High-throughput intensity diffraction tomography with a computational microscope.

We demonstrate a motion-free intensity diffraction tomography technique that enables the direct inversion of 3D phase and absorption from intensity-only measurements for weakly scattering samples. We derive a novel linear forward model featuring slice-wise phase and absorption transfer functions using angled illumination. This new framework facilitates flexible and efficient data acquisition, enabling arbitrary sampling of the illumination angles. The reconstruction algorithm performs 3D synthetic aperture using a robust computation and memory efficient slice-wise deconvolution to achieve resolution up to the incoherent limit. We demonstrate our technique with thick biological samples having both sparse 3D structures and dense cell clusters. We further investigate the limitation of our technique when imaging strongly scattering samples. Imaging performance and the influence of multiple scattering is evaluated using a 3D sample consisting of stacked phase and absorption resolution targets. This computational microscopy system is directly built on a standard commercial microscope with a simple LED array source add-on, and promises broad applications by leveraging the ubiquitous microscopy platforms with minimal hardware modifications.

Quantitative two-photon microscopy imaging analysis of human skin to evaluate enhanced transdermal delivery by hybrid-type multi-lamellar nanostructure.

Transdermal skin delivery is a method to transport various topical formulations to a deeper skin layer non-invasively. Permeability analysis of many delivering agents has been mostly conducted by a simple tape stripping method. However, it cannot reveal a detailed depth-dependent distribution profile of transdermally delivered agents in the skin. In this work, we achieved a cellular-level depth-defined visualization of fluorophore-labelled human epidermal growth factor (EGF) transdermally delivered to human skin by using encapsulation with common liposomes and newly fabricated multi-lamellar nanostructures using a custom-design two-photon microscopy system. It was able to generate 3D reconstructed images displaying the distribution of human EGF inside the human skin sample with high-resolution. Based on a depthwise fluorescence intensity profile showing the permeation of human EGF, a quantitative analysis was performed to assess the transdermal delivery efficacy achieved by each formulation, showing a significant improvement of the efficacy with the utilization of multi-lamellar nanostructure.

Optical volumetric projection for fast 3D imaging through circularly symmetric pupil engineering.

Monitoring and manipulating neuronal activities with optical microscopy desires a method where light can be focused or projected over a long axial range so that large brain tissues (>100 [Formula: see text] thick) can be simultaneously imaged, and specific brain regions can be optogenetically stimulated without the need for slow optical refocusing. However, the micron-scale resolution required in neuronal imaging yields a depth of field of less than 10 [Formula: see text] in conventional imaging systems. We propose to use a circularly symmetric phase mask to extend the depth of field. A numerical study shows that our method maintains both the peak and the shape of the point spread function vs the axial position better than current methods. Imaging of a 3D bead suspension and sparsely labelled thick brain tissue confirms the feasibility of the system for fast volumetric imaging.

A handheld microscope integrating photoacoustic microscopy and optical coherence tomography.

The combination of optical resolution photoacoustic microscopy (ORPAM) and optical coherence tomography (OCT) is capable of providing complementary imaging contrasts. Unfortunately, the miniaturization of ORPAM remains a major challenge in the development of a handheld dual-modality imaging microscope with OCT. Here, we report the design and evaluation of an integrated ORPAM and OCT imaging probe using a two-dimensional MEMS (micro-electro-mechanical-system)-based optical scanner. This microscope, weighting 35.4 g, has an ultracompact size of 65×30×18 mm3, and an effective imaging area of 2×2 mm2. The experimental lateral resolutions are 3.7 µm (ORPAM) and 5.6 µm (OCT), and the axial resolutions are measured as 120 µm (ORPAM) and 7.3 µm (OCT). Besides phantom and animal experiments, we carried out oral imaging of a healthy volunteer to show the clinical feasibility of this technique.

Three-dimensional tomographic microscopy technique with multi-frequency combination with partially coherent illuminations.

We demonstrate a three-dimensional (3D) optical diffraction tomographic technique with multi-frequency combination (MFC-ODT) for the 3D quantitative phase imaging of unlabeled specimens. Three sets of through-focus intensity images are captured under an annular aperture and two circular apertures with different coherence parameters. The 3D phase optical transfer functions (POTF) corresponding to different illumination apertures are combined to obtain a synthesized frequency response, achieving high-quality, low-noise 3D reconstructions with imaging resolution up to the incoherent diffraction limit. Besides, the expression of 3D POTF for arbitrary illumination pupils is derived and analyzed, and the 3D imaging performance of annular illumination is explored. It is shown that the phase-contrast washout effect in high-NA circular apertures can be effectively addressed by introducing a complementary annular aperture, which strongly boosts the phase contrast and improves the imaging resolution. By incorporating high-NA illumination as well as high-NA detection, MFC-ODT can achieve a theoretical transverse resolution up to 200 nm and an axial resolution of 645 nm. To test the feasibility of the proposed MFC-ODT technique, the 3D refractive index reconstruction results are based on a simulated 3D resolution target and experimental investigations of micro polystyrene bead and unstained biological samples are presented. Due to its capability for high-resolution 3D phase imaging as well as the compatibility with a widely available commercial microscope, the MFC-ODT is expected to find versatile applications in biological and biomedical research.

