Anisha Thayil

In vivo, pixel-resolution mapping of thick filaments' orientation in nonfibrilar muscle using polarization-sensitive second harmonic generation microscopy

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.

Adaptive harmonic generation microscopy of mammalian embryos

Adaptive optics is implemented in a harmonic generation microscope using a wavefront sensorless correction scheme. Both the second- and third-harmonic intensity signals are used as the optimization metric. Aberration correction is performed to compensate both system- and specimen-induced aberrations by using an efficient optimization routine based upon Zernike polynomial modes. Images of live mouse embryos show an improved signal level and resolution.

Characterisation of the dynamic behaviour of lipid droplets in the early mouse embryo using adaptive harmonic generation microscopy.

BackgroundLipid droplets (LD) are organelles with an important role in normal metabolism and disease. The lipid content of embryos has a major impact on viability and development. LD in Drosophila embryos and cultured cell lines have been shown to move and fuse in a microtubule dependent manner. Due to limitations in current imaging technology, little is known about the behaviour of LD in the mammalian embryo. Harmonic generation microscopy (HGM) allows one to image LD without the use of exogenous labels. Adaptive optics can be used to correct aberrations that would otherwise degrade the quality and information content of images.ResultsWe have built a harmonic generation microscope with adaptive optics to characterise early mouse embryogenesis. At fertilization, LD are small and uniformly distributed, but in the implanting blastocyst, LD are larger and enriched in the invading giant cells of the trophectoderm. Time-lapse studies reveal that LD move continuously and collide but do not fuse, instead forming aggregates that subsequently behave as single units. Using specific inhibitors, we show that the velocity and dynamic behaviour of LD is dependent not only on microtubules as in other systems, but also on microfilaments. We explore the limits within which HGM can be used to study living embryos without compromising viability and make the counterintuitive finding that 16 J of energy delivered continuously over a period of minutes can be less deleterious than an order of magnitude lower energy delivered dis-continuously over a period of hours.ConclusionsLD in pre-implantation mouse embryos show a previously unappreciated complexity of behaviour that is dependent not only on microtubules, but also microfilaments. Unlike LD in other systems, LD in the mouse embryo do not fuse but form aggregates. This study establishes HGM with adaptive optics as a powerful tool for the study of LD biology and provides insights into the photo-toxic effects of imaging embryos.

Three-dimensional imaging of direct-written photonic structures

Third-harmonic generation microscopy has been used to analyze the morphology of photonic structures created using the femtosecond laser direct-write technique. Three-dimensional waveguide arrays and waveguide-Bragg gratings written in fused-silica and doped phosphate glass were investigated. A sensorless adaptive-optical system was used to correct the optical aberrations occurring in the sample and microscope system, which had a lateral resolution of less than 500 nm. This nondestructive testing method creates volume reconstructions of photonic devices and reveals details invisible to other linear microscopy and index profilometry techniques.

Long-term imaging of mouse embryos using adaptive harmonic generation microscopy.

We present a detailed description of an adaptive harmonic generation (HG) microscope and culture techniques that permit long-term, three-dimensional imaging of mouse embryos. HG signal from both pre- and postimplantation stage (0.5-5.5 day-old) mouse embryos are fully characterized. The second HG images reveal central spindles during cytokinesis whereas third HG images show several features, such as lipid droplets, nucleoli, and plasma membranes. The embryos are found to develop normally during one-day-long discontinuous HG imaging, permitting the observation of several dynamic events, such as morula compaction and blastocyst formation.

The influence of aberrations in third harmonic generation microscopy

We present an analysis of the effects of aberrations in third harmonic generation (THG) microscopy by considering different specimen geometries. Numerical simulations show the general trend that signal intensity and resolution are reduced as aberrations increase in amplitude. It is also shown that there are certain combinations of specimen structure and focusing position for which the presence of aberrations results in an increase in the image intensity. This occurs, for example, when there are several interfaces near the focal volume. The axial spreading of the excitation focal volume increases the characteristic coherence length for THG signal build-up, resulting in a significant contribution to the image brightness from axial planes near the focal plane. These results have important consequences for the interpretation of THG microscope images and for image based aberration correction in adaptive optics THG microscopy.

Adaptive optics for two-photon and harmonic generation microscopy

Specimen-induced aberrations frequently affect image quality in high-resolution microscopes. We apply adaptive optics to correct aberrations in two-photon fluorescence, and second and third harmonic microscopes. In particular, this is applied to imaging of mouse embryos.

Adaptive optics for microscopy and photonic fabrication

The image resolution and contrast of microscopes are often detrimentally affected by aberrations that are introduced when focusing deep into specimens. These aberrations arise from spatial differences in optical properties of the specimen or refractive index mismatches. This is particularly problematic in multiphoton microscopy, where short pulsed lasers are used to generate contrast through non-linear optical effects, such as two-photon fluorescence or third harmonic generation. The non-linear nature of the signal generation process means that the signal level is strongly affected by changes in the focal spot intensity. We have applied the techniques of adaptive optics to measure and correct the aberrations, restoring image quality. In particular, this has been demonstrated in harmonic generation microscopy of developing mouse embryos. Similar aberration problems affect the resolution and efficiency of three-dimensional optical fabrication systems, such those used for the manufacture of photonic crystals or optical waveguides. These systems are based around microscope optics and use short pulsed laser illumination to induce localized multiphoton effects in a fabrication substrate. In this case, significant aberrations are introduced when focusing deep into the substrate. We report on the development of adaptive optics systems for these applications and discuss the specific challenges for wave front sensing and correction that are presented by these systems.

Adaptive multiphoton and harmonic generation microscopy for developmental biology

Specimen-induced aberrations often affect microscopes, particularly when high numerical aperture lenses are used to image deep into biological specimens. These aberrations cause a reduction in resolution and focal intensity. This is particularly problematic in multiphoton microscopy, where the non-linear nature of the signal generation process means that the signal level is strongly affected by changes in the focal spot intensity. We have applied the techniques of adaptive optics to correct aberrations in two-photon fluorescence and harmonic generation microscopes, restoring image quality. In particular we have used these microscopes for studies in developmental biology and for the imaging of mammalian embryos.