KEYWORDS: Red blood cells, Oxygenation, Microscopy, Tissues, Absorption, Third harmonic generation, Sum frequency generation, Spatial resolution, Resonance enhancement, Multiphoton microscopy
We 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.
We propose a framework to quantify photodamage in multiphoton light-sheet microscopy. Using cardiac imaging in live zebrafish embryos, we demonstrate an order of magnitude signal enhancement is safely obtained by adjusting the laser repetition rate.
Light-sheet illumination enables major increase in multiphoton imaging speed for in vivo studies. 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 balance 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 in vivo imaging.
Light-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-of-magnitude 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 3- photon 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.
The application range of P-THG microscopy has been so far restricted to studies on molecular order and anisotropy of static specimen removed from their biological environment. Slow polarization commutation limits the investigation of highly dynamic systems because of motion artifacts. Here we have developed a new fast-P-THG microscope enabling efficient in vivo studies in dynamic biological samples. Our P-THG scheme benefits from a built-in EOM that switch polarization states at kilohertz between image lines to provide artefact-free P-THG images with micrometric resolution. Furthermore, we have developed a fast Fourier analysis enabling rapid P-THG processing to quantify lipid order and angular maps. We demonstrated that fast-P-THG is suitable in two major applications. Using first a linear polarization configuration, fast P-THG imaging revealed molecular order changes in MLVs undergoing phase-transition upon heating despite sample distortions. Anisotropy properties of small endogenous microparticles swimming in the otolith cavity embryos were also reported in early zebrafish embryos. A second configuration with linear-circular polarization commutation enabled efficient detection of birefringent media such as anisotropic vesicles in C-elegans gut cells.
Large-scale microscopy approaches are transforming brain imaging, but currently lack efficient multicolor contrast modalities. We address this issue by introducing chromatic multiphoton serial (ChroMS) microscopy, a method combining multicolor multiphoton excitation through wavelength mixing and microtome-assisted serial block-face image acquisition. This approach delivers large-scale micrometric imaging of spectrally distinct fluorescent proteins with constant micrometer-scale resolution and sub-micron channel registration over the entire imaged volume. We achieve multicolor 3D imaging over several cubic millimeters and brain-wide serial 2D multicolor imaging. We illustrate the potential of this method for several novel types of measurements interesting for region-scale or whole brain studies: (i) color-based analysis of astrocyte morphology and spatial interactions in the mouse cerebral cortex, (ii) tracing of densely labeled neurons, and (iii) brain-wide mapping of axonal projections labeled with distinct tracers.
Over the past decade the range of available excitation wavelengths used in nonlinear microscopy has continuously extended within the near infrared window. Nowadays, excitation wavelengths ranging from 750 nm to 1300 nm are routinely used to perform multi-parametric imaging [1,2] and multiple wavelength excitation are used in many techniques, including multicolor 2-photon excited fluorescence imaging, Coherent Anti-stokes Raman Scattering (CARS), Stimulated Raman Scattering (SRS) or sum-frequency generation. While this trend opens new perspectives and applications in the biomedical sciences, it also raises new technical issues. For instance, it calls for new standards for quantifying and comparing the performances of nonlinear microscopes over a broad range of wavelengths. In particular, microscopes equipped with multiple femtosecond sources spanning the entire near-infrared wavelength range are often problematic to characterize with current approaches based on fluorescent probes.
In this study, we present a new and straightforward method to quantify the imaging properties of nonlinear microscopes over a broad range of excitation wavelengths [3]. We show that harmonic generation nanoprobes are a unique tool to map the spatial resolution, field curvature and chromatic aberrations of nonlinear microscopes with a precision below the diffraction limit, across the whole field of view, and with a single calibration sample. We analyze and compare measurements obtained with several microscope objectives designed for multiphoton microscopy over the 850-1100nm wavelength range. Finally, we discuss strategies to minimize the impact of chromatic aberrations during multicolor imaging and we show how our metrology can be used for the post-acquisition correction of chromatic aberrations.
[1] Mahou et al. Nat. Methods 9, 815-18 (2012).
[2] Alexander et al. Curr. Opin. Cell Biol. 25 , 659-71 (2013)
[3] Mahou et al., submitted.
Two-photon laser scanning microscopy has become a standard to map thick and live tissues. However, its application for fast and multicolor imaging remains challenging. To address this issue, we report on the implementation of mixed wavelength excitation in a two-photon light-sheet microscope. We illustrate the potential of the technique by recording sustained multicolor two-photon movies of the beating heart in zebrafish embryos with negligible photobleaching at 28 million pixels/second. In particular, 3D reconstructions of the heart periodic motion are obtained with sufficient spatiotemporal resolution to track the fast movements of individual cells during a cardiac cycle.
