Profile for Pablo Loza-Alvarez

Pixel resolution polarization-sensitive second harmonic generation (PSHG) imaging has been recently shown as a promising imaging modality, by largely enhancing the capabilities of conventional intensity-based SHG microscopy. PSHG is able to obtain structural information from the elementary SHG active structures, which play an important role in many biological processes. Although the technique is of major interest, acquiring such information requires long offline processing, even with current computers. In this paper, we present an approach based on Fourier analysis of the anisotropy signature that allows processing the PSHG images in less than a second in standard single core computers. This represents a temporal improvement of several orders of magnitude compared to conventional fitting algorithms. This opens up the possibility for fast PSHG information with the subsequent benefit of potential use in medical applications.

Live microscopy techniques (i.e., differential interference contrast, confocal microscopy, etc.) have enabled the understanding of the mechanisms involved in cells and tissue formation. In long-term studies, special care must be taken in order to avoid sample damage, restricting the applicability of the different microscopy techniques. We demonstrate the potential of using third-harmonic generation (THG) microscopy for morphogenesis/embryogenesis studies in living Caenorhabditis elegans (C. elegans). Moreover, we show that the THG signal is obtained in all the embryo development stages, showing different tissue/structure information. For this research, we employ a 1550-nm femtosecond fiber laser and demonstrate that the expected water absorption at this wavelength does not severely compromise sample viability. Additionally, this has the important advantage that the THG signal is emitted at visible wavelengths (516 nm). Therefore, standard collection optics and detectors operating near maximum efficiency enable an optimal signal reconstruction. All this, to the best of our knowledge, demonstrates for the first time the noninvasiveness and strong potential of this particular wavelength to be used for high-resolution four-dimensional imaging of embryogenesis using unstained C. elegans in vivo samples.

We demonstrate a simple scanless two-photon (2p) excited fluorescence microscope based on selective plane illumination microscopy (SPIM). Optical sectioning capability is presented and depth-resolved imaging of cameleon protein in C. elegans pharyngeal muscle is implemented.

In this study we present for the first time the use of confocal microscopy and laser scanning brightfield microscopy (LSBF) for real time imaging of femtosecond laser nanosurgery and its dynamics in C. elegans. A single multimodal optical workstation that provides the ability to perform femtosecond laser nanosurgery and simultaneous confocal and LSBF imaging was used for the purpose. With this tool several dynamic phenomena concomitant with laser nanosurgery in C. elegans were observed and imaged. Some of these dynamic phenomena, like muscular contraction and single muscle cell stimulation, have been imaged for the first time during nano-neurosurgery of C. elegans.

In this work we show that a pulsed laser light placed at a distance is able to modulate the growth of axons of primary neuronal cell cultures. In our experiments continuous wave (CW), chopped CW and modelocked fs (FS) laser light was focused through a microscope objective to a point placed at a distance of about 15 degrees mum from the growth cone. We found that CW light does not produce any significant influence on the axon growth. In contrast, when using pulsed light (chopped CW light or FS pulses), the beam was able to modify the trajectory of the axons, attracting approximately 45% of the observed cases to the beam spot. Such effect could possibly indicate the capacity of neurons to interpret the pulsating NIR light as the source of other nearby cells, resulting in extension of processes in the direction of the source.

In this paper we provide, for the first time to our knowledge, the effective orientation of the SHG source in cultured cortical neuronal processes in vitro. This is done by the use of the polarization sensitive second harmonic generation (PSHG) imaging microscopy technique. By performing a pixel-level resolution analysis we found that the SHG dipole source has a distribution of angles centered at thetae =33.96 degrees , with a bandwidth of Deltathetae = 12.85 degrees . This orientation can be related with the molecular geometry of the tubulin heterodimmer contained in microtubules.

In this work we propose and build a multimodal optical workstation that extends a commercially available confocal microscope (Nikon Confocal C1-Si) to include nonlinear/multiphoton microscopy and optical manipulation/stimulation tools such as nanosurgery. The setup allows both subsystems (confocal and nonlinear) to work independently and simultaneously. The workstation enables, for instance, nanosurgery along with simultaneous confocal and brightfield imaging. The nonlinear microscopy capabilities are added around the commercial confocal microscope by exploiting all the flexibility offered by this microscope and without need for any mechanical or electronic modification of the confocal microscope systems. As an example, the standard differential interference contrast condenser and diascopic detector in the confocal microscope are readily used as a forward detection mount for second harmonic generation imaging. The various capabilities of this workstation, as applied directly to biology, are demonstrated using the model organism Caenorhabditis elegans.

