersion, we adjust the Raman frequency probed by each pair in an intrinsically stable way. The resulting CARS intensities are detected simultaneously by a single photomultiplier as sum and difference using lock-in detection. We demonstrate imaging of living cells with strongly suppressed non-resonant background. We also show D-CARS using a single femtosecond laser source.

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. We demonstrate imaging of polymer beads and living cells with suppressed nonresonant CARS background and improved chemical sensitivity.

We have developed a home-built CARS microscope which exploits linearly-chirped ultrafast laser pulses. By using glass of high group-velocity dispersion, Stokes and Pump pulses of 150 fs duration Fourier-limited are equally chirped to pulse durations in the 0.5 ps-2.8 ps range. In this way we reduce the spectral width of the instantaneous frequency difference to the Fourier limit of the chirped pulse duration (spectral focussing). As a proof of principle, CARS spectroscopy with high spectral resolution is demonstrated on polystyrene beads. We also show, both theoretically and experimentally, that for chirped pulse durations shorter than or comparable to the Raman coherence time, maximum CARS signal occurs for a Pump arriving after the Stokes pulse. Furthermore, we demonstrate the applicability of our CARS microscope to biological sciences by performing CARS microspectroscopy on different live cells and fixed tissue samples.

We demonstrate coherent anti-Stokes Raman scattering (CARS) microspectroscopy using a single Ti:sapphire laser oscillator and simple passive optical elements. Spectral selection by dichroic mirrors and linear chirping by glass elements creates a vibrational excitation tuneable over a large spectral range ( ∼ 800–2200 cm−1) with adjustable spectral resolution ( ∼ 10–100 cm−1). We furthermore demonstrate the applicability of differential CARS, enhancing the chemical selectivity, with the proposed single-laser configuration.

We demonstrate experimentally that coherent anti-Stokes Raman microspectroscopy with high spectral resolution is achieved using femtosecond laser pulses chirped up to a few picoseconds by glass elements of known group-velocity dispersion without significant intensity losses. By simply choosing the length of the glass, the chirp of Stokes and pump pulses is tailored to obtain a spectral resolution given by the Fourier limit of the chirped pulse duration. We show that for chirped pulse durations shorter than or comparable to the Raman coherence time, maximum signal occurs for a pump arriving after the Stokes pulse, a time-ordering effect confirmed by numerical simulations.

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. The method is validated by measuring disorder in heated fascicles using SHG and showing that results are significantly correlated with morphometric determination. Applicability of the method to tissue pathology is demonstrated by analysis of a mouse model of intervertebral disk injury. Disks were subjected to tensile or compressive forces in vivo for one week. Structural disorder in the annulus fibrosus was measured by SHG scanning and by standard morphometric analysis. Values for disorder obtained by SHG scanning were significantly correlated with values obtained by morphometry (p

We present a new method of light amplification based on vectorial two-wave mixing in cubic crystal via an unshifted photorefractive grating. The energy transfer between interacting waves is achieved in an experimental configuration that provides the amplitudes of modulation of refractive indices of opposite signs, Dn(1)=-Dn(2), for two orthogonal polarization modes of the interacting waves. The configuration demonstrates bidirectional amplification; i.e., the weak wave is always amplified, independent of its direction with respect to the strong pump beam. We present the results of numerical analysis along with the experimental results for a Bi(12)TiO(20) crystal with a dc applied electric field.

The molecular origins of second order nonlinear effects in collagen fibril assemblies have been identified with sum-frequency generation, infrared and Raman vibrational spectroscopies.

Transitions in order/disorder phase states of biological tissues were analyzed using polarization modulated second harmonic generation imaging. The degree of structural disorder was significantly correlated with severity of pathological changes.

The noncentrosymmetric molecular structures responsible for the second order nonlinear effects of collagen fibrils are identified by sum frequency vibrational spectroscopy. CH 2 and C=O functional groups dominate the achiral and chiral spectra, respectively.

