Vascular bifurcation mapping with photoacoustic microscopy.

The early detection of microvascular changes in cancer diagnosis is needed in the clinic. A change in the vascular bifurcation density is a biomarker for the sprouting activity. Here, Optical-Resolution PhotoAcoustic Microscopy is used for quantitative vascular bifurcation mapping in 2D after the creation of Virtual Tubes out of Bifurcations. In stacks of OR-PAM images of the hemoglobin distribution, bifurcations become tubes and are selected by the 3D tubeness filter. These fast analyses will be compared to a classical approach and are easier to implement for functional analysis of the vascular bifurcation density in healthy and diseased tissues.

Photo-acoustic tomography based on laser optical feedback imaging of surface displacements.

We present how a laser optical feedback imaging (LOFI) setup can be used for the optical detection of ultrasound in photo-acoustic tomography (PAT). A PAT image is reconstructed by an inversion algorithm using surface displacement measurements made at several locations with our LOFI setup and following the optical irradiation with a pulsed Nd:YAG laser of a sample with absorbing inclusions. The width of the reconstructed inclusions and the signal-to-noise ratio (SNR) of the reconstructed images are first studied on the numerical model of a sample with three absorbing inclusions (i.e., with three acoustic punctual sources). Finally, an experimental PAT image of a phantom composed of two polyamide tubes with an internal diameter of 800 µm filled with red ink and submerged at -3.5 mm depth in a tank filled with water is reconstructed. Experimentally, the water surface displacement measurements have been made with our LOFI vibrometer, which provides an amplitude sensitivity of 1 nm (for a single-shot measurement) in a detection bandwidth of roughly 1 MHz adapted to the detection of the polyamide tubes. Under our experimental conditions, the surface energy densities of the LOFI focalized beam for the detection and of the pulsed Nd:YAG laser used for the irradiation, are compatible with the maximum permissive exposure for future biomedical measurements. The SNR and the resolution of the reconstructed PAT images are in good agreement with the theoretical predictions.

Multi-wavelength photo-acoustic microscopy in the frequency domain for simultaneous excitation and detection of dyes.

An optical-resolution photoacoustic microscope with modulated CW laser diodes allowing multi-channel imaging is presented that can be used for both imaging biological tissues and for targeted photo-dynamic therapy (PDT) varying the optical power and exposure time. The effects of this therapy are immediately monitored in order to optimize the time of irradiation. After the description of the experimental setup, in vitro and in vivo applications are presented on a synthetic sample and on the mouse ear using hemoglobin as endogenous and methylene blue as exogenous dye for imaging and PDT, respectively.

Ultrasound vibration measurements based on laser optical feedback imaging.

This paper examines the detection of ultrasound vibrations with nanometric amplitude by using a laser optical feedback imaging (LOFI) setup. By means of numerical simulations, we show typical examples of ultrasound vibrations having different temporal shapes (harmonic and transient), extracted from the laser output power modulation induced by the frequency-shifted optical feedback. Considering the laser quantum noise dynamic and the detection noise separately, we show that the simulated vibration noise is in good agreement with the theoretical prediction. Also, we demonstrate that ultra-high frequencies (in the gigahertz range) can be detected by using a usual LOFI setup with a low-power laser (few mW) and a conventional detection with a usual white noise level. Then we show how the noise of a short transient vibration can be reduced by the reconstruction of its wide vibration spectrum by concatenation. Finally, the experimental detection of transient-harmonics ultrasound vibrations propagating in water and detected at the air/water interface is presented.

Prediction performance of reservoir computing systems based on a diode-pumped erbium-doped microchip laser subject to optical feedback.

Reservoir computing (RC) systems are computational tools for information processing that can be fully implemented in optics. Here, we experimentally and numerically show that an optically pumped laser subject to optical delayed feedback can yield similar results to those obtained for electrically pumped lasers. Unlike with previous implementations, the input data are injected at a time interval that is much larger than the time-delay feedback. These data are directly coupled to the feedback light beam. Our results illustrate possible new avenues for RC implementations for prediction tasks.

Nonlinear laser dynamics induced by frequency shifted optical feedback: application to vibration measurements.

