Search

Brillouin microscopy can assess mechanical properties of biological samples in a three-dimensional (3D), all-optical and hence non-contact fashion, but its weak signals often lead to long imaging times and require an illumination dosage harmful for living organisms. Here, we present a high-resolution line-scanning Brillouin microscope for multiplexed and hence fast 3D imaging of dynamic biological processes with low phototoxicity. The improved background suppression and resolution, in combination with fluorescence light-sheet imaging, enables the visualization of the mechanical properties of cells and tissues over space and time in living organism models such as fruit flies, ascidians and mouse embryos.

Fluorescence microscopy is a ubiquitous technique in the life sciences that uses fluorescent molecules to visualize specific components of biological specimens. This powerful tool has revolutionized biology, and it represents a perfect example of the advancements enabled by biophysical research and technology development. However, despite its central role in contemporary research, fluorescence is hardly covered in typical secondary school curricula, with few hands-on “entry-level” materials available for secondary school teachers to introduce this important method to their students. Furthermore, most commercially available fluorescence microscopes are prohibitively costly and often appear as “black boxes.” To address this gap, we introduce here an experimental, research-grade fluorescence microscopy kit and educational resource targeted at secondary school students and teachers. Microscope in Action is an interdisciplinary resource based on active learning that combines concepts from both optics and biology. The students assemble a functional microscope from basic optical, mechanical, and electronic parts, thereby testing and understanding the function of each component “hands-on.” We also present sample preparation and imaging activities that can be incorporated to enable an exploration of biological topics with the assembled microscope and exercises in which students actively learn and practice scientific thinking by collecting and analyzing data. Although the resource was developed with secondary schools in mind, the variety of available protocols and the adjustable module lengths make it suitable for different age groups and topics, from middle school to PhD level, from short workshops to courses spanning several days.

The cnidarian

Brillouin microscopy is an emerging optical elastography technique capable of assessing mechanical properties of biological samples in a 3D, all-optical and hence non-contact fashion. The typically weak Brillouin scattering signal can be substantially enhanced via a stimulated photon-phonon process, which improves the signal-to-background ratio (SBR) as well as provides higher spectral resolution. However, current implementations of stimulated Brillouin spectroscopy (SBS) require high pump powers, which prohibit applications in many areas of biology, especially when studying photosensitive samples, or when live-imaging in 3D and/or over extended time periods. Here, we present a pulsed SBS scheme that takes full advantage of the non-linearity of the pump-probe interaction in SBS. In particular, we show that through quasi-pulsing and diligent optimization of signal detection parameters, the required pump laser power can be decreased ~20-fold without affecting the signal levels or spectral precision. Moreover, we devise a custom analysis approach that facilitates the analysis of complex, multi-peaked Brillouin spectra in order to harness the high spectral resolution of SBS for the specific identification of biomechanical components inside the point-spread function of the microscope. We then demonstrate the low-phototoxicity and high-specificity of our pulsed SBS approach by imaging sensitive single cells, zebrafish larvae, and mouse embryos as well as adultC. eleganswith sub-cellular detail. Furthermore, our method permits observing the mechanics of organoids andC. elegansembryos over time. We expect that the substantially lower photo-burden and improved SBR of pulsed SBS will facilitate studying biomechanics in 3D at high spatio-temporal resolution in living biological specimens in a non-invasive manner, opening up exciting new possibilities for the field of mechanobiology.

Development is a highly dynamic process in which organisms often experience changes in both form and behavior, which are typically coupled to each other. However, little is known about how organismal-scale behaviors such as body contractility and motility impact morphogenesis. Here, we use the cnidarian Nematostella vectensis as a developmental model to uncover a mechanistic link between organismal size, shape, and behavior. Using quantitative live imaging in a large population of developing animals, combined with molecular and biophysical experiments, we demonstrate that the muscular-hydraulic machinery that controls body movement also drives larva-polyp morphogenesis. We show that organismal size largely depends on cavity inflation through fluid uptake, whereas body shape is constrained by the organization of the muscular system. The generation of ethograms identifies different trajectories of size and shape development in sessile and motile animals, which display distinct patterns of body contractions. With a simple theoretical model, we conceptualize how pressures generated by muscular hydraulics can act as a global mechanical regulator that coordinates tissue remodeling. Altogether, our findings illustrate how organismal contractility and motility behaviors can influence morphogenesis. ispartof: CURRENT BIOLOGY vol:32 issue:21 pages:4707-+ ispartof: location:England status: published