Volumetric optical coherence microscopy with a high space-bandwidth-time product enabled by hybrid adaptive optics.

Optical coherence microscopy (OCM) is a promising modality for high resolution imaging, but has limited ability to capture large-scale volumetric information about dynamic biological processes with cellular resolution. To enhance the throughput of OCM, we implemented a hybrid adaptive optics (hyAO) approach that combines computational adaptive optics with an intentionally aberrated imaging beam generated via hardware adaptive optics. Using hyAO, we demonstrate the depth-equalized illumination and collection ability of an astigmatic beam compared to a Gaussian beam for cellular-resolution imaging. With this advantage, we achieved volumetric OCM with a higher space-bandwidth-time product compared to Gaussian-beam acquisition that employed focus-scanning across depth. HyAO was also used to perform volumetric time-lapse OCM imaging of cellular dynamics over a 1mm × 1mm × 1mm field-of-view with 2 µm isotropic spatial resolution and 3-minute temporal resolution. As hyAO is compatible with both spectral-domain and swept-source beam-scanning OCM systems, significant further improvements in absolute volumetric throughput are possible by use of ultrahigh-speed swept sources.

Quantization of collagen organization in the stroma with a new order coefficient.

Many optical and biomechanical properties of the cornea, specifically the transparency of the stroma and its stiffness, can be traced to the degree of order and direction of the constituent collagen fibers. To measure the degree of order inside the cornea, a new metric, the order coefficient, was introduced to quantify the organization of the collagen fibers from images of the stroma produced with a custom-developed second harmonic generation microscope. The order coefficient method gave a quantitative assessment of the differences in stromal collagen arrangement across the cornea depths and between untreated stroma and cross-linked stroma.

Quantitative three-dimensional evaluation of immunofluorescence staining for large whole mount spheroids with light sheet microscopy.

Three-dimensional cell biology and histology of tissue sections strongly benefit from advanced light microscopy and optimized staining procedures to gather the full three-dimensional information. In particular, the combination of optical clearing with light sheet-based fluorescence microscopy simplifies fast high-quality imaging of thick biological specimens. However, verified in toto immunostaining protocols for large multicellular spheroids or for tissue sections have not been published. We present a method for the verification of immunostaining in three-dimensional spheroids. The analysis relies on three criteria to evaluate the immunostaining quality: quality of the antibody stain specificity, signal intensity achieved by the staining procedure and the correlation of the signal intensity with that of a homogeneously dispersed fluorescent dye. We optimized and investigated variations of five immunostaining protocols for three-dimensional cell biology. Our method is an important contribution to three-dimensional cell biology and the histology of tissues since it allows to evaluate the efficiency of immunostaining protocols for large three-dimensional specimens, and to study the distribution of protein expression and cell types within spheroids and spheroid-specific morphological structures without the need of physical sectioning.

Label-free photoacoustic imaging of the cardio-cerebrovascular development in the embryonic zebrafish.

Zebrafish play an important role in biology, pharmacology, toxicology, and medicine. The cardio-cerebrovascular development of zebrafish is particularly critical to understand both brain disorders and cardiovascular diseases in human. In this paper, we applied optical resolution photoacoustic microscopy (ORPAM) to image the whole-body vasculature of the embryonic zebrafish with a special focus on the development of the cardio-cerebrovascular system. Using the intrinsic optical absorption contrast of the embryo, we successfully visualized the formation of the cardio-cerebrovascular network in high-resolution using a 10 × objective, and monitored the whole-body vascular development using a 4 × objective. In addition, we evaluated the impact of the eggshell and pigment inhibitor on the image quality. Our results suggest that ORPAM is capable of studying the cardio-cerebrovascular development of zebrafish in the embryonic stage, and thus has the potential to investigate the cardiovascular and cerebrovascular diseases of human in the future.

Three-dimensional side-view endomicroscope for tracking individual cells in vivo.