In this article, we study the nonlinear dynamics of a laser subjected to frequency shifted optical reinjection coming back from a vibrating target. More specifically, we study the nonlinear dynamical coupling between the carrier and the vibration signal. The present work shows how the nonlinear amplification of the vibration spectrum is related to the strength of the carrier and how it must be compensated to obtain accurate (i.e., without bias) vibration measurements. The theoretical predictions, confirmed by numerical simulations, are in good agreement with the experimental data. The main motivation of this study is the understanding of the nonlinear response of a laser optical feedback imaging sensor for quantitative phase measurements of small vibrations in the case of strong optical feedback.

The unexpected structure of the designed protein Octarellin V.1 forms a challenge for protein structure prediction tools.

Despite impressive successes in protein design, designing a well-folded protein of more 100 amino acids de novo remains a formidable challenge. Exploiting the promising biophysical features of the artificial protein Octarellin V, we improved this protein by directed evolution, thus creating a more stable and soluble protein: Octarellin V.1. Next, we obtained crystals of Octarellin V.1 in complex with crystallization chaperons and determined the tertiary structure. The experimental structure of Octarellin V.1 differs from its in silico design: the (αßα) sandwich architecture bears some resemblance to a Rossman-like fold instead of the intended TIM-barrel fold. This surprising result gave us a unique and attractive opportunity to test the state of the art in protein structure prediction, using this artificial protein free of any natural selection. We tested 13 automated webservers for protein structure prediction and found none of them to predict the actual structure. More than 50% of them predicted a TIM-barrel fold, i.e. the structure we set out to design more than 10years ago. In addition, local software runs that are human operated can sample a structure similar to the experimental one but fail in selecting it, suggesting that the scoring and ranking functions should be improved. We propose that artificial proteins could be used as tools to test the accuracy of protein structure prediction algorithms, because their lack of evolutionary pressure and unique sequences features.

The Role of Active Site Flexible Loops in Catalysis and of Zinc in Conformational Stability of Bacillus cereus 569/H/9 ß-Lactamase.

Metallo-ß-lactamases catalyze the hydrolysis of most ß-lactam antibiotics and hence represent a major clinical concern. The development of inhibitors for these enzymes is complicated by the diversity and flexibility of their substrate-binding sites, motivating research into their structure and function. In this study, we examined the conformational properties of the Bacillus cereus ß-lactamase II in the presence of chemical denaturants using a variety of biochemical and biophysical techniques. The apoenzyme was found to unfold cooperatively, with a Gibbs free energy of stabilization (ΔG(0)) of 32 ± 2 kJ·mol(-1) For holoBcII, a first non-cooperative transition leads to multiple interconverting native-like states, in which both zinc atoms remain bound in an apparently unaltered active site, and the protein displays a well organized compact hydrophobic core with structural changes confined to the enzyme surface, but with no catalytic activity. Two-dimensional NMR data revealed that the loss of activity occurs concomitantly with perturbations in two loops that border the enzyme active site. A second cooperative transition, corresponding to global unfolding, is observed at higher denaturant concentrations, with ΔG(0) value of 65 ± 1.4 kJ·mol(-1) These combined data highlight the importance of the two zinc ions in maintaining structure as well as a relatively well defined conformation for both active site loops to maintain enzymatic activity.

Phases of Talbot patterns in angular self-imaging.

The original Talbot (self-imaging) effect is observed in the vicinity of a grating of slits shined with a plane wave, and results in periodic images of the initial diffraction pattern (integer Talbot effect) and the appearance of images with a periodicity reduced by an integer factor (fractional Talbot effect). Most of the studies on Talbot effect so far have focused on the distribution of the intensity of the diffracted light. However, the phases of the Talbot images, obtained in both the integer and fractional self-imaging cases, can be calculated in a closed form and display interesting auto-correlation properties. This paper reports what is, to the best of our knowledge, the first experimental investigation of the phases of Talbot images beyond the integer self-imaging case. We address the problem of experimental measurement of the phases of the Talbot images in the equivalent frame of the angular Talbot effect, a recently reported manifestation of the Talbot effect in the far field. The phases of the Talbot images are measured by far-field holography, and the obtained results are in excellent agreement with theoretical calculations. They also suggest the possibility of using the scheme for a precise "fractional ruler" aimed at distances' measurements.

Control of the differential interference contrast in reinjected bimode laser.