The mammalian inferior colliculus (IC) is responsible for encoding sound location. However, the underlying neuronal population code at single-trial basis remains unclear due to the absence of large-scale simultaneous recordings. To address this knowledge gap, here we performed volumetric two-photon calcium imaging in awake mice and simultaneously recorded the activity of dorsal IC populations (∼1,000 cells in size) in response to sounds delivered from 13 different frontal horizontal locations (azimuths). The recorded population responses revealed substantial trial-to-trial variability and noise correlations. Nevertheless, noise correlations helped decode stimulus azimuth effectively from these responses, reaching an accuracy matching that of the animal’s discrimination ability. These findings demonstrate how correlated neuronal noise can improve the accuracy of a noisy population code, and support that single-trial population responses in the dorsal mammalian IC robustly encode sound location despite the inherent variation in responses from trial to trial.

Visualizing dynamic processes over large, three-dimensional fields of view at high speed is essential for many applications in the life sciences. Light-field microscopy (LFM) has emerged as a tool for fast volumetric image acquisition, but its effective throughput and widespread use in biology has been hampered by a computationally demanding and artifact-prone image reconstruction process. Here, we present a framework for artificial intelligence–enhanced microscopy, integrating a hybrid light-field light-sheet microscope and deep learning–based volume reconstruction. In our approach, concomitantly acquired, high-resolution two-dimensional light-sheet images continuously serve as training data and validation for the convolutional neural network reconstructing the raw LFM data during extended volumetric time-lapse imaging experiments. Our network delivers high-quality three-dimensional reconstructions at video-rate throughput, which can be further refined based on the high-resolution light-sheet images. We demonstrate the capabilities of our approach by imaging medaka heart dynamics and zebrafish neural activity with volumetric imaging rates up to 100 Hz. A deep learning–based algorithm enables efficient reconstruction of light-field microscopy data at video rate. In addition, concurrently acquired light-sheet microscopy data provide ground truth data for training, validation and refinement of the algorithm.

Aggregation-induced emission nanoparticles serve as promising fluorescence probes for multi-photon excitation microscopy due to the large absorption cross-section in the NIR-IIb region. Here we present organic AIE nanoparticles that feature a high absorption cross-section under three-photon excitation. We show that these enable ultra-deep NIR-IIa excited three-photon imaging in the in vivo mouse brain.

Several important questions in biology require non-invasive and three-dimensional imaging techniques with an appropriate spatiotemporal resolution that permits live organisms to move in an unconstrained fashion over an extended field-of-view. While selective-plane illumination microscopy (SPIM) has emerged as a powerful method to observe live biological specimens at high spatio-temporal resolution, typical implementations often necessitate constraining sample mounting or lack the required volumetric speed. Here, we report on an open-top, dual-objective oblique plane microscope (OPM) capable of observing millimeter-sized, freely moving animals at cellular resolution. We demonstrate the capabilities of our mesoscopic OPM (MesOPM) by imaging the behavioral dynamics of the sea anemone Nematostella vectensis over 1.56 × 1.56 × 0.25 mm at 1.5 × 2.8 × 5.3 µm resolution and 0.5 Hz volume rate.

Brillouin microscopy (BM) can be used to assess the mechanical properties of biological samples in a 3D, all-optical, and hence non-contact fashion, but its weak signals require long imaging times and illumination dosages harmful to living organisms. Here, we present a line-scanning Brillouin microscope optimized for fast and high-resolution live-imaging of dynamic biological processes with low photo-toxicity. In combination with fluorescence light-sheet imaging, we demonstrate the capabilities of our microscope to visualize the mechanical properties of cells and tissues over space and time in living model organisms such as fruit flies, ascidians, and mouse embryos.