In this study, the second harmonic generation (SHG) response to polarization and subsequent data analysis is used to discriminate, in the same image, different SHG source architectures with pixel resolution. This is demonstrated in a mammalian tissue containing both skeletal muscle and fibrilar collagen. The SHG intensity variation with the input polarization (PSHG) is fitted pixel by pixel in the image using an algorithm based on a generalized biophysical model. The analysis provides the effective orientation, theta(e), of the different SHG active structures (harmonophores) at every pixel. This results in a new image in which collagen and muscle are clearly differentiated. In order to quantify the SHG response, the distribution of theta(e) for every harmonophore is obtained. We found that for collagen, the distribution was centered at theta(e) = 42.7 degrees with a full width at half maximum of theta = 5.9 degrees while for muscle theta(e) = 65.3 degrees , with theta = 7.7 degrees . By comparing these distributions, a quantitative measurement of the discrimination procedure is provided.

The polarization dependence of second harmonic generation (SHG) microscopy is used to uncover structural information in different muscle cells in a living Caenorhabditis elegans (C. elegans) nematode. This is done by using a generalized biophysical model in which element ratios for the associated second-order nonlinear tensor and angular orientations for thick filaments are retrieved using a pixel-by-pixel fitting algorithm. As a result, multiple arbitrary orientations of thick filaments, at the pixel-resolution level, are revealed in the same image. The validity of our method is first corroborated in well-organized thick filaments such as the nonfibrilar body wall muscles. Next, a region of the nonstriated muscular cells of the pharynx is analyzed by showing different regions with homogenous orientations of thick filament as well as their radial distribution. As a result, different sets of the nonstriated muscle cell groups in the pharynx of this nematode were exposed. This methodology is presented as a filtering mechanism to uncover biological information unreachable by common intensity SHG microscopy. Finally, a method to experimentally retrieve the distribution of the effective orientation of active SHG molecules is proposed and tested.

A 3 mW focused femtosecond laser spot at a distance (>15 mum) has shown to attract the fillopodia from growth cones of primary neuronal cell cultures (mice E15). The phenomenological behavior of fillopodia is studied under short durations (~40 min) and different laser light conditions. The analysis of the fillopodia movement showed that they become significantly attracted towards the focused femtosecond laser light. In contrast, the use of continuous wave under the same conditions did not generate the same effect, the results of which were indistinguishable from when there was no laser light present (control condition). These results suggest the possible existence of an optically-induced signaling mechanism in growth cones.

We report for the first time the use of two photon fluorescence as detection method of affinity binding reactions. We use a resonant grating waveguide structure as platform enhancement for detecting the interaction between fluorescent labeled Boldenone, a non-natural androgenic hormone, and a specific anti-anabolic antibody. We were able to detect a surface coverage of approximately 0.7 ng/mm(2).

In this paper we discuss in detail the underlying theory of a novel method that allows the characterizing of ultrashort laser pulses to be achieved in an analytical way. MEFISTO, (measuring the electric field by interferometric spectral trace observation) is based on a Fourier analysis of the information contained in a spectrally resolved interferometric correlation and can be applied to both situations: the characterization of an unknown pulse (MEFISTO) or to the simultaneous characterization of two different unknowns pulses (Blind-MEFISTO). The theoretical development and experimental practical implications are discussed in both situations.

We present a new methodology that obtains, in an analytical way, the complex electric field of ultrashort pulses. This methodology is based only on Fourier analysis of the frequency components of spectrally resolved interferometric collinear autocorrelations. We present an experimental demonstration of this technique and the results are compared with the conventional second-harmonic generation frequency-resolved optical gating technique.

We report the use of starch as an ideal nonlinear medium with which to perform collinear frequency-resolved optical gating measurements of ultrashort pulses at the focal plane of a high-numerical-aperture (NA) lens. We achieved these measurements by simply sandwiching starch granules (suspended in water) between two coverslips and placing them within the focal plane of a high-NA lens. The natural nonlinear characteristics of starch allow the correct phase matching of pulses at the focal plane of a high-NA lens at different wavelengths. This elegant arrangement overcomes all the complexity and problems that were previously associated with pulse characterization within a multiphoton microscope.

We outline criteria for fast and accurate acquisition of collinear FROG (CFROG) trace and how it can be transformed into the more traditional noncollinear FROG trace. The CFROG has an intrinsically simple geometry that provides greater versatility as well as the ability for built-in delay calibration and enhanced error-checking. The procedure, based on data processing, allows conventional SHG-FROG retrieval algorithms to be used. This technique is tested numerically and experimentally giving excellent results. This work represents an attractive alternative to the traditional, more complex non-collinear FROG technique while, at the same time, extending its use to experiments where collinear geometry is imposed.

We report our observations of the transient self-narrowing of light beams mediated by dominant Kerr nonlinearities and two-photon absorption in a bulk potassium titanyl phosphate crystal near conditions for second-harmonic generation. Drastic differences between upconversion and downconversion processes are highlighted.