[Show abstract][Hide abstract] ABSTRACT: Light-induced toxicity is a fundamental bottleneck in microscopic imaging of live embryos. In this article, after a review of photodamage mechanisms in cells and tissues, we assess photo-perturbation under illumination conditions relevant for point-scanning multiphoton imaging of live Drosophila embryos. We use third-harmonic generation (THG) imaging of developmental processes in embryos excited by pulsed near-infrared light in the 1.0-1.2 µm range. We study the influence of imaging rate, wavelength, and pulse duration on the short-term and long-term perturbation of development and define criteria for safe imaging. We show that under illumination conditions typical for multiphoton imaging, photodamage in this system arises through 2- and/or 3-photon absorption processes and in a cumulative manner. Based on this analysis, we derive general guidelines for improving the signal-to-damage ratio in two-photon (2PEF/SHG) or THG imaging by adjusting the pulse duration and/or the imaging rate. Finally, we report label-free time-lapse 3D THG imaging of gastrulating Drosophila embryos with sampling appropriate for the visualisation of morphogenetic movements in wild-type and mutant embryos, and long-term multiharmonic (THG-SHG) imaging of development until hatching.
PLoS ONE 01/2014; 9(8):e104250. · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Nonlinear optical microscopy is a biocompatible avenue for probing
ordered molecular assemblies in biological tissues. As in linear optics,
the nonlinear optical response from ordered systems is
polarization-sensitive. This dependence can be used to identify and
characterize local molecular ordering with micrometer-scale 3D
resolution in a nonlinear microscope. In particular, third-harmonic
generation (THG) microscopy is a nonlinear optical modality sensitive to
the electronic nonlinear susceptibility χ(3) of a
material. THG microscopy can be used to map χ(3) spatial
variations (i.e. material interfaces), and to probe birefringence. In
principle, polarization-resolved THG (P-THG) can therefore be used to
probe ordered molecular arrays. However, the orientation, distribution,
and nonlinear optical properties of the molecules near the beam focus
all affect the detected signal. It is therefore necessary to develop a
theoretical method which decouples these effects and permits the
extraction of orientational information from P-THG images. In this
report, we first present P-THG images of model systems (lipid droplets,
multilamellar lipid vesicles) and biological tissues (human skin biopsy)
which establish that P-THG is sensitive to lipid ordering and that it is
maximized when excitation polarization is parallel to the ordered lipid
molecules, giving impetus for the development of a thorough theoretical
analysis. We then outline a multiscale model spanning the molecular (nm)
and ensemble (μm) scales predicting the PTHG signal, consisting of
three main steps: (i) calculation of the molecular electronic
hyperpolarizability; (ii) determination of the anisotropic
χ(3) for various molecular distribution parameters; and
(iii) numerical calculations of the P-THG signal from lipid-water
interfaces. This analysis links the measured P-THG response to lipid
molecular structure and ordering.
[Show abstract][Hide abstract] ABSTRACT: Ordered lipid assemblies are responsible for important physiological functions including skin barrier and axon conductivity. However, techniques commonly used to probe molecular order such as X-ray scattering and nuclear magnetic resonance are not suited for in-situ tissue studies. Here, we identify and characterize a novel contrast mechanism in nonlinear optical microscopy which is sensitive to molecular ordering in multilamellar lipid vesicles (MLVs) and in samples obtained from human skin biopsy: polarized third-harmonic generation (P-THG). We develop a multiscale theoretical framework to calculate the anisotropic, nonlinear optical response of lipid arrays as a function of molecular order. This analysis reveals that conserved carbon-carbon bond and aliphatic tail directionality are the atomic- and molecular-scale sources of the observed P-THG response, respectively. Agreement between calculations and experiments on lipid droplets and MLVs validates the use of P-THG as a probe of lipid ordering. Finally, we show that P-THG can be used to map molecular ordering in the multilamellar, intercorneocyte lipid matrix of the stratum corneum of human skin. These results provide the foundation for the use of P-THG in probing molecular order and highlight a novel biomedical application of multiphoton microscopy in an optically accessible tissue relevant to monitoring lipid-related disorder.
[Show abstract][Hide abstract] ABSTRACT: We study theoretically and numerically third-harmonic generation (THG) from model geometries (interfaces, slabs, periodic media) illuminated by Bessel beams produced by focusing an annular intensity profile. Bessel beams exhibit a phase and intensity distribution near focus different from Gaussian beams, resulting in distinct THG phase matching properties and coherent scattering directions. Excitation wave vectors are controlled by adjusting the bounding aperture angles of the Bessel beam. In addition to extended depth-of-field imaging, this opens interesting perspectives for coherent nonlinear microscopy, such as extracting sample spatial frequencies in the λ/8 - λ range in the case of organized media.