Highly sensitive detection is crucial for all-optical photoacoustic (PA) imaging. However, free-space optical detectors are prone to optical aberrations, which can degrade the pressure sensitivity and result in deteriorated image quality. While spatial mode-filtering has been proposed to alleviate these problems in Fabry-Pérot-based pressure sensors, their real functional advantage has never been properly investigated.We rigorously and quantitatively compare the performance of free-space and fiber-coupled detectors for Fabry-Pérot-based pressure sensors.We develop and characterize a quantitative correlative setup capable of simultaneous PA imaging using a free space and a fiber-coupled detector.We found that fiber-coupled detectors are superior in terms of both signal level and image quality in realistic all-optical PA tomography settings.Our study has important practical implications in the field of PA imaging, as for most applications and implementations fiber-coupled detectors are relatively easy to employ since they do not require modifications to the core of the system but only to the peripherally located detector.

Optical resonators are some of the most promising optical devices for manufacturing high-performance pressure sensors for photoacoustic imaging. Among these, Fabry–Perot (FP)-based pressure sensors have been successfully used for a multitude of applications. However, critical performance aspects of FP-based pressure sensors have not been studied extensively, including the effects that system parameters such as beam diameter and cavity misalignment have on transfer function shape. Here, we discuss the possible origins of the transfer function asymmetry, ways to correctly estimate the FP pressure sensitivity under practical experimental conditions, as well as show the importance of proper assessments for real-world applications.

SummaryDuring development, organisms interact with their natural habitats while undergoing morphological changes, yet it remains unclear whether the interplay between developing systems and their environments impacts animal morphogenesis. Here, we use the cnidarian Nematostella vectensis as a developmental model to uncover a mechanistic link between organism size, shape and behavior. Using quantitative live imaging, including extensive behavioral profiling, combined with molecular and biophysical experiments, we demonstrate that the muscular hydraulic machinery that controls body movement directly drives larva-polyp morphogenesis. Unexpectedly, size and shape development are differentially controlled by antagonistic muscles. A simple theoretical model shows how a combination of slow-priming and fast-pumping pressures generated by muscular hydraulics acts as a global mechanical regulator that coordinates tissue remodeling. Altogether, our findings illuminate how dynamic behavioral modes in the environment can be harnessed to drive morphogenetic trajectories, establishing ethology as a critical component of organismal morphogenesis – termed ethology of morphogenesis.

Zernike polynomials are one of the most widely used mathematical descriptors of optical aberrations in the fields of imaging and adaptive optics. Their mathematical orthogonality as well as isomorphisms with experimentally observable aberrations make them a very powerful tool in solving numerous problems in beam optics, most notably the recent developments of adaptive optics for correcting beam aberrations. However, Zernike aberrations show cross coupling between individual modes when used in combination with Gaussian beams, which are ubiquitous in most practical applications, an effect that has not been extensively studied. Here we propose a novel framework that is capable of explaining the fundamental cross-compensation of Zernike type aberrations, in both low-aberration and high-aberration regimes. Our approach is based on analyzing the coupling between Zernike modes and different classes of Laguerre–Gauss modes, which allows investigating aberrated beams not only on a single transverse plane but also during their 3D propagation.

All-optical ultrasound detection bears a number of unique advantages for photoacoustic tomography, including the ability for high resolution sampling of the acoustic field and its compatibility with a wide variety of other optical modalities. However, optical schemes based on miniaturized cavities are sensitive to optical aberrations as well as manufacturing-induced cavity imperfections which degrade sensor sensitivity and deteriorate photoacoustic image quality. Here we present an experimental method based on adaptive optics that is capable of enhancing the overall sensitivity of Fabry-Perot based photoacoustic sensors. We experimentally observe clear improvements in photoacoustic signal detection as well as overall image quality after photoacoustic tomography reconstructions when applied to mammalian tissues in vivo.

