A mechanical transition from tension to buckling underlies the jigsaw puzzle shape morphogenesis of histoblasts in the Drosophila epidermis.

The polygonal shape of cells in proliferating epithelia is a result of the tensile forces of the cytoskeletal cortex and packing geometry set by the cell cycle. In the larval Drosophila epidermis, two cell populations, histoblasts and larval epithelial cells, compete for space as they grow on a limited body surface. They do so in the absence of cell divisions. We report a striking morphological transition of histoblasts during larval development, where they change from a tensed network configuration with straight cell outlines at the level of adherens junctions to a highly folded morphology. The apical surface of histoblasts shrinks while their growing adherens junctions fold, forming deep lobules. Volume increase of growing histoblasts is accommodated basally, compensating for the shrinking apical area. The folded geometry of apical junctions resembles elastic buckling, and we show that the imbalance between the shrinkage of the apical domain of histoblasts and the continuous growth of junctions triggers buckling. Our model is supported by laser dissections and optical tweezer experiments together with computer simulations. Our analysis pinpoints the ability of histoblasts to store mechanical energy to a much greater extent than most other epithelial cell types investigated so far, while retaining the ability to dissipate stress on the hours time scale. Finally, we propose a possible mechanism for size regulation of histoblast apical size through the lateral pressure of the epidermis, driven by the growth of cells on a limited surface. Buckling effectively compacts histoblasts at their apical plane and may serve to avoid physical harm to these adult epidermis precursors during larval life. Our work indicates that in growing nondividing cells, compressive forces, instead of tension, may drive cell morphology.

Frequency-encoded two-photon excited fluorescence microscopy.

Two-photon excited fluorescence (2PEF) microscopy is the most popular non-linear imaging method of biomedical samples. State-of-the art 2PEF microscopes use multiple detectors and spectral filter sets to discriminate different fluorophores based on their distinct emission behavior (emission discrimination). One drawback of 2PEF is that fluorescence photons outside the filter transmission range are inherently lost, thereby reducing the imaging efficiency and speed. Furthermore, emission discrimination of different fluorophores may fail if their emission profiles are too similar. Here, we present an alternative 2PEF method that discriminates fluorophores based on their excitation spectra (excitation discrimination). For excitation we use two lasers of different wavelengths (ω1, ω2) resulting in excitation energies at 2ω1, 2ω2, and the mixing energy ω1+ω2. Both lasers are frequency encoded (FE) by an intensity modulation at distinct frequencies while all 2PEF emission is collected on a single detector. The signal is fed into a lock-in-amplifier and demodulated at various frequencies simultaneously. A customized nonnegative matrix factorization (NNMF) then generates fluorescence images that are free of cross talk. Combining FE-2PEF with multiple detectors has the potential to enable the simultaneous imaging of an unprecedented number of fluorophores.

An adaptive microscope for the imaging of biological surfaces.

Scanning fluorescence microscopes are now able to image large biological samples at high spatial and temporal resolution. This comes at the expense of an increased light dose which is detrimental to fluorophore stability and cell physiology. To highly reduce the light dose, we designed an adaptive scanning fluorescence microscope with a scanning scheme optimized for the unsupervised imaging of cell sheets, which underly the shape of many embryos and organs. The surface of the tissue is first delineated from the acquisition of a very small subset (~0.1%) of sample space, using a robust estimation strategy. Two alternative scanning strategies are then proposed to image the tissue with an improved photon budget, without loss in resolution. The first strategy consists in scanning only a thin shell around the estimated surface of interest, allowing high reduction of light dose when the tissue is curved. The second strategy applies when structures of interest lie at the cell periphery (e.g. adherens junctions). An iterative approach is then used to propagate scanning along cell contours. We demonstrate the benefit of our approach imaging live epithelia from Drosophila melanogaster. On the examples shown, both approaches yield more than a 20-fold reduction in light dose -and up to more than 80-fold- compared to a full scan of the volume. These smart-scanning strategies can be easily implemented on most scanning fluorescent imaging modality. The dramatic reduction in light exposure of the sample should allow prolonged imaging of the live processes under investigation.

Shot-noise limited tunable dual-vibrational frequency stimulated Raman scattering microscopy.

We present a shot-noise limited SRS implementation providing a >200 mW per excitation wavelength that is optimized for addressing two molecular vibrations simultaneously. As the key to producing a 3 ps laser of different colors out of a single fs-laser (15 nm FWHM), we use ultra-steep angle-tunable optical filters to extract 2 narrow-band Stokes laser beams (1-2 nm & 1-2 ps), which are separated by 100 cm-1. The center part of the fs-laser is frequency doubled to pump an optical parametric oscillator (OPO). The temporal width of the OPO's output (1 ps) is matched to the Stokes beams and can be tuned from 650-980 nm to address simultaneously two Raman shifts separated by 100 cm-1 that are located between 500 cm-1 and 5000 cm-1. We demonstrate background-free SRS imaging of C-D labeled biological samples (bacteria and Drosophila). Furthermore, high quality virtual stimulated Raman histology imaging of a brain adenocarcinoma is shown for pixel dwell times of 16 µs.