[Show abstract][Hide abstract] ABSTRACT: We demonstrate a simple method for mapping optical aberrations with 3D resolution within thick samples. The method relies on the local measurement of the variation in image quality with externally applied aberrations. We discuss the accuracy of the method as a function of the signal strength and of the aberration amplitude and we derive the achievable resolution for the resulting measurements. We then report on measured 3D aberration maps in human skin biopsies and mouse brain slices. From these data, we analyse the consequences of tissue structure and refractive index distribution on aberrations and imaging depth in normal and cleared tissue samples. The aberration maps allow the estimation of the typical aplanetism region size over which aberrations can be uniformly corrected. This method and data pave the way towards efficient correction strategies for tissue imaging applications.
[Show abstract][Hide abstract] ABSTRACT: We achieve simultaneous two-photon excitation of three chromophores with distinct absorption spectra using synchronized pulses from a femtosecond laser and an optical parametric oscillator. The two beams generate separate multiphoton processes, and their spatiotemporal overlap provides an additional two-photon excitation route, with submicrometer overlay of the color channels. We report volume and live multicolor imaging of 'Brainbow'-labeled tissues as well as simultaneous three-color fluorescence and third-harmonic imaging of fly embryos.
[Show abstract][Hide abstract] ABSTRACT: Multiphoton microscopy is a powerful tool in neuroscience, promising to deliver important data on the spatiotemporal activity within individual neurons as well as in networks of neurons. A major limitation of current technologies is the relatively slow scan rates along the z direction compared to the kHz rates obtainable in the x and y directions. Here, we describe a custom-built microscope system based on an architecture that allows kHz scan rates over hundreds of microns in all three dimensions without introducing aberration. We further demonstrate how this high-speed 3D multiphoton imaging system can be used to study neuronal activity at millisecond resolution at the subcellular as well as the population level.
Proceedings of the National Academy of Sciences 02/2012; 109(8):2919-24. · 9.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Accurate control over the phase and amplitude modulation in an adaptive
microscope is essential to the quality of aberration correction that can
be achieved. In this paper we present a robust and compact method for
characterising such amplitude and phase modulation in the pupil plane of
the focussing objective. This method, based on phase diversity, permits
calibrating the microscope as a whole and thus avoids errors in the
alignment of the wavefront shaping device after calibration and the
resulting imprecision in the induced modulation: by acquiring three 2D
images of the point spread function at different distances from the
focal plane, we show that the electric field distribution at the pupil
plane can be retrieved using an iterative algorithm. We have applied
this technique to the characterisation of the phase modulation induced
by a deformable mirror when conjugated with the entrance pupil of
different objectives, which permits accurate evaluation of the
performance of the mirror for subsequent aberration correction.
[Show abstract][Hide abstract] ABSTRACT: We investigate the parameters governing the accuracy of correction in
modal sensorless adaptive optics for microscopy. In this paper we focus
on the case of two-photon excited fluorescence. Using analytical,
numerical and experimental results, we show that using a suitable number
of measurements, accurate correction can be achieved for up to 2 rad rms
initial aberrations even without optimisation of the correction modes.
We demonstrate that this correction can be achieved using low light
levels to minimise photobleaching and toxicity, and we provide examples
of such optimised correction.
[Show abstract][Hide abstract] ABSTRACT: Modal sensorless adaptive optics relies on the use of an image quality
metric to estimate the amplitude of aberrations, and of a well-suited
set of aberration modes to describe the aberration. This set is chosen
so that aberration of one mode does not influence correction in another
mode. In this paper, we show how these modes can be derived
experimentally, and investigate the influence of imperfect crosstalk
removal on the accuracy of correction. We show that the resulting error
can be mitigated using appropriate algorithms that can incorporate
knowledge of the influence of the modes on the metric and, if available,
partial knowledge of the aberrations. Finally, we derive from these
results the minimum time required for correction in various situations.
[Show abstract][Hide abstract] ABSTRACT: We investigate theoretically and experimentally the parameters governing the accuracy of correction in modal sensorless adaptive optics for microscopy. On the example of two-photon fluorescence imaging, we show that using a suitable number of measurements, precise correction can be obtained for up to 2 radians rms aberrations without optimising the aberration modes used for correction. We also investigate the number of photons required for accurate correction when signal acquisition is shot-noise limited. We show that only 10(4) to 10(5) photons are required for complete correction so that the correction process can be implemented with limited extra-illumination and associated photoperturbation. Finally, we provide guidelines for implementing an optimal correction algorithm depending on the experimental conditions.
[Show abstract][Hide abstract] ABSTRACT: Over the last decade, researchers have applied adaptive optics—a technology
that was originally conceived for telescopes—to high-resolution microscopy in order
to overcome the problems caused by specimen-induced aberrations.