To capture highly dynamic biological processes at cellular resolution is a recurring challenge in biology. Here we show that combining selective-volume illumination with simultaneous acquisition of orthogonal light fields yields three-dimensional images with high, isotropic spatial resolution and a significant reduction of reconstruction artefacts, thereby overcoming current limitations of light-field microscopy implementations. We demonstrate medaka heart and blood flow imaging at single-cell resolution and free of motion artefacts at volume rates of up to 200 Hz. Iso-LFM enables rapid, instantaneous volumetric imaging of biological processes with isotropic and improved resolution by simultaneously capturing orthogonal light fields.

We present our work in modelling the effects of optical aberrations on Fabry-Perot pressure sensor performance, as well as experimental strategies to tackle said aberrations using adaptive optics and laser mode filtering.

Light field microscopy is a powerful tool for fast volumetric image acquisition in biology which requires a computationally demanding and artefact-prone image reconstruction process. I will present a novel framework consisting of a hybrid light-field light-sheet microscope and deep learning-based volume reconstruction, where single light-sheet acquisitions continuously serve as training data and validation for the convolutional neural network reconstructing the LFM volume. Our framework produces video-rate reconstructions; their fidelity can be verified on demand and the network can be fine-tuned as necessary.

In order to study dynamic biological processes in-vivo in mammalian organisms techniques are required which enable non-invasive imaging at large tissue depth with sub-cellular resolution. However, optical aberrations and scattering in biological tissue lead to signal loss and a degradation of both spatial resolution and penetration depth. Here, we combine two powerful optical techniques, multi-photon microscopy and adaptive optics, to push the depth limit further while retaining diffraction limited resolution. We apply these techniques to open questions in the field of neuron and glia biology. By utilizing three-photon excitation at the 1300 nm spectral excitation window we achieve highresolution imaging of GFP-labeled neurons up to a depth of 1.2 mm in the in-vivo mouse brain. Furthermore, we have combined our approach with indirect modal-based wavefront correction and synchronization of our microscope to the animal’s heart pulsation. This allowed us to improve the resolution up to ~6-fold and achieve synaptic resolution throughout an entire cortical column. With adaptive optics correction, small structures such a dendritic branches thus become clearly visible at over 1mm depth in the hippocampus. Furthermore, our adaptive three-photon microscope system enabled, for the first time, to investigate calcium dynamics of fibrous astrocytes which reside in the corpus callosum and whose dynamics have previously not been possible to image.

Brillouin microscopy is a fully-optical technique that relies on the interaction between spontaneous, thermal generated soundwaves (phonons) and monochromatic laser light. Mechanical properties of biological samples can be inferred in 3D from the spectrum of the scattered light. A state-of-the-art Brillouin microscope consists of a confocal microscope coupled with a spectrometer that features an acquisition time ~

AIE particles for multi-photon imaging

We explore the link between on-going neuronal activity at primary motor cortex (M1) and face movement in awake mice. By combining custom-made behavioral sequencing analysis and fast volumetric Ca2+-imaging, we simultaneously tracked M1 population activity during many different facial motor sequences. We show that a facial area of M1 displays distinct trajectories of neuronal population dynamics across different spontaneous facial motor sequences, suggesting an underlying population dynamics code.Significance statementHow our brain controls a seemingly limitless diversity of body movements remains largely unknown. Recent research brings new light into this subject by showing that neuronal populations at the primary motor cortex display different dynamics during forelimb reaching movements versus grasping, which suggests that different motor sequences could be associated with distinct motor cortex population dynamics. To explore this possibility, we designed an experimental paradigm for simultaneously tracking the activity of neuronal populations in motor cortex across many different motor sequences. Our results support the concept that distinct population dynamics encode different motor sequences, bringing new insight into the role of motor cortex in sculpting behavior while opening new avenues for future research.