Physics of growing biological tissues: the complex cross-talk between cell activity, growth and resistance.

For many organisms, shapes emerge from growth, which generates stresses, which in turn can feedback on growth. In this review, theoretical methods to analyse various aspects of morphogenesis are discussed with the aim to determine the most adapted method for tissue mechanics. We discuss the need to work at scales intermediate between cells and tissues and emphasize the use of finite elasticity for this. We detail the application of these ideas to four systems: active cells embedded in tissues, brain cortical convolutions, the cortex of Caenorhabditis elegans during elongation and finally the proliferation of epithelia on extracellular matrix. Numerical models well adapted to inhomogeneities are also presented. This article is part of the theme issue 'Rivlin's legacy in continuum mechanics and applied mathematics'.

Mechanical Forces and Growth in Animal Tissues.

Mechanical forces shape biological tissues. They are the effectors of the developmental programs that orchestrate morphogenesis. A lot of effort has been devoted to understanding morphogenetic processes in mechanical terms. In this review, we focus on the interplay between tissue mechanics and growth. We first describe how tissue mechanics affects growth, by influencing the orientation of cell divisions and the signaling pathways that control the rate of volume increase and proliferation. We then address how the mechanical state of a tissue is affected by the patterns of growth. The forward and reverse interactions between growth and mechanics must be investigated in an integrative way if we want to understand how tissues grow and shape themselves. To illustrate this point, we describe examples in which growth homeostasis is achieved by feedback mechanisms that use mechanical forces.

Dynamic clonal analysis based on chronic in vivo imaging allows multiscale quantification of growth in the Drosophila wing disc.

In the course of morphogenesis, tissues change shape and grow. How this is orchestrated is largely unknown, partly owing to the lack of experimental methods to visualize and quantify growth. Here, we describe a novel experimental approach to investigate the growth of tissues in vivo on a time-scale of days, as employed to study the Drosophila larval imaginal wing disc, the precursor of the adult wing. We developed a protocol to image wing discs at regular intervals in living anesthetized larvae so as to follow the growth of the tissue over extended periods of time. This approach can be used to image cells at high resolution in vivo. At intermediate scale, we tracked the increase in cell number within clones as well as the changes in clone area and shape. At scales extending to the tissue level, clones can be used as landmarks for measuring strain, as a proxy for growth. We developed general computational tools to extract strain maps from clonal shapes and landmark displacements in individual tissues, and to combine multiple datasets into a mean strain. In the disc, we use these to compare properties of growth at the scale of clones (a few cells) and at larger regional scales.

A global pattern of mechanical stress polarizes cell divisions and cell shape in the growing Drosophila wing disc.

Organismal development is under genetic control. Ultimately, mechanical forces shape embryos. If we want to understand the precise regulation of size and shape in animals, we must dissect how forces are distributed in developing tissues, and how they drive cell behavior to shape organs. This has not been addressed fully in the context of growing tissues. As cells grow and divide, they exert a pressure on their neighbors. How these local stresses add up or dissipate as the tissue grows is an unanswered question. We address this issue in the growing wing imaginal disc of Drosophila larvae, the precursor of the adult wing. We used a quantitative approach to analyze the strains and stresses of cells of the wing pouch, and found a global pattern of stress whereby cells in the periphery of the tissue are mechanically stretched and cells in the center are compressed. This pattern has important consequences on cell shape in the wing pouch: cells respond to it by polarizing their acto-myosin cortex, and aligning their divisions with the main axis of cell stretch, thereby polarizing tissue growth. Ectopic perturbations of tissue growth by the Hippo signaling pathway reorganize this pattern in a non-autonomous manner, suggesting a synergy between tissue mechanics and growth control during wing disc morphogenesis.

Crystallization of fluorescent quantum dots within a three-dimensional bio-organic template of actin filaments and lipid membranes.

Biological molecules and molecular self-assemblies are promising templates to organize well-defined inorganic nanostructures. We demonstrate the ability of a self-assembled three-dimensional crystal template of helical actin protein filaments and lipids bilayers to generate a hierarchical self-assembly of quantum dots. Functionnalized tricystein peptidic quantum dots (QDs) are incorporated during the dynamical self-assembly of this actin/lipid template resulting in the formation of crystalline fibers. The crystal parameters, 26.5×18.9×35.5 nm3, are imposed by the membrane thickness, the diameter, and the pitch of the actin self-assembly. This process ensures the high quality of the crystal and results in unexpected fluorescence properties. This method of preparation offers opportunities to generate crystals with new symmetries and a large range of distance parameters.