An experimental method for quantifying disorder within the anulus fibrosus is described based on polarization-modulated second harmonic generation imaging (PM-SHG-I). This method is demonstrated by imaging the anular lamellar architecture of a mouse model of compressive loading. Results were consistent with those obtained in an earlier study where organization was quantified directed secants image analysis on photomicrographs. In this study the orientation within individual lamellia is quantified by average orientation of the collagen molecules within a defined volume of a single lamellar as measured by the PM-SHG-I. Lamellar boundaries can be identified through the SHG intensity images, and confirmed through co-registration with photomicrographs of the same region. The orientation within the lamellar is quantified by the polarization angle of the maximum second harmonic intensity. PM-SHG-I offers several advantages as compared with the method of directed secants: first, it is nondestructive, allowing repeated measurements of the same tissue; second, images are captured on the order of seconds and capable of obtaining information up to a depth of 200-300 microns, thus allowing for real-time assessment of load damage; third, organization is measured at a much higher resolution, as it is based on disorder within the molecular arrays of a single lamella.

An analytical solution is presented to the vectorial coupled wave equations for the steady state amplitudes of two waves interacting in cubic photorefractive crystals. The solution accounts for pump depletion as well as an arbitrary phase shift between the interference pattern and photorefractive grating. It is shown that bidirectional vectorial amplification and polarization orthogonalization of the interacting beams take place in photorefractive crystal with diffusion, drift or mixed mechanism of space-charge formation.

Summary form only given. Space charge waves (SCW) may be generated in a material with large mobility-lifetime product in the presence of electric field. When excited with the light fringes the SCW may give rise to such exciting phenomena like spatial subharmonics generation and/or resonant photorefractive wave coupling. The SCW can be excited under homogeneous illumination, too., In present work we propose and implement a new to our knowledge technique of resonant enhancement of photorefraction, which is based on parametric interaction of light induced grating with self-excited SCW. To achieve SCW self-excitation the CdTe sample is illuminated by low-coherent radiation of 980 nm laser.

We present the results of a theoretical and experimental investigation of the photorefractive effect under a revolving external electric field in BSO and BTO crystals. We show that the revolving field enhances 90°-shifted with respect to the interference pattern space-charge field, which does not depend on the recorded grating orientation. We analyze in detail the effect of the external applied field on the two-wave mixing and show that the direction of maximum momentary two-wave mixing gain changes synchronously with the revolving applied field. We propose a configuration that demonstrates an isotropic polar pattern of the time average two-wave mixing gain.

We present a numerical and experimental study of vectorial two-wave mixing in cubic photorefractive crystals with nonlocal gratings in the case of strong wave coupling when the grating is not uniform along the propagation direction. We demonstrate that, in important experimental configurations, vectorial two-wave mixing leads to equal intensities and orthogonal polarizations of the interacting waves at the output of the photorefractive crystal. We analyze a bidirectional vectorial light amplification and discuss the effects of the crystal orientation and optical activity. Experimental results of two-wave and multiwave mixing in a Bi12TiO20 crystal with an ac applied electric field are presented.

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. In contrast, the stretching vibration of the carbonyl groups associated with the amide I band results in a strong chiral contribution to the optical second-order nonlinear effect. The length scale of these chiral effects ranges from the molecular to the supramolecular.

Summary form only given.We report a detailed experimental study of photo-EMF in Bi/sub 12/TiO/sub 20/ (BTO) crystal in the presence of a dc transverse external electric field. We demonstrate the possibility of enhancement of the photo-emf signal by the transverse field in comparison with the longitudinal configuration.

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. This work is supported by the Generalitat de Catalunya grant 2009-SGR-159, the Spanish government grants TEC2009-09698 and SAF2008-00211, the EU project STELUM (FP7-PEOPLE-2007-3-1-IAPP, 217997), the Swiss National Science Foundation (SNSF) Sinergia project grant CRSII3_125447, the FP7-NMP VIBRANT project grant CP-IP 228933-2, the NoE P4L and Laserlab optobio. Jacob Licea-Rodriguez acknowledges CONACYT Mexico for supporting his stay at ICFO through a mixed fellowship. This research has been partially supported by Fundació Cellex Barcelona and was mainly conducted at ICFO’s “Super-Resolution Light Microscopy and Nanoscopy Facility”

