Dynamic Optical Tweezers using Acousto-Optic Modulators

Treballs Finals de Grau de Física, Facultat de Física, Universitat de Barcelona, Any: 2015, Tutors: Estela Martín Badosa i Mario Montes UsateguiThis work consists of the study, characterisation and set-up of an Acousto-Optic Light Modulator made up of two TeO2 crystals, to use it in an optical tweezers system. We have performed the following tasks: optical system assembly and alignment, optimization of the first diffraction order efficiency of the device and, finally, deflection analysis. Furthermore, we have developed a control system based on a multi-function data acquisition board and a software using LabVIEW that allows the user to dynamically control the position of the laser spot, as well as its amplitude. Lastly, we have improved the program making possible the use of time-sharing trapping

Fast in vivo multiphoton light-sheet microscopy with optimal pulse frequency

International audienceImproving the imaging speed of multiphoton microscopy is an active research field. Among recent strategies, light-sheet illumination holds distinctive advantages for achieving fast imaging in vivo. However, photoperturbation in multiphoton light-sheet microscopy remains poorly investigated. We show here that the heart beat rate of zebrafish embryos is a sensitive probe of linear and nonlinear photoperturbations. By analyzing its behavior with respect to laser power, pulse frequency and wavelength, we derive guidelines to find the best balance between signal and photoperturbation. We then demonstrate one order-of-magnitude signal enhancement over previous implementations by optimizing the laser pulse frequency. These results open new opportunities for fast live tissue imaging

Fast in vivo multiphoton microscopy using optimized light-sheet illumination

International audienceLight-sheet fluorescence microscopy is a method of choice for multiscale live imaging. Indeed, its orthogonal geometry results in high acquisition speed, large field-of-view and low photodamage. Its combination with multiphoton excited fluorescence improves its imaging depth in biological tissues. However, it appears femtosecond laser sources commonly used in multiphoton microscopy at an 80 MHz repetition rate may not be optimized to take full advantage of light-sheet illumination during live imaging. Hence, we investigated the nature of induced photodamage in multiphoton light-sheet microscopy and the influence of laser parameters on the signal-to-photodamage ratio. To this end, we used zebrafish embryonic heart beat rate and fluorophore photobleaching as sensitive reporters of photoperturbations. We characterized linear and nonlinear disruptions depending on laser parameters such as laser mean power, pulse frequency or wavelength, and determine their order and relative impact. We found an optimal pulse frequency of ~10 MHz for imaging mCherry labeled beating hearts at 1030 nm excitation wavelength. Thus, we achieved high-speed imaging without inducing additional linear heating or reaching nonlinear photodamage compared to previous implementation. We reach an order-ofmagnitude enhancement in two-photon excited fluorescence signal by optimizing the laser pulse frequency while maintaining low both the laser average power and its peak irradiance. It is possible to reach even larger enhancement of 3photon excited fluorescence using such laser parameters. More generally, using low laser pulse frequency in multiphoton light-sheet microscopy results in a drastic improvement in signal level without compromising live sample, which opens new opportunities for fast in vivo imaging

Intravital deep-tumor single-beam 3-photon, 4-photon, and harmonic microscopy

International audienceThree-photon excitation has recently been demonstrated as an effective method to perform intravital microscopy in deep, previously inaccessible regions of the mouse brain. The applicability of 3-photon excitation for deep imaging of other, more heterogeneous tissue types has been much less explored. In this work, we analyze the benefit of high-pulse-energy 1 MHz pulse-repetition-rate infrared excitation near 1300 and 1700 nm for in-depth imaging of tumorous and bone tissue. We show that this excitation regime provides a more than 2-fold increased imaging depth in tumor and bone tissue compared to the illumination conditions commonly used in 2-photon excitation, due to improved excitation confinement and reduced scattering. We also show that simultaneous 3- and 4-photon processes can be effectively induced with a single laser line, enabling the combined detection of blue to far-red fluorescence together with second and third harmonic generation without chromatic aberration, at excitation intensities compatible with live tissue imaging. Finally, we analyze photoperturbation thresholds in this excitation regime and derive setpoints for safe cell imaging. Together, these results indicate that infrared high-pulse-energy low-repetition-rate excitation opens novel perspectives for intravital deep-tissue microscopy of multiple parameters in strongly scattering tissues and organs

Label-free imaging of red blood cells and oxygenation with color third-order sum-frequency generation microscopy

International audienceAbstract Mapping red blood cells (RBCs) flow and oxygenation is of key importance for analyzing brain and tissue physiology. Current microscopy methods are limited either in sensitivity or in spatio-temporal resolution. In this work, we introduce a novel approach based on label-free third-order sum-frequency generation (TSFG) and third-harmonic generation (THG) contrasts. First, we propose a novel experimental scheme for color TSFG microscopy, which provides simultaneous measurements at several wavelengths encompassing the Soret absorption band of hemoglobin. We show that there is a strong three-photon (3P) resonance related to the Soret band of hemoglobin in THG and TSFG signals from zebrafish and human RBCs, and that this resonance is sensitive to RBC oxygenation state. We demonstrate that our color TSFG implementation enables specific detection of flowing RBCs in zebrafish embryos and is sensitive to RBC oxygenation dynamics with single-cell resolution and microsecond pixel times. Moreover, it can be implemented on a 3P microscope and provides label-free RBC-specific contrast at depths exceeding 600 µm in live adult zebrafish brain. Our results establish a new multiphoton contrast extending the palette of deep-tissue microscopy

Label-free imaging of red blood cells and oxygenation with color third-order sum-frequency generation microscopy

International audienceMapping red blood cells (RBCs) flow and oxygenation is of key importance for analyzing brain and tissue physiology. Current microscopy methods are limited either in sensitivity or in spatio-temporal resolution. In this work, we introduce a novel approach based on label-free third-order sum-frequency generation (TSFG) and third-harmonic generation (THG) contrasts. First, we propose a novel experimental scheme for color TSFG microscopy, which provides simultaneous measurements at several wavelengths encompassing the Soret absorption band of hemoglobin. We show that there is a strong three-photon (3P) resonance related to the Soret band of hemoglobin in THG and TSFG signals from zebrafish and human RBCs, and that this resonance is sensitive to RBC oxygenation state. We demonstrate that our color TSFG implementation enables specific detection of flowing RBCs in zebrafish embryos and is sensitive to RBC oxygenation dynamics with single-cell resolution and microsecond pixel times. Moreover, it can be implemented on a 3P microscope and provides label-free RBC-specific contrast at depths exceeding 600 µm in live adult zebrafish brain. Our results establish a new multiphoton contrast extending the palette of deep-tissue microscopy

Color TSFG microscopy of red blood cells and oxygenation

International audienceWe present color third-order sum-frequency generation (color TSFG) microscopy, a multiphoton imaging strategy based on the simultaneous detection of several third-order coherent signals produced by two synchronized femtosecond pulse trains. We demonstrate that it can be used to obtain red blood cell (RBC)specific label-free contrast in live zebrafish and is a promising tool for probing RBC oxygenation

Source laser multicolore dans le SWIR pour la microscopie non-linéaire à 3 photons

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Source laser multicolore dans le SWIR pour la microscopie non-linéaire à 3 photons

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Advances in fast multiphoton microscopy using light-sheet illumination

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