We have demonstrated, both theoretically and experimentally, that it is possible to control (i.e., to enhance or cancel) the contrast of the interference pattern appearing in the intensity images obtained with a laser optical feedback imaging (LOFI) setup using a bimode laser. The laser is composed of two coupled orthogonally polarized states that interact (i.e., interfere) through the cross saturation laser dynamics. We created the contrast control by choosing the frequency shift (i.e., the beating frequency) between the feedback electric fields and the intracavity electric fields. We have shown that the interference contrast of the output power modulation of the laser total intensity is independent from the frequency shift and is always maximal. On the other hand, the interference contrast of each polarization state is frequency dependent. We obtained the maximal contrast when the frequency shift was equal to one of the resonance frequencies of the bimode dynamics, and was very low (and almost cancels) for an intermediate frequency located at the intersection of the two resonance curves.

Laser optical feedback imaging controlled by an electronic feedback loop.

In autodyne interferometry, the beating between the reference beam and the signal beam takes place inside the laser cavity and therefore the laser fulfills simultaneously the roles of emitter and detector of photons. In these conditions, the laser relaxation oscillations play a leading role, both in the laser quantum noise, which determines the signal-to-noise ratio (SNR), and also in the laser dynamics, which determines the response time of the interferometer. In the present study, we have experimentally analyzed the SNR and the response time of a laser optical feedback imaging (LOFI) interferometer based on a Nd(3+) microchip laser, with a relaxation frequency in the megahertz range. More precisely, we have compared the image quality obtained when the laser dynamics is free and when it is controlled by a stabilizing electronic feedback loop using a differentiator. From this study, we can conclude that when the laser time response is shorter (i.e., the LOFI gain is lower), the image quality can be better (i.e., the LOFI SNR can be higher) and that the use of an adapted electronic feedback loop allows high-speed LOFI with a shot-noise limited sensitivity. Despite the critical stability of the electronic feedback loop, the obtained experimental results are in good agreement with the theoretical predictions.

Demonstration of a plenoptic microscope based on laser optical feedback imaging.

A new kind of plenoptic imaging system based on Laser Optical Feedback Imaging (LOFI) is presented and is compared to another previously existing device based on microlens array. Improved photometric performances, resolution and depth of field are obtained at the price of a slow point by point scanning. Main properties of plenoptic microscopes such as numerical refocusing on any curved surface or aberrations compensation are both theoretically and experimentally demonstrated with a LOFI-based device.

Optimization of an autodyne laser interferometer for high-speed confocal imaging.

In autodyne interferometry, the beating between the reference beam and the signal beam takes place inside the laser cavity and therefore the laser fulfills simultaneously the roles of the emitter and the detector of photons. In these conditions, the laser relaxation oscillations play a leading role, both in the laser quantum noise that determines the signal-to-noise ratio (SNR) and also in the laser dynamics that determine the response time of the interferometer. In the present study, we have theoretically analyzed the SNR and the response time of a laser optical feedback imaging (LOFI) setup based on an autodyne interferometer. More precisely, we have compared the image quality of two lasers having the same output power and the same relaxation frequency, but having two different values of the LOFI gain induced by two different values of the laser response time. From this study, we have finally determined the best laser dynamical parameters and the best experimental conditions for high-speed imaging at the shot-noise limit. Finally, we conclude that a laser diode with a very short response time (in the nanosecond range) seems to be an interesting candidate compared to solid-state microchip laser with a response time of several tens of microseconds. Analytical predictions are confirmed by numerical simulations.

Novel Cold-Adapted Esterase MHlip from an Antarctic Soil Metagenome.

An Antarctic soil metagenomic library was screened for lipolytic enzymes and allowed for the isolation of a new cytosolic esterase from the a/b hydrolase family 6, named MHlip. This enzyme is related to hypothetical genes coding esterases, aryl-esterases and peroxydases, among others. MHlip was produced, purified and its activity was determined. The substrate profile of MHlip reveals a high specificity for short p-nitrophenyl-esters. The apparent optimal activity of MHlip was measured for p-nitrophenyl-acetate, at 33 °C, in the pH range of 6-9. The MHlip thermal unfolding was investigated by spectrophotometric methods, highlighting a transition (Tm) at 50 °C. The biochemical characterization of this enzyme showed its adaptation to cold temperatures, even when it did not present evident signatures associated with cold-adapted proteins. Thus, MHlip adaptation to cold probably results from many discrete structural modifications, allowing the protein to remain active at low temperatures. Functional metagenomics is a powerful approach to isolate new enzymes with tailored biophysical properties (e.g., cold adaptation). In addition, beside the ever growing amount of sequenced DNA, the functional characterization of new catalysts derived from environment is still required, especially for poorly characterized protein families like α/b hydrolases.