Coherent Anti-Stokes Raman Scattering (CARS) microscopy has recently emerged as a new technique for imaging in cell biology, offering chemical specificity without the need of staining and fluorescence-tagging. We have developed a home-built CARS microscope [1] which exploits linearly-chirped ultrafast laser pulses to achieve spectral focussing of the vibrational excitation [2]. CARS Stokes pulses are generated by a Ti:Sa laser (835nm, 100fs), which pumps an optical parametric oscillator (OPO). The intra-cavity frequency-doubled signal of the OPO provides the CARS Pump. Both pulses are equally chirped with glass blocks of calibrated group-velocity dispersion and we studied the effect of linear chirp corresponding to pulse durations from 600fs to 2.5ps [1]. As a proof-of principle, we investigated polystyrene beads (0.2–3µm diameter) in 2% agar solution by detecting the forward-collected CARS at the C-H aromatic band (3054cm−1). By varying the delay time between Pump and Stokes their instantaneous frequency difference (IFD) changes so that CARS spectra can be measured. This is shown in Fig.1a for three different chirped pulse durations τ. The measured spectral width of the aromatic C-H vibration in polystyrene is decreasing with increasing τ, as expected since the spectral width σ D of the vibrational excitation decreases [1]. The peak intensity of the resonant CARS response keeps essentially constant as long as σD is larger than the probed Raman line-width 2T 2 −1∼27 cm−1. Vice versa, CARS from the agarose gel, dominated by the spectrally non-resonant CARS response of water, is broadening in delay time and decreasing in peak intensity with increasing pulse duration τ. The ratio between resonant and non-resonant CARS is accordingly increasing with τ and saturates for τ ≫ T 2 ∼ 0.4 ps, as shown in the inset of Fig.1a. A good compromise between a high ratio improving the selectivity and increasing with τ, and a high resonant CARS intensity improving the sensitivity and decreasing with τ, is τ∼T 2 . For durations comparable or less than T 2 , we also found that the maximum CARS is not created for zero delay between Pump and Stokes pulses. In fact, since CARS is generated modulating the Pump by the induced coherent vibration, a Pump pulse arriving after the Stokes pulse (by about 20% of the pulse duration for τ ∼T 2 ) optimizes the CARS signal and reduces the nonresonant CARS background [1]. It is therefore better to set the frequency difference between Pump and Stokes to be larger than the vibrational resonance (by about 20% of the pulse spectral width for τ ∼T 2 ), so that the IFD matches the resonance at the optimum delay. Our microscope also features two-photon fluorescence, second harmonic generation, and differential interference contrast imaging modalities. We have demonstrated its applicability to cell imaging on a range of live unstained cells including 3T3-L1 fibroblasts, HeLa cells (see Fig.1b), HepG2 liver cells, and in fixed tissues (mouse mammary gland and intestine). Current progress towards a differential detection modality with improved signal-to-background ratio will also be presented.