All-optical ultrasound detection bears a number of unique advantages for photoacoustic tomography, including the ability for high resolution sampling of the acoustic field and its compatibility with a wide variety of other optical modalities. However, optical schemes based on miniaturized cavities are sensitive to optical aberrations as well as manufacturing-induced cavity imperfections which degrade sensor sensitivity and deteriorate photoacoustic image quality. Here we present an experimental method based on adaptive optics that is capable of enhancing the overall sensitivity of Fabry-Pérot based photoacoustic sensors. We experimentally observe clear improvements in photoacoustic signal detection as well as overall image quality after photoacoustic tomography reconstructions when applied to mammalian tissues in vivo.

Multiphoton microscopy has become a powerful tool with which to visualize the morphology and function of neural cells and circuits in the intact mammalian brain. However, tissue scattering, optical aberrations and motion artifacts degrade the imaging performance at depth. Here we describe a minimally invasive intravital imaging methodology based on three-photon excitation, indirect adaptive optics (AO) and active electrocardiogram gating to advance deep-tissue imaging. Our modal-based, sensorless AO approach is robust to low signal-to-noise ratios as commonly encountered in deep scattering tissues such as the mouse brain, and permits AO correction over large axial fields of view. We demonstrate near-diffraction-limited imaging of deep cortical spines and (sub)cortical dendrites up to a depth of 1.4 mm (the edge of the mouse CA1 hippocampus). In addition, we show applications to deep-layer calcium imaging of astrocytes, including fibrous astrocytes that reside in the highly scattering corpus callosum., Three-photon microscopy in combination with adaptive optics-based aberration correction and ECG-triggered gating allows high-resolution imaging of neurons and astrocytes up to a depth of 1.4 mm in the mouse brain.

Zernike polynomials are widely used mathematical models of experimentally observed optical aberrations. Their useful mathematical properties, in particular their orthogonality, make them a ubiquitous basis set for solving various problems in beam optics. Thus they have found widespread use in adaptive optics realizations that are used to correct wavefront aberrations. However, Zernike aberrations lose their orthogonality when used in combination with Gaussian beams, which are omnipresent in real-world optical applications. As a consequence, Zernike aberrations in Gaussian beams start to cross-couple between each other, a phenomenon that does not occur for Zernike aberrations in plane waves. Here, we describe how the aberration radius influences this cross-coupling of Zernike aberrations. Furthermore, we propose that this effect can actually be harnessed to allow efficient compensation of higher-order aberrations using only low-order Zernike modes. This finding has important practical implications, as it suggests the possibility of using adaptive optics devices with low element numbers to compensate aberrations which would normally require more complex and expensive devices., Comment: 12 pages, 3 figures, including Supplementary Information

Aggregation-induced emission nanoparticles serve as promising fluorescence probes for multi-photon excitation microscopy due to the large absorption cross-section at NIR-IIb region. Here we present organic AIE nanoparticles that feature high aborption cross-section under three-photon excitation. We show that these enable ultra-deep NIR-IIa excited three-photon imaging in the in-vivo mouse brain.

Preclinical breast tumor models are an invaluable tool to systematically study tumor progression and treatment response, yet methods to non-invasively monitor the involved molecular and mechanistic properties under physiologically relevant conditions are limited. Here we present an intravital mesoscopic fluorescence molecular tomography (henceforth IFT) approach that is capable of tracking fluorescently labeled tumor cells in a quantitative manner inside the mammary gland of living mice. Our mesoscopic approach is entirely non-invasive and thus permits prolonged observational periods of several months. The relatively high sensitivity and spatial resolution further enable inferring the overall number of oncogene-expressing tumor cells as well as their tumor volume over the entire cycle from early tumor growth to residual disease following the treatment phase. We find that sheer tumor volume, as commonly assessed by other imaging modalities, is not well correlated to tumor cell quantity, hence our IFT approach is a promising new method for studying tumor growth dynamics in a quantitative and longitudinal fashion in-vivo.

