The microscopic environment inside a metazoan organism is highly crowded. Whether individual cells can tailor their behavior to the limited space remains unclear. In this study, we found that cells measure the degree of spatial confinement by using their largest and stiffest organelle, the nucleus. Cell confinement below a resting nucleus size deforms the nucleus, which expands and stretches its envelope. This activates signaling to the actomyosin cortex via nuclear envelope stretch-sensitive proteins, up-regulating cell contractility. We established that the tailored contractile response constitutes a nuclear ruler–based signaling pathway involved in migratory cell behaviors. Cells rely on the nuclear ruler to modulate the motive force that enables their passage through restrictive pores in complex three-dimensional environments, a process relevant to cancer cell invasion, immune responses, and embryonic development.

During metazoan development, immune surveillance and cancer dissemination, cells migrate in complex three-dimensional microenvironments 1-3. These spaces are crowded by cells and extracellular matrix, generating mazes with differently sized gaps that are typically smaller than the diameter of the migrating cell 4,5. Most mesenchymal and epithelial cells and some, but not all, cancer cells actively generate their migratory path using pericellular tissue proteolysis 6. By contrast, amoeboid cells such as leukocytes use non-destructive strategies of locomotion 7 , raising Reprints and permissions information is available at http://www.nature.com/reprints.

Previous implementations of structured-illumination microscopy (SIM) were slow or designed for one-color excitation, sacrificing two unique and extremely beneficial aspects of light microscopy: live-cell imaging in multiple colors. This is especially unfortunate because, among the resolution-extending techniques, SIM is an attractive choice for live-cell imaging; it requires no special fluorophores or high light intensities to achieve twice diffraction-limited resolution in three dimensions. Furthermore, its wide-field nature makes it lightefficient and decouples the acquisition speed from the size of the lateral field of view, meaning that high frame rates over large volumes are possible. Here, we report a previously undescribed SIM setup that is fast enough to record 3D two-color datasets of living whole cells. Using rapidly programmable liquid crystal devices and a flexible 2D grid pattern algorithm to switch between excitation wavelengths quickly, we show volume rates as high as 4 s in one color and 8.5 s in two colors over tens of time points. To demonstrate the capabilities of our microscope, we image a variety of biological structures, including mitochondria, clathrin-coated vesicles, and the actin cytoskeleton, in either HeLa cells or cultured neurons.extended resolution | frequency mixing | multicolor | patterned excitation F luorescence microscopy allows noninvasive 3D imaging of the interior of living specimens with molecular specificity, and is therefore an invaluable resource to the biological sciences. Unfortunately, the resolving power of fluorescence microscopy is fundamentally limited by the diffraction of light.Lately, many techniques have been introduced to extend the resolution beyond the classic diffraction limit (1-8). Although the improvement of spatial resolution is impressive, the practical impact of these new techniques will largely depend on whether they can keep the two key advantages of fluorescence microscopy, namely, the capability of imaging living cells in three dimensions and multicolor labeling. Live-cell imaging over many time points has been demonstrated for some resolution-extending techniques (9-15) but, to our knowledge, not for multicolor 3D imaging of whole cells.Stimulated emission depletion (STED) microscopy increases the spatial resolution by suppressing the fluorescence emission on the rim of a focused laser spot, theoretically enabling unlimited resolution (16). STED microscopy has been demonstrated for live imaging at high frame rates (12, 15), but its point-scanning nature makes it unsuitable for fast volumetric imaging over large fields of view. Furthermore, to achieve high spatial resolution, very high power densities (38-540 MW/cm 2 ) (12) are necessary for the STED laser beam, which may cause increased photobleaching and phototoxicity, thus limiting the application of live-cell STED microscopy.In localization-based microscopy, only sparse subsets of photoswitchable fluorophores in a sample are imaged at a time, which allows the isolation and precise localizati...