El dopaje de materiales con elementos de tierras raras como el Erbio (Er3+) y el Yterbio (Yb3+), mejora las propiedades de luminiscencia de los materiales, como consecuencia de la fluorescencia inducida por un proceso de conversión ascendente. Para el caso de materiales co-dopados con Er3+ /Yb3+ la eficiencia en la excitación de la fluorescencia se incrementa considerablemente y, además, el espectro de emisión fluorescente posee bandas de energía térmicamente acopladas, por lo que dichos materiales se pueden utilizar como sensores de temperatura. Esto es posible mediante la medición de la razón de intensidades de fluorescencia (FIR, por sus siglas en inglés) de dichas bandas acopladas. La FIR ha sido utilizada exitosamente para medir la temperatura en células de una manera remota o bien para la fabricación de sensores de temperatura. No obstante, la obtención de la FIR radica en una medición puntual del espectro de fluorescencia mediante el uso de espectrómetros, con los cuales se obtiene la intensidad de cada una de las bandas acopladas. Esto inevitablemente imposibilita una medición bidimensional de la temperatura, a menos que se escanee la muestra. En esta tesis se proponen dos metodologías experimentales para medir la distribución bidimensional de la temperatura en medios sensores compuestos por polidimetilsiloxano dopado con Er3+ e Yb3+ (PDMS-Yb3+/Er3)). Se demuestra que esto es posible mediante la adquisición de imágenes de la fluorescencia de conversión ascendente (FCA) para medir directamente, o mediante un cálculo aritmético, la razón de intensidades (FIR); esto es, empleando únicamente una cámara (monocromática o a color) sin la necesidad de utilizar un espectrómetro. Rare earth elements doping, such as Erbium (Er3+) and Ytterbium (Yb3+), improves materials' luminescence properties. In particular, the fluorescence excitation efficiency, induced by an up-conversion process in materials co-doped with Er3+ / Yb3+, increases considerably. Since the fluorescent emission spectrum of Er3+ has thermally coupled energy bands, such co-doped with Er3+ / Yb3 materials can be used as temperature sensors. This is possible by measuring the Fluorescence Intensity Ratio (FIR) of such coupled bands. FIR measurement has proven to be a successful way to measure the temperature in cells remotely and to manufacture temperature sensors. However, FIR measurements are commonly performed pointwise, i.e. measured over a small spatial region, using spectrometers to obtain each corresponding coupled band's intensity. Such a pointwise measurements makes impossible the rapid acquisition of a two-dimensional temperature measurements unless the sample is scanned. This thesis proposes two experimental methodologies to measure the two-dimensional temperature distribution in a sensor composed of polydimethylsiloxane doped with Er3+ and Yb3+ (PDMS-Yb3+ / Er3+). We demonstrate that the FIR measurement is possible directly via either the image acquisition and processing of the up-conversion fluorescence (UC) signal on the specific coupled band or by an arithmetic calculation over a single image taken over the hyperspectral UC image, using a single (monochrome or color) camera instead of a spectrometer.

La biofotónica es un área de investigación relativamente nueva que ha tenido un gran progreso en tan solo dos décadas, con la introducción de nuevas técnicas fotónicas en microbiología y biomedicina. Particularmente para el estudio y la manipulación de sistemas microscópicos tales como las macro moléculas, las células y el tejido biológico (Prasad, 2003). En esta tesis se llevó a cabo una investigación para implementar y caracterizar la técnica de enfoque espectral utilizando el proceso de generación de suma de frecuencias ópticas. Dicha técnica de enfoque espectral es el paso intermedio requerido para desarrollar micro-espectroscopía óptica no lineal de esparcimiento Raman estimulado conocida como CARS (Esparcimiento Raman Anti-Stokes Coherente); técnica de imagen de gran interés por su capacidad de obtener información de contraste molecular sin marcaje fluorescente. La técnica de enfoque espectral consiste en dilatar temporalmente dos pulsos de femto-segundos de diferente frecuencia, propagándolos a través de ventanas ópticas de alta dispersión de velocidad de grupo. Con ello se modula linealmente el contenido espectral de ambos haces a lo largo de la duración temporal de los pulsos (técnica conocida como “chirp”). El “enfoque” se obtiene al concentrar, mediante un traslape espacio-temporal, la energía de los dos espectros modulados de manera tal que la diferencia entre sus frecuencias ópticas es constante en el tiempo de traslape. Excitando única y exclusivamente una frecuencia de vibración molecular Raman. Dicha excitación coherente es instantánea y más eficiente comparada con la excitación Raman espontánea. Dando como resultado la posibilidad de tomar imágenes Raman en tiempo real. La ventaja particular del enfoque espectral presentado en este trabajo es el uso de una sola fuente láser de pulsos ultracortos en el régimen femto-segundos (fs) con un contenido espectral ancho (>100 nm), el cual se divide ópticamente para obtener los dos haces requeridos. Asimismo, la caracterización de esta técnica utilizando el proceso de suma de frecuencias ópticas, y no la diferencia (como comúnmente se utiliza), es una contribución original de este estudio. Resultando ser más sencilla experimentalmente pero con ciertas dificultades de interpretación teórica las cuales se discuten en este trabajo de tesis. Bio photonics is a new research area having an enormous and rapid progress in the last two decades, involving the development and application of new photonic techniques in biology and biomedicine; in particular, for the study and manipulation of microscopic systems, such as macro molecules, cells and biologic tissue (Prasad, 2003). In this thesis an experimental research work to implement and characterize the spectral focusing technique using nonlinear optical sum frequency generation is presented. This technique is a preliminary step to implement nonlinear stimulated Raman scattering micro-spectroscopy technique known as CARS (Coherent Anti- Stokes Raman Spectroscopy); an attractive technique due to its ability to obtain molecular contrast without the need of fluorescent markers. Spectral focusing consists on pulse stretching two different frequency femto- second pulses using high group velocity dispersion glass windows. In order to modulate linearly the spectral content of both pulses along the pulse duration (technique known as chirp). By overlapping spatially and temporally the energy of the two chirped pulses produce a constant difference frequency during the time overlap of both pulses. In that way the energy of both pulses is focused to excite exclusively one vibrational Raman frequency, and resulting in a more efficient Raman excitation if compared with the spontaneous Raman excitation. The end result is the possibility of taking molecular contrast (Raman) imaging in real time. The main advantage of spectral focusing shown in this thesis is the use of a single femto-second laser source with a wide spectral content (>100 nm), in order to obtain the two beams required by using passive optical filters. It is important to remark that the characterization of this technique using sum of optic frequencies (and not the difference) is an original contribution of this study, resulting simpler experimentally but with some difficulties in the theoretical interpretation which are discussed in this thesis.