Visualizing dynamic processes over large, three-dimensional fields of view at high speed is essential for many applications in the life sciences. Light-field microscopy (LFM) has emerged as a tool for fast volumetric image acquisition, but its effective throughput and widespread use in biology has been hampered by a computationally demanding and artifact-prone image reconstruction process. Here, we present a framework for artificial intelligence-enhanced microscopy, integrating a hybrid light-field light-sheet microscope and deep learning-based volume reconstruction. In our approach, concomitantly acquired, high-resolution two-dimensional light-sheet images continuously serve as training data and validation for the convolutional neural network reconstructing the raw LFM data during extended volumetric time-lapse imaging experiments. Our network delivers high-quality three-dimensional reconstructions at video-rate throughput, which can be further refined based on the high-resolution light-sheet images. We demonstrate the capabilities of our approach by imaging medaka heart dynamics and zebrafish neural activity with volumetric imaging rates up to 100 Hz.

Many important biological functions and processes are reflected in cell and tissue mechanical properties such as elasticity and viscosity. However, current techniques used for measuring these properties have major limitations, such as that they can often not measure inside intact cells and/or require physical contact—which cells can react to and change. Brillouin light scattering offers the ability to measure mechanical properties in a non-contact and label-free manner inside of objects with high spatial resolution using light, and hence has emerged as an attractive method during the past decade. This new approach, coined “Brillouin microscopy,” which integrates highly interdisciplinary concepts from physics, engineering, and mechanobiology, has led to a vibrant new community that has organized itself via a European funded (COST Action) network. Here we share our current assessment and opinion of the field, as emerged from a recent dedicated workshop. In particular, we discuss the prospects towards improved and more bio-compatible instrumentation, novel strategies to infer more accurate and quantitative mechanical measurements, as well as our current view on the biomechanical interpretation of the Brillouin spectra.

Preclinical breast tumor models are an invaluable tool to systematically study tumor progression and treatment response, yet methods to non-invasively monitor the involved molecular and mechanistic properties under physiologically relevant conditions are limited. Here we present an intravital mesoscopic fluorescence molecular tomography (henceforth IFT) approach that is capable of tracking fluorescently labeled tumor cells in a quantitative manner inside the mammary gland of living mice. Our mesoscopic approach is entirely non-invasive and thus permits prolonged observational periods of several months. The relatively high sensitivity and spatial resolution further enable inferring the overall number of oncogene-expressing tumor cells as well as their tumor volume over the entire cycle from early tumor growth to residual disease following the treatment phase. Our IFT approach is a promising method for studying tumor growth dynamics in a quantitative and longitudinal fashion in-vivo., Mehmet S. Ozturk et al. present an intravital mesoscopic fluorescence tomography (IFT) technique that is capable of tracking fluorescently labeled tumor cells inside living mice over several months. It demonstrates a promising new method for studying tumor growth dynamics in a quantitative and longitudinal fashion in-vivo.

Light-field microscopy has emerged as a particularly elegant and powerful technique for fast and instantaneous three-dimensional imaging in biology, as it captures a 3D image with a single camera snapshot. In this talk I will present recent work in which we tackled the main challenges of establishing this technique for applications in the life sciences. In particular I will show how innovative approaches have allowed us to increase spatial resolution and contrast while reducing the presence of artefacts, all of which were imperative for imaging fast biological processes such as neuronal and cardiovascular dynamics.

Summary The vasculature is innervated by a network of peripheral afferents that sense and regulate blood flow. Here, we describe a system of non-peptidergic sensory neurons with cell bodies in the spinal ganglia that regulate vascular tone in the distal arteries. We identify a population of mechanosensitive neurons, marked by tropomyosin receptor kinase C (TrkC) and tyrosine hydroxylase in the dorsal root ganglia, which projects to blood vessels. Local stimulation of TrkC neurons decreases vessel diameter and blood flow, whereas systemic activation increases systolic blood pressure and heart rate variability via the sympathetic nervous system. Ablation of the neurons provokes variability in local blood flow, leading to a reduction in systolic blood pressure, increased heart rate variability, and ultimately lethality within 48 h. Thus, a population of TrkC+ sensory neurons forms part of a sensory-feedback mechanism that maintains cardiovascular homeostasis through the autonomic nervous system., Graphical abstract, Highlights • TrkC+/Th+ DRG neurons project to blood vessels • Local stimulation of TrkC+ DRG neurons decreases vessel diameter and blood flow • Systemic activation of TrkC+ DRG neurons increases blood pressure and heart rate • Ablation of TrkC+ neurons dysregulates cardiovascular homeostasis and is lethal, Morelli et al. identify a subpopulation of peripheral sensory neurons marked by TrkC and Th that projects to distal blood vessels. They demonstrate that these neurons regulate peripheral perfusion, blood pressure, and heart rate.