Limitations of synthetic aperture laser optical feedback imaging.

In this paper we study the origin and the effect of amplitude and phase noise on laser optical feedback imaging associated with a synthetic aperture (SA) imaging system. Amplitude noise corresponds to photon noise and acts as an additive noise; it can be reduced by increasing the global measurement time. Phase noise can be divided in three families: random, sinusoidal, and drift phase noise; we show that it acts as a multiplicative noise. We explain how we can reduce phase noise by making oversampling or multiple measurements depending on its type. This work can easily be extended to all SA systems (radar, laser, or terahertz), especially when raw holograms are acquired point by point.

Synthetic aperture laser optical feedback imaging using a translational scanning with galvanometric mirrors.

In this paper we present an experimental setup based on laser optical feedback imaging (LOFI) and on synthetic aperture with translational scanning by galvanometric mirrors for the purpose of making deep and resolved images through scattering media. We provide real two-dimensional optical synthetic aperture image of a fixed scattering target with a moving aperture and an isotropic resolution. We demonstrate theoretically and experimentally that we can keep microscope resolution beyond the working distance. A photometric balance is made, and we show that the number of photons participating in the final image decreases with the square of the reconstruction distance. This degradation is partially compensated by the high sensitivity of LOFI.

Sensitivity of synthetic aperture laser optical feedback imaging.

In this paper, we compare the sensitivity of two imaging configurations, both based on laser optical feedback imaging (LOFI). The first one is direct imaging, which uses conventional optical focalization on target, and the second one is made by a synthetic aperture (SA) laser, which uses numerical focalization. We show that SA configuration allows us to obtain good resolutions with high working distance and that the drawback of SA imagery is that it has a worse photometric balance in comparison to a conventional microscope. This drawback is partially compensated by the important sensitivity of LOFI. Another interest of SA relies on the capacity of getting three-dimensional information in a single x-y scan.

Heterodyne beatings between frequency-shifted feedback lasers.

Frequency-shifted feedback (FSF) lasers are potential candidates for long distance telemetry due to the appearance of beatings in the noise spectrum at the output of a homodyne interferometer: the frequencies of these beatings vary linearly with the path delay. In this Letter we demonstrate that these beatings also occur in the heterodyne mixing of two identical, but distinct, FSF lasers. This phenomenon is explained by the passive cavity model and is exploited to characterize the time-spectrum properties of FSF lasers. Consequences on telemetry with FSF lasers are presented.

Experimental comparison of autodyne and heterodyne laser interferometry using an Nd:YVO4 microchip laser.

Using an Nd:YVO4 microchip laser with a relaxation frequency in the megahertz range, we have experimentally compared a heterodyne interferometer based on a Michelson configuration with an autodyne interferometer based on the laser optical feedback imaging (LOFI) method regarding their signal-to-noise ratios. In the heterodyne configuration, the beating between the reference beam and the signal beam is realized outside the laser cavity, while in the autodyne configuration, the wave beating takes place inside the laser cavity, and the relaxation oscillations of the laser intensity then play an important part. For a given laser output power, object under investigation, and detection noise level, we have determined the amplification gain of the LOFI interferometer compared to the heterodyne interferometer. LOFI interferometry is demonstrated to show higher performance than heterodyne interferometry for a wide range of laser powers and detection levels of noise. The experimental results are in good agreement with the theoretical predictions.

Comparative study of autodyne and heterodyne laser interferometry for imaging.

For given laser output power, object under investigation, and photodiode noise level, we have theoretically compared the signal-to-noise ratios of a heterodyne scanning imager based on a Michelson interferometer and of an autodyne setup based on the laser optical feedback imaging (LOFI) technique. In both cases, the image is obtained point by point. In the heterodyne configuration, the beating between the reference beam and the signal beam is realized outside the laser cavity (i.e., directly on the detector), while in the autodyne configuration, the wave beating takes place inside the laser cavity and therefore is indirectly detected. In the autodyne configuration, where the laser relaxation oscillations play a leading role, we have compared one-dimensional scans obtained by numerical simulations with different lasers' dynamical parameters. Finally, we have determined the best laser for LOFI applications and the experimental conditions for which the LOFI detection setup (autodyne interferometer) is competitive compared to a heterodyne interferometer.