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El área de la biofotónica involucra el desarrollo y la aplicación de técnicas ópticas para manipular y/o estudiar moléculas, células y tejido biológico (Prasad, 2003). En esta tesis se presenta un trabajo experimental en el área de la microscopía nolineal, la cual cae dentro del espectro de la biofotónica. En particular se construyó un microscopio no lineal en base al proceso óptico no lineal (de allí su nombre) de segundo orden conocido como generación de segundo armónico (SHG, por sus siglas en inglés). Dicho microscopio no lineal se utilizó para estudiar la no linealidad óptica de segundo orden en fibras de colágeno generadas in vitro. El microscopio fue construido en el CICESE (microscopio no comercial)y está conformado por tres sistemas: i)el sistema de iluminación: compuesto por un láser de pulsos ultracortos en el régimen de femtosegundos como fuente de excitación; ii)el sistema de barrido óptico: compuesto por un par de espejos galvano-métricos, un arreglo telescópico y un objetivo de microscopio que en su conjunto barren el haz de luz láser en el plano focal, y iii)el sistema de detección: compuesto por una lente, un filtro interferométrico y un tubo fotomultiplicador, a través de los cuales se colecta y detecta únicamente la señal armónica generada. Para la generación de fibras de colágeno se utilizó una solución con monómeros de colágeno la cual se preparó químicamente y se llevó a condiciones de temperatura y pH adecuados para para inducir la fibrogénesis. Este proyecto de tesis inicia con la calibración del barrido láser y la resolución del sistema óptico, pasando por la metodología para preparar fibras de colágeno mediante la inducción de fibrogénesis in vitro y termina con un análisis de la señal de segundo armónico generada en las muestras de fibras de colágeno crecidas in vitro. The biophotonics field involves the development and application of optical techniques to manipulate and/or study molecules, cells and biological tissue (Prasad, 2003). This thesis presents an experimental work in the area of nonlinear microscopy, a field considered a major branch of biophotonics. In particular, a nonlinear microscope based on the second-order nonlinear optical process known as second harmonic generation (SHG) was built. This nonlinear microscope was used to study the second order nonlinearity in collagen fibers grown in vitro. The microscope was built in CICESE (microscope non commercial) and is divided in three systems: i)the illumination system: consisting of an ultrashort laser pulse (in the femtosecond range) as the excitation source; ii)the optical scanning system: including galvanometric mirrors, a telescopic array and a microscope objective which together scan the laser beam in the focal plane; and iii)the detection system: consisting on a lens, an interferometric filter and a photomultiplier tube, through which the harmonic signal generated is collected, filtered and detected, respectively. To generate collagen fibers a solution containing collagen monomers was prepared and brought to temperature and pH conditions suitable for fibrogenesis ignition. This thesis work started with the calibration of the optical scanning and optical resolution; followed by mastering the methodology to prepare collagen fibers obtained from fibrogenesis induced in vitro; and finally concludes with the analysis and discussion of the second harmonic signal generated from the collagen fibers generated.