We demonstrate a side-view endomicroscope using a monolithic 3-axis scanner placed in the post-objective position that performs either tilt or piston motion to achieve either optical scan angles >10° or large vertical displacements, respectively. This configuration allows for scaling down of instrument dimensions for high maneuverability and accurate positioning in vivo. Images exceeded either 700 × 600 µm2 in the horizontal plane or vertical depths of 200 µm. Resolution of 1.19 and 3.46 µm was obtained in the horizontal and oblique planes, respectively. Optical sections were collected from dysplastic colonic epithelium in vivo in mice that express tdTomato at 10 Hz to visualize individual cells.

Visible spectrum extended-focus optical coherence microscopy for label-free sub-cellular tomography.

We present a novel extended-focus optical coherence microscope (OCM) attaining 0.7 µm axial and 0.4 µm lateral resolution maintained over a depth of 40 µm, while preserving the advantages of Fourier domain OCM. Our system uses an ultra-broad spectrum from a supercontinuum laser source. As the spectrum spans from near-infrared to visible wavelengths (240 nm in bandwidth), we call the system visOCM. The combination of such a broad spectrum with a high-NA objective creates an almost isotropic 3D submicron resolution. We analyze the imaging performance of visOCM on microbead samples and demonstrate its image quality on cell cultures and ex-vivo brain tissue of both healthy and alzheimeric mice. In addition to neuronal cell bodies, fibers and plaques, visOCM imaging of brain tissue reveals fine vascular structures and sub-cellular features through its high spatial resolution. Sub-cellular structures were also observed in live cells and were further revealed through a protocol traditionally used for OCT angiography.

Looking inside the heart: a see-through view of the vascular tree.

The ability to acquire 3D images of the heart and its vasculature at cellular resolution facilitates a more detailed study of many heart diseases. Here, we describe a novel technique to image in 3D the heart vasculature by combining the CUBIC clearing protocol combined with in vivo administration of fluorescent-labeled lectin. The use of these techniques in combination with Selective Plane Illumination Microscopy (SPIM) made it possible to obtain high resolution 3D images of the cardiac vascular tree. This methodological approach may enhance the visualization of 3D images of the cardiac vasculature remodeling associated with coronary disease.

Measurement of dynamic cell-induced 3D displacement fields in vitro for traction force optical coherence microscopy.

Traction force microscopy (TFM) is a method used to study the forces exerted by cells as they sense and interact with their environment. Cell forces play a role in processes that take place over a wide range of spatiotemporal scales, and so it is desirable that TFM makes use of imaging modalities that can effectively capture the dynamics associated with these processes. To date, confocal microscopy has been the imaging modality of choice to perform TFM in 3D settings, although multiple factors limit its spatiotemporal coverage. We propose traction force optical coherence microscopy (TF-OCM) as a novel technique that may offer enhanced spatial coverage and temporal sampling compared to current methods used for volumetric TFM studies. Reconstructed volumetric OCM data sets were used to compute time-lapse extracellular matrix deformations resulting from cell forces in 3D culture. These matrix deformations revealed clear differences that can be attributed to the dynamic forces exerted by normal versus contractility-inhibited NIH-3T3 fibroblasts embedded within 3D Matrigel matrices. Our results are the first step toward the realization of 3D TF-OCM, and they highlight the potential use of OCM as a platform for advancing cell mechanics research.

Serial optical coherence scanning reveals an association between cardiac function and the heart architecture in the aging rodent heart.

Normal aging is accompanied by structural changes in the heart architecture. To explore this remodeling, we used a serial optical coherence tomography scanner to image entire mouse hearts at micron scale resolution. Ex vivo hearts of 7 young (4 months) and 5 old (24 months) C57BL/6 mice were acquired with the imaging platform. OCT of the myocardium revealed myofiber orientation changing linearly from the endocardium to the epicardium. In old mice, this rate of change was lower when compared to young mice while the average volume of old mice hearts was significantly larger (p<0.05). Myocardial wall thickening was also accompanied by extracellular spacing in the endocardium, resulting in a lower OCT attenuation coefficient in old mice endocardium (p<0.05). Prior to serial sectioning, cardiac function of the same hearts was imaged in vivo using MRI and revealed a reduced ejection fraction with aging. The use of a serial optical coherence tomography scanner allows new insight into fine age-related changes of the heart associated with changes in heart function.

Fast label-free microscopy technique for 3D dynamic quantitative imaging of living cells.

