3D time-lapse imaging of a mouse embryo using intensity diffraction tomography embedded inside a deep learning framework.

We present a compact 3D diffractive microscope that can be inserted directly in a cell incubator for long-term observation of developing organisms. Our setup is particularly simple and robust, since it does not include any moving parts and is compatible with commercial cell culture containers. It has been designed to image large specimens (>100×100×100µm3) with subcellular resolution. The sample's optical properties [refractive index (RI) and absorption] are reconstructed in 3D from intensity-only images recorded with different illumination angles produced by an LED array. The reconstruction is performed using the beam propagation method embedded inside a deep-learning network where the layers encode the optical properties of the object. This deep neural network is trained for a given multiangle intensity acquisition. After training, the weights of the neural network deliver the 3D distribution of the optical properties of the sample. The effect of spherical aberrations due to the sample holder/air interfaces are taken into account in the forward model. Using this approach, we performed time-lapse 3D imaging of preimplantation mouse embryos over six days. Images of embryos from a single cell (low-scattering regime) to the blastocyst stage (highly scattering regime) were successfully reconstructed. Due to its subcellular resolution, our system can provide quantitative information on the embryos' development and viability. Hence, this technology opens what we believe to be novel opportunities for 3D label-free live-cell imaging of whole embryos or organoids over long observation times.

Phase and fluorescence imaging with a surprisingly simple microscope based on chromatic aberration.

We propose a simple and compact microscope combining phase imaging with multi-color fluorescence using a standard bright-field objective. The phase image of the sample is reconstructed from a single, approximately 100 µm out-of-focus image taken under semi-coherent illumination, while fluorescence is recorded in-focus in epi-fluorescence geometry. The reproducible changes of the focus are achieved with specifically introduced chromatic aberration in the imaging system. This allows us to move the focal plane simply by changing the imaging wavelength. No mechanical movement of neither sample nor objective or any other part of the setup is therefore required to alternate between the imaging modality. Due to its small size and the absence of motorized components the microscope can easily be used inside a standard biological incubator and allows long-term imaging of cell culture in physiological conditions. A field-of-view of 1.2 mm2 allows simultaneous observation of thousands of cells with micro-meter spatial resolution in phase and multi-channel fluorescence mode. In this manuscript we characterize the system and show a time-lapse of cell culture in phase and multi-channel fluorescence recorded inside an incubator. We believe that the small dimensions, easy usage and low cost of the system make it a useful tool for biological research.

Confinement-Induced Transition between Wavelike Collective Cell Migration Modes.

The structural and functional organization of biological tissues relies on the intricate interplay between chemical and mechanical signaling. Whereas the role of constant and transient mechanical perturbations is generally accepted, several studies recently highlighted the existence of long-range mechanical excitations (i.e., waves) at the supracellular level. Here, we confine epithelial cell monolayers to quasi-one-dimensional geometries, to force the establishment of tissue-level waves of well-defined wavelength and period. Numerical simulations based on a self-propelled Voronoi model reproduce the observed waves and exhibit a phase transition between a global and a multinodal wave, controlled by the confinement size. We confirm experimentally the existence of such a phase transition, and show that wavelength and period are independent of the confinement length. Together, these results demonstrate the intrinsic origin of tissue oscillations, which could provide cells with a mechanism to accurately measure distances at the supracellular level.

Localisation microscopy with quantum dots using non-negative matrix factorisation.

We propose non-negative matrix factorisation with iterative restarts (iNMF) to model a noisy dataset of highly overlapping fluorophores with intermittent intensities. We can recover high-resolution images of individual sources from the optimised model, despite their high mutual overlap in the original data. Each source can have an arbitrary, unknown shape of the PSF and blinking behaviour. This allows us to use quantum dots as bright and stable fluorophores for localisation microscopy. We compare the iNMF results to CSSTORM, 3B and bSOFI. iNMF shows superior performance in the challenging task of super-resolution imaging using quantum dots. We can also retrieve axial localisation of the sources from the shape of the recovered PSF.

Phase optimisation for structured illumination microscopy.

Structured illumination microscopy can achieve super-resolution in fluorescence imaging. The sample is illuminated with periodic light patterns, and a series of images are acquired for different pattern positions, also called phases. From these a super-resolution image can be computed. However, for an artefact-free reconstruction it is important that the pattern phases be known with very high precision. If the necessary precision cannot be guaranteed experimentally, the phase information has to be retrieved a posteriori from the acquired data. We present a fast and robust algorithm that iteratively determines these phases with a precision of typically below λ/100. Our method, which is based on cross-correlations, allows optimisation of pattern phase even when the pattern itself is too fine for detection, in which case most other methods inevitably fail. We analyse the performance of this method using simulated data from a synthetic 2D sample as well as experimental single-slice data from a 3D sample and compare it with another previously published approach.

Line scan--structured illumination microscopy super-resolution imaging in thick fluorescent samples.

Structured illumination microscopy in thick fluorescent samples is a challenging task. The out-of-focus fluorescence background deteriorates the illumination pattern and the reconstructed images suffer from influence of noise. We present a combination of structured illumination microscopy with line scanning. This technique reduces the out-of-focus fluorescence background, which improves the modulation and the quality of the illumination pattern and therefore facilitates the reconstruction. We present super-resolution, optically sectioned images of a thick fluorescent sample, revealing details of the specimen's inner structure.

t-SNARE protein conformations patterned by the lipid microenvironment.

The spatial distribution of the target (t-)SNARE proteins (syntaxin and SNAP-25) on the plasma membrane has been extensively characterized. However, the protein conformations and interactions of the two t-SNAREs in situ remain poorly defined. By using super-resolution optical techniques and fluorescence lifetime imaging microscopy, we observed that within the t-SNARE clusters syntaxin and SNAP-25 molecules interact, forming two distinct conformations of the t-SNARE binary intermediate. These are spatially segregated on the plasma membrane with each cluster exhibiting predominantly one of the two conformations, representing the two- and three-helical forms previously observed in vitro. We sought to explain why these two t-SNARE intermediate conformations exist in spatially distinct clusters on the plasma membrane. By disrupting plasma membrane lipid order, we found that all of the t-SNARE clusters now adopted a single conformational state corresponding to the three helical t-SNARE intermediates. Together, our results define spatially distinct t-SNARE intermediate states on the plasma membrane and how the conformation adopted can be patterned by the underlying lipid environment.

Structured illumination microscopy of a living cell.

Due to diffraction, the resolution of imaging emitted light in a fluorescence microscope is limited to about 200 nm in the lateral direction. Resolution improvement by a factor of two can be achieved using structured illumination, where a fine grating is projected onto the sample, and the final image is reconstructed from a set of images taken at different grating positions. Here we demonstrate that with the help of a spatial light modulator, this technique can be used for imaging slowly moving structures in living cells.

Lens-free microscopy for 3D + time acquisitions of 3D cell culture.

Thanks to a novel three-dimensional imaging platform based on lens-free microscopy, it is possible to perform multi-angle acquisitions and holographic reconstructions of 3D cell cultures directly into the incubator. Being able of reconstructing volumes as large as ~5 mm3 over a period of time covering several days, allows us to observe a broad range of migration strategies only present in 3D environment, whether it is single cell migration, collective migrations of cells and dispersal of cells. In addition we are able to distinguish new interesting phenomena, e.g. large-scale cell-to-matrix interactions (>1 mm), fusion of cell clusters into large aggregate (~10,000 µm2) and conversely, total dissociation of cell clusters into clumps of migrating cells. This work on a novel 3D + time lens-free microscopy technique thus expands the repertoire of phenomena that can be studied within 3D cell cultures.