[Show abstract][Hide abstract] ABSTRACT: Investigating cell dynamics during early zebrafish embryogenesis requires specific image acquisition and analysis strategies. Multiharmonic microscopy, i.e., second- and third-harmonic generations, allows imaging cell divisions and cell membranes in unstained zebrafish embryos from 1- to 1000-cell stage. This paper presents the design and implementation of a dedicated image processing pipeline (tracking and segmentation) for the reconstruction of cell dynamics during these developmental stages. This methodology allows the reconstruction of the cell lineage tree including division timings, spatial coordinates, and cell shape until the 1000-cell stage with minute temporal accuracy and micrometer spatial resolution. Data analysis of the digital embryos provides an extensive quantitative description of early zebrafish embryogenesis.
[Show abstract][Hide abstract] ABSTRACT: Nonlinear microscopy can be used to probe the intrinsic optical properties of biological tissues. Using femtosecond pulses, third-harmonic generation (THG) and four-wave mixing (FWM) signals can be efficiently produced and detected simultaneously. Both signals probe a similar parameter, i.e. the real part of the third-order nonlinear susceptibility χ((3)). However THG and FWM images result from different phase matching conditions and provide complementary information. We analyze this complementarity using calculations, z-scan measurements on water and oils, and THG-FWM imaging of cell divisions in live zebrafish embryos. The two signals exhibit different sensitivity to sample size and clustering in the half-wavelength regime. Far from resonance, THG images reveal spatial variations |Δχ((3))(-3ω;ω,ω,ω)| with remarkable sensitivity while FWM directly reflects the distribution of χ((3))(-2ω(1) + ω(2);ω(1), -ω(2), ω(1)). We show that FWM images provide χ((3)) maps useful for proper interpretation of cellular THG signals, and that combined imaging carries additional structural information. Finally we present simultaneous imaging of intrinsic THG, FWM, second-harmonic (SHG) and two-photon-excited fluorescence (2PEF) signals in live Caenorhabditis elegans worms illustrating the information provided by multimodal nonlinear imaging of unstained tissue.
[Show abstract][Hide abstract] ABSTRACT: Multiphoton imaging is a promising approach for addressing current issues in systems biology and high-content investigation of embryonic development. Recent advances in multiphoton microscopy, including light-sheet illumination, optimized laser scanning, adaptive and label-free strategies, open new opportunities for embryo imaging. However, the literature is often unclear about which microscopy technique is most adapted for achieving specific experimental goals. In this review, we describe and discuss the key concepts of imaging speed, imaging depth, photodamage, and nonlinear contrast mechanisms in the context of recent advances in live embryo imaging. We illustrate the potentials of these new imaging approaches with a selection of recent applications in developmental biology.
Current opinion in genetics & development 09/2011; 21(5):538-48. · 8.99 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We present a simple and versatile scheme for characterising amplitude and phase modulation by an active element, such as a deformable mirror, in the pupil plane of a high NA microscope. By placing a mirror in the vicinity of the focal plane of the objective and recording images of the reflected focal spot on a camera, we show that reliable measurements of the influence function of the mirror actuators in the pupil plane of the objective can be obtained using an iterative electric field retrieval algorithm. Compared to direct wavefront sensors, the proposed method allows characterisation for a variety of objectives with different NA and pupil sizes without modification of the setup, requires minimal space inside the microscope, and can be used with pulsed sources such as used for multiphoton microscopy. In order to validate our method, we compare our data to the results obtained with a Shack-Hartmann wavefront sensor, and show that comparable precision is achieved.
Journal of Microscopy 08/2011; 244(2):136-43. · 1.63 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The reconstruction of the cell lineage tree of early zebrafish embryogenesis requires the use of in-vivo microscopy imaging and image processing strategies. Second (SHG) and third harmonic generation (THG) microscopy observations in unstained zebrafish embryos allows to detect cell divisions and cell membranes from 1-cell to 1K-cell stage. In this article, we present an ad-hoc image processing pipeline for cell tracking and cell membranes segmentation enabling the reconstruction of the early zebrafish cell lineage tree until the 1K-cell stage. This methodology has been used to obtain digital zebrafish embryos allowing to generate a quantitative description of early zebrafish embryogenesis with minute temporal accuracy and μm spatial resolution.
Proceedings of the 8th IEEE International Symposium on Biomedical Imaging: From Nano to Macro, ISBI 2011, March 30 - April 2, 2011, Chicago, Illinois, USA; 01/2011
[Show abstract][Hide abstract] ABSTRACT: Quantifying cell behaviors in animal early embryogenesis remains a challenging issue requiring in toto imaging and automated image analysis. We designed a framework for imaging and reconstructing unstained whole zebrafish embryos for their first 10 cell division cycles and report measurements along the cell lineage with micrometer spatial resolution and minute temporal accuracy. Point-scanning multiphoton excitation optimized to preferentially probe the innermost regions of the embryo provided intrinsic signals highlighting all mitotic spindles and cell boundaries. Automated image analysis revealed the phenomenology of cell proliferation. Blastomeres continuously drift out of synchrony. After the 32-cell stage, the cell cycle lengthens according to cell radial position, leading to apparent division waves. Progressive amplification of this process is the rule, contrasting with classical descriptions of abrupt changes in the system dynamics.