SummaryThe vasculature is innervated by a network of peripheral afferents that sense and regulate blood flow. Here, we describe a system of non-peptidergic sensory neurons with cell bodies in the spinal ganglia that regulate vascular tone in the distal arteries. We identify a population of mechanosensitive neurons marked by TrkC and Tyrosine hydroxylase in the dorsal root ganglia that project to blood vessels. Local stimulation of these neurons decreases vessel diameter and blood flow, while systemic activation increases systolic blood pressure and heart rate variability via the sympathetic nervous system. Chemogenetic inactivation or ablation of the neurons provokes variability in local blood flow leading to a reduction in systolic blood pressure, increased heart rate variability and ultimately lethality within 48 hours. Thus, TrkC/Tyrosine hydroxylase positive sensory neurons form part of a sensory feedback mechanism that maintains cardiovascular homeostasis through the autonomic nervous system.

Scientific reports 10(1), 3142 (2020). doi:10.1038/s41598-020-59791-w, Published by Macmillan Publishers Limited, part of Springer Nature, [London]

Cancer prognosis and treatment efficacy are assessed by evaluating the hallmarks of a tumor which are volume change of the tumor and the vascular network reformation around a tumor. Non-invasive quantitative assessment of those indications, in-vivo, is still a challenge for traditional imaging modalities, owing to a large interrogation area and deep-seated molecular (fluorescence) signal in highly scattering, anatomically complex skin tissue. Currently available techniques either utilize surgically implanted imaging windows or conduct terminal experiments for each time point. The former is prone to inflammation at the implantation site thus interfering with the tumor microenvironment. The latter one is prone to sample variability thus results in a pseudo-longitudinal outcome for tumor development. Here, we combine Intravital Fluorescence Tomography (IFT) and Spectral Domain Optical Coherence Tomography (SD-OCT) for tumor imaging with a Non-invasive Intravital Imaging Window (NIIW) for tissue stabilization. This platform enabled us to follow tumor development non-invasively covering tumor initiation, development and regression on the same animal over months-long period. IFT-OCT multimodal imaging not only reveals tumor volume change but also skin anatomical features, and it is capable of revealing neo-vascularization around the tumor site. This platform thus serves as a useful noninvasive tool to explore future research questions pertaining to cancer biology in common fluorescence-based mouse models, such as tumor progression or treatment efficacy. In addition, our multi-modal platform alleviates the burden put on animals while imaging and reduce the experimental cost significantly.

The role and importance of mechanical properties of cells and tissues in cellular function, development and disease has widely been acknowledged, however standard techniques currently used to assess them exhibit intrinsic limitations. Recently, Brillouin microscopy, a type of optical elastography, has emerged as a non-destructive, label- and contact-free method that can probe the viscoelastic properties of biological samples with diffraction-limited resolution in 3D. This led to increased attention amongst the biological and medical research communities, but it also sparked debates about the interpretation and relevance of the measured physical quantities. Here, we review this emerging technology by describing the underlying biophysical principles and discussing the interpretation of Brillouin spectra arising from heterogeneous biological matter. We further elaborate on the technique’s limitations, as well as its potential for gaining insights in biology, in order to guide interested researchers from various fields. This Review discusses the principles, advantages and limitations of Brillouin microscopy, a non-invasive tool for measuring mechanical properties of biological samples in three dimensions.