Orange-red fluorescent proteins (FPs) are widely used in biomedical research for multiplexed epifluorescence microscopy with GFP-based probes, but their different excitation requirements make multiplexing with new advanced microscopy methods difficult. Separately, orange-red FPs are useful for deep-tissue imaging in mammals due to the relative tissue transmissibility of orange-red light, but their dependence on illumination limits their sensitivity as reporters in deep tissues. Here we describe CyOFP1, a bright engineered orange-red FP that is excitable by cyan light. We show that CyOFP1 enables single-excitation multiplexed imaging with GFP-based probes in single-photon and two-photon microscopy, including time-lapse imaging in light-sheet systems. CyOFP1 also serves as an efficient acceptor for resonance energy transfer from the highly catalytic blue-emitting luciferase NanoLuc. An optimized fusion of CyOFP1 and NanoLuc, called Antares, functions as a highly sensitive bioluminescent reporter in vivo, producing substantially brighter signals from deep tissues than firefly luciferase and other bioluminescent proteins.

Fluorescence imaging has revolutionized biomedical research over the past three decades. Its high molecular specificity and unrivaled single molecule level sensitivity have enabled breakthroughs in a variety of research fields. For in vivo applications, its major limitation is the superficial imaging depth as random scattering in biological tissues causes exponential attenuation of the ballistic component of a light wave. Here we present fluorescence imaging beyond the ballistic regime by combining single cycle pulsed ultrasound modulation and digital optical phase conjugation. We demonstrate a near isotropic 3D localized sound-light interaction zone. With the exceptionally high optical gain provided by the digital optical phase conjugation system, we can deliver sufficient optical power to a focus inside highly scattering media for not only fluorescence imaging but also a variety of linear and nonlinear spectroscopy measurements. This technology paves the way for many important applications in both fundamental biology research and clinical studies.

The use of propagation invariant Bessel beams has enabled high-resolution subcellular light sheet fluorescence microscopy. However, the energy within the concentric side lobe structure of Bessel beams increases significantly with propagation length, generating unwanted out-of-focus fluorescence that enforces practical limits on the imaging field of view size. Here, we present a light sheet fluorescence microscope that achieves 390 nm isotropic resolution and high optical sectioning strength (i.e., out-of-focus blur is strongly suppressed) over large field of views, without the need for structured illumination or deconvolution-based postprocessing. We demonstrate simultaneous dual-color, high-contrast, and high-dynamic-range time-lapse imaging of migrating cells in complex three-dimensional microenvironments, three-dimensional tracking of clathrin-coated pits, and long-term imaging spanning >10 h and encompassing >2600 time points.

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.

We present cleared tissue Axially Swept Light-Sheet Microscopy (ctASLM), which enables isotropic, subcellular resolution, high optical sectioning capability, and large field of view imaging over a broad range of immersion media. ctASLM can image live, expanded, and both aqueous and organic chemically cleared tissue preparations. Depending on the optical configuration, ctASLM provides up to 260 nm axial resolution, an improvement over confocal and other reported cleared tissue light-sheet microscopes by a factor 3-10. We image millimeter-scale tissues with subcellular 3D resolution, which enabled us to automatically detect with computer vision multicellular tissue architectures, individual cells, synaptic spines, and rare cell-cell interactions.Human tissues are composed of multiple polarized cell types organized in well-defined three-dimensional architectures. Interestingly, it has been shown that rare subsets of cells play a crucial role in disease progression, 1 and interdisciplinary efforts now aim to generate comprehensive atlases of human cells in diverse tissue types. To date, this has largely relied on massively parallel sequencing and machine learning-based analyses to identify unique sub-populations of cells. Combined with advanced imaging, such efforts could not only shed light on the diversity of cell types, but the biological context in which each population operates. However, imaging large tissues with subcellular resolution remains challenging due to the heterogeneous refractive index and composition of tissues, which results in complex aberrations and an increased scattering coefficient, both of which decrease spatial resolution and limit imaging depth. 2