The refractive index (RI) is an important optical characteristic that is often exploited in label-free microscopy for analysis of biological objects. A technique for 3D RI reconstruction of living cells has to be fast enough to capture the cell dynamics and preferably needs to be compatible with standard wide-field microscopes. To solve this challenging problem, we present a technique that provides fast measurement and processing of data required for real-time 3D visualization of the object RI. Specifically, the 3D RI is reconstructed from the measurement of bright-field intensity images, axially scanned by a high-speed focus tunable lens mounted in front of a sCMOS camera, by using a direct deconvolution approach designed for partially coherent light microscopy in the non-paraxial regime. Both the measurement system and the partially coherent illumination, that provides optical sectioning and speckle-noise suppression, enable compatibility with wide-field microscopes resulting in a competitive and affordable alternative to the current holographic laser microscopes. Our experimental demonstrations show video-rate 3D RI visualization of living bacteria both freely swimming and optically manipulated by using freestyle laser traps allowing for their trapping and transport along 3D trajectories. These results prove that is possible to conduct simultaneous 4D label-free quantitative imaging and optical manipulation of living cells, which is promising for the study of the cell biophysics and biology.

Observation of live chromatin dynamics in cells via 3D localization microscopy using Tetrapod point spread functions.

We report the observation of chromatin dynamics in living budding yeast (Saccharomyces cerevisiae) cells, in three-dimensions (3D). Using dual color localization microscopy and employing a Tetrapod point spread function, we analyze the spatio-temporal dynamics of two fluorescently labeled DNA loci surrounding the GAL locus. From the measured trajectories, we obtain different dynamical characteristics in terms of inter-loci distance and temporal variance; when the GAL locus is activated, the 3D inter-loci distance and temporal variance increase compared to the inactive state. These changes are visible in spite of the large thermally- and biologically-driven heterogeneity in the relative motion of the two loci. Our observations are consistent with current euchromatin vs. heterochromatin models.

Statistical parametric mapping of stimuli evoked changes in total blood flow velocity in the mouse cortex obtained with extended-focus optical coherence microscopy.

Functional magnetic resonance (fMRI) imaging is the current gold-standard in neuroimaging. fMRI exploits local changes in blood oxygenation to map neuronal activity over the entire brain. However, its spatial resolution is currently limited to a few hundreds of microns. Here we use extended-focus optical coherence microscopy (xfOCM) to quantitatively measure changes in blood flow velocity during functional hyperaemia at high spatio-temporal resolution in the somatosensory cortex of mice. As optical coherence microscopy acquires hundreds of depth slices simultaneously, blood flow velocity measurements can be performed over several vessels in parallel. We present the proof-of-principle of an optimised statistical parametric mapping framework to analyse quantitative blood flow timetraces acquired with xfOCM using the general linear model. We demonstrate the feasibility of generating maps of cortical hemodynamic reactivity at the capillary level with optical coherence microscopy. To validate our method, we exploited 3 stimulation paradigms, covering different temporal dynamics and stimulated limbs, and demonstrated its repeatability over 2 trials, separated by a week.

Diattenuation of brain tissue and its impact on 3D polarized light imaging.

3D-polarized light imaging (3D-PLI) reconstructs nerve fibers in histological brain sections by measuring their birefringence. This study investigates another effect caused by the optical anisotropy of brain tissue - diattenuation. Based on numerical and experimental studies and a complete analytical description of the optical system, the diattenuation was determined to be below 4 % in rat brain tissue. It was demonstrated that the diattenuation effect has negligible impact on the fiber orientations derived by 3D-PLI. The diattenuation signal, however, was found to highlight different anatomical structures that cannot be distinguished with current imaging techniques, which makes Diattenuation Imaging a promising extension to 3D-PLI.

Rapid adaptive remote focusing microscope for sensing of volumetric neural activity.

The ability to record neural activity in the brain of a living organism at cellular resolution is of great importance for defining the neural circuit mechanisms that direct behavior. Here we present an adaptive two-photon microscope optimized for extraction of neural signals over volumes in intact Drosophila brains, even in the presence of specimen motion. High speed volume imaging was made possible through reduction of spatial resolution while maintaining the light collection efficiency of a high resolution, high numerical aperture microscope. This enabled simultaneous recording of odor-evoked calcium transients in a defined volume of mushroom body Kenyon cell bodies in a live fruit fly.

Fast 3D visualization of endogenous brain signals with high-sensitivity laser scanning photothermal microscopy.

A fast, high-sensitivity photothermal microscope was developed by implementing a spatially segmented balanced detection scheme into a laser scanning microscope. We confirmed a 4.9 times improvement in signal-to-noise ratio in the spatially segmented balanced detection compared with that of conventional detection. The system demonstrated simultaneous bi-modal photothermal and confocal fluorescence imaging of transgenic mouse brain tissue with a pixel dwell time of 20 µs. The fluorescence image visualized neurons expressing yellow fluorescence proteins, while the photothermal signal detected endogenous chromophores in the mouse brain, allowing 3D visualization of the distribution of various features such as blood cells and fine structures probably due to lipids. This imaging modality was constructed using compact and cost-effective laser diodes, and will thus be widely useful in the life and medical sciences.