Embryonic development strictly depends on fluid dynamics. As a consequence, understanding biological fluid dynamic
is essential since it is unclear how flow affects development. For example, the specification of the left-right axis in
vertebrates depends on fluid flow where beating cilia generate a directional flow necessary for breaking the embryonic
symmetry in the so-called left-right organizer. To investigate flow dynamics in vivo proper labeling methods necessitate
approaches that are compatible with both normal biology and in vivo imaging. In this study, we describe a strategy for
labeling and analyzing microscopic fluid flows in vivo that meets this challenge. We developed an all-optical approach
based on three steps. First we used sub-cellular femtosecond laser ablation to generate fluorescent micro-debris to label
the flow. The non-linear effect used in this technique allows a high spatial confinement and a low invasiveness, thus
permitting the targeting of sub-cellular regions deep inside the embryo. Then, we used fast confocal imaging and 3D-particle
tracking were used to image and quantify the seeded flow. This approach was used to investigate the flow
generated within zebrafish left-right organizer, a micrometer scale ciliated vesicle located deep inside the embryo and
involved in breaking left-right embryonic symmetry. We mapped the velocity field within the vesicle and surrounding a
single beating cilium, and showed that this method can address the dynamics of cilia-driven flows at multiple length
scales. We could validate the flow features as predicted from previous simulations. Such detailed descriptions of fluid
movements will be valuable in unraveling the relationships between cilia-driven flow and signal transduction. More
generally, this all-optical approach opens new opportunities for investigating microscopic flow in living tissues.
Third-harmonic generation (THG) microscopy can provide structural information from unstained biological samples such as developing embryos. However, the contrast mechanisms in THG imaging need to be better characterized in order to develop practical applications. We studied experimentally and theoretically the influence of sample structure and excitation NA (Rayleigh length) on THG signals for various cases (spheres, interfaces). Because the third-harmonic signal critically depends on the spatial distribution of the Gouy shift, the effect of changing the excitation NA depends on the sample geometry within the focal volume. This phenomenon can be used to highlight certain structures within a complex system. Finally, we measured the nonlinear optical properties of several liquids, and we identified lipid bodies as an important source of contrast in biological THG imaging. We show that the technique can be used to characterize lipid accumulation in a variety of cells and tissues.
The regulation of morphogenetic movements that shape an embryo during its development remains a challenging issue in developmental biology, and may in certain cases involve mechanical sensitivity. Addressing this issue requires novel experimental approaches. We show that the combination of femtosecond laser pulse-induced ablation and multiphoton microcopy can be used to modulate and quantify morphogenetic movements in Drosophila embryos. We characterized the effects of focused nanoJoule pulse trains in developing embryos. We used targeted ablations to locally modify the embryo structural integrity and modulate morphogenesis. Femtosecond-pulse induced ablation was combined with nonlinear microscopy based on two-photon-excited fluorescence (2PEF) and third-harmonic generation (THG).Correlation-based analysis of microscopy data allowed us to track the outcome of ablations and to analyze tissue deformations. These experiments provided insight into the interplay between gene expression and tissue deformations in developing embryos.
Animal embryo development exhibits a complex ensemble of cell movements that are tightly regulated by developmental gene expression. It was proposed recently that mechanical factors may also play an important role during development. Investigating these dynamical processes is technically challenging and requires novel in vivo investigation methods. We show that multiphoton microscopy can be used for both perturbing and analyzing morphogenetic movements in vivo. (i) nonlinear microscopy is well adapted for the sustained imaging of early Drosophila embryos despite their highly scattering nature; (ii) femtosecond pulse-induced ablation can be used to process specific tissues in vivo. Combining this approach with multimodal microscopy (two-photon-excited fluorescence (2PEF) and third-harmonic generation (THG)), we report the successful quantitative modulation of morphogenetic movements in vivo. Our data provides insight to the issue of morphogenesis regulation.
Animal embryo development exhibits a complex choreography of cell movements highly regulated both in time and space. This sequence of morphogenetic movements is initiated at gastrulation and is tightly controlled by a cascade of developmental gene expression. We have recently reported that developmental gene expression can in turn be mechanically regulated by morphogenetic movements during Drosophila melanogaster early development. In order to study this phenomenon of mechanically induced gene expression, it is necessary to develop new techniques of in vivo investigation. We show that the combination of femtosecond pulse intratissue surgery and two-photon-excitation fluorescence (2PEF) microscopy is a powerful tool for (i) disrupting natural morphogenetic movements and (ii) imaging native and disrupted morphogenetic movements during Drosophila development. (i) First, non-linear-absorption-mediated photo-disruption makes it possible to perform controlled intra-vital micro-dissections resulting in the modulation of morphogenetic movements and subsequent mechano-sensitive gene expression. (ii) Second, in vivo 2PEF microscopy of transgenic GFP systems appears to be an excellent technique for long-term in vivo imaging of the complex morphogenetic movements involved in normal or perturbed Drosophila gastrulation. Together, these two techniques provide a powerful novel approach to study embryo development.
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