Activation of ILC2s through constitutive IFNγ signaling reduction leads to spontaneous pulmonary fibrosis.

Pulmonary fibrosis (PF), a condition characterized by inflammation and collagen deposition in the alveolar interstitium, causes dyspnea and fatal outcomes. Although the bleomycin-induced PF mouse model has improved our understanding of exogenous factor-induced fibrosis, the mechanism governing endogenous factor-induced fibrosis remains unknown. Here, we find that Ifngr1-/-Rag2-/- mice, which lack the critical suppression factor for group 2 innate lymphoid cells (ILC2), develop PF spontaneously. The onset phase of fibrosis includes ILC2 subpopulations with a high Il1rl1 (IL-33 receptor) expression, and fibrosis does not develop in ILC-deficient or IL-33-deficient mice. Although ILC2s are normally localized near bronchioles and blood vessels, ILC2s are increased in fibrotic areas along with IL-33 positive fibroblasts during fibrosis. Co-culture analysis shows that activated-ILC2s directly induce collagen production from fibroblasts. Furthermore, increased IL1RL1 and decreased IFNGR1 expressions are confirmed in ILC2s from individuals with idiopathic PF, highlighting the applicability of Ifngr1-/-Rag2-/- mice as a mouse model for fibrosis research.

High-Throughput 3D Imaging Flow Cytometry of Suspended Adherent 3D Cell Cultures.

3D cell cultures are indispensable in recapitulating in vivo environments. Among the many 3D culture methods, culturing adherent cells on hydrogel beads to form spheroid-like structures is a powerful strategy for maintaining high cell viability and functions in the adherent states. However, high-throughput, scalable technologies for 3D imaging of individual cells cultured on the hydrogel scaffolds are lacking. This study reports the development of a high throughput, scalable 3D imaging flow cytometry platform for analyzing spheroid models. This platform is realized by integrating a single objective fluorescence light-sheet microscopy with a microfluidic device that combines hydrodynamic and acoustofluidic focusing techniques. This integration enabled unprecedentedly high-throughput and scalable optofluidic 3D imaging, processing 1310 spheroids consisting of 28 117 cells min-1 . The large dataset obtained enables precise quantification and comparison of the nuclear morphology of adhering and suspended cells, revealing that the adhering cells have smaller nuclei with less rounded surfaces. This platform's high throughput, robustness, and precision for analyzing the morphology of subcellular structures in 3D culture models hold promising potential for various biomedical analyses, including image-based phenotypic screening of drugs with spheroids or organoids.

Label-free mid-infrared photothermal live-cell imaging beyond video rate.

Advancement in mid-infrared (MIR) technology has led to promising biomedical applications of MIR spectroscopy, such as liquid biopsy or breath diagnosis. On the contrary, MIR microscopy has been rarely used for live biological samples in an aqueous environment due to the lack of spatial resolution and the large water absorption background. Recently, mid-infrared photothermal (MIP) imaging has proven to be applicable to 2D and 3D single-cell imaging with high spatial resolution inherited from visible light. However, the maximum measurement rate has been limited to several frames s-1, limiting its range of use. Here, we develop a significantly improved wide-field MIP quantitative phase microscope with two orders-of-magnitude higher signal-to-noise ratio than previous MIP imaging techniques and demonstrate live-cell imaging beyond video rate. We first derive optimal system design by numerically simulating thermal conduction following the photothermal effect. Then, we develop the designed system with a homemade nanosecond MIR optical parametric oscillator and a high full-well-capacity image sensor. Our high-speed and high-spatial-resolution MIR microscope has great potential to become a new tool for life science, in particular for live-cell analysis.

Adaptive dynamic range shift (ADRIFT) quantitative phase imaging.

Quantitative phase imaging (QPI) with its high-contrast images of optical phase delay (OPD) maps is often used for label-free single-cell analysis. Contrary to other imaging methods, sensitivity improvement has not been intensively explored because conventional QPI is sensitive enough to observe the surface roughness of a substrate that restricts the minimum measurable OPD. However, emerging QPI techniques that utilize, for example, differential image analysis of consecutive temporal frames, such as mid-infrared photothermal QPI, mitigate the minimum OPD limit by decoupling the static OPD contribution and allow measurement of much smaller OPDs. Here, we propose and demonstrate supersensitive QPI with an expanded dynamic range. It is enabled by adaptive dynamic range shift through a combination of wavefront shaping and dark-field QPI techniques. As a proof-of-concept demonstration, we show dynamic range expansion (sensitivity improvement) of QPI by a factor of 6.6 and its utility in improving the sensitivity of mid-infrared photothermal QPI. This technique can also be applied for wide-field scattering imaging of dynamically changing nanoscale objects inside and outside a biological cell without losing global cellular morphological image information.

Sequentially timed all-optical mapping photography boosted by a branched 4f system with a slicing mirror.

We present sequentially timed all-optical mapping photography (STAMP) with a slicing mirror in a branched 4f system for an increased number of frames without sacrificing pixel resolution. The branched 4f system spectrally separates the laser light path into multiple paths by the slicing mirror placed in the Fourier plane. Fabricated by an ultra-precision end milling process, the slicing mirror has 18 mirror facets of differing mirror angles. We used the boosted STAMP to observe dynamics of laser ablation with two image sensors which captured 18 subsequent frames at a frame rate of 126 billion frames per second, demonstrating this technique's potential for imaging unexplored ultrafast non-repetitive phenomena.

Label-free detection of Giardia lamblia cysts using a deep learning-enabled portable imaging flow cytometer.

We report a field-portable and cost-effective imaging flow cytometer that uses deep learning and holography to accurately detect Giardia lamblia cysts in water samples at a volumetric throughput of 100 mL h-1. This flow cytometer uses lens free color holographic imaging to capture and reconstruct phase and intensity images of microscopic objects in a continuously flowing sample, and automatically identifies Giardia lamblia cysts in real-time without the use of any labels or fluorophores. The imaging flow cytometer is housed in an environmentally-sealed enclosure with dimensions of 19 cm × 19 cm × 16 cm and weighs 1.6 kg. We demonstrate that this portable imaging flow cytometer coupled to a laptop computer can detect and quantify, in real-time, low levels of Giardia contamination (e.g., <10 cysts per 50 mL) in both freshwater and seawater samples. The field-portable and label-free nature of this method has the potential to allow rapid and automated screening of drinking water supplies in resource limited settings in order to detect waterborne parasites and monitor the integrity of the filters used for water treatment.

Quantitative phase imaging with molecular vibrational sensitivity.

Quantitative phase imaging (QPI) quantifies the sample-specific optical-phase-delay enabling objective studies of optically transparent specimens such as biological samples but lacks chemical sensitivity, limiting its application to a morphology-based diagnosis. We present wide-field molecular vibrational (MV) microscopy realized in the framework of QPI utilizing a mid-infrared (MIR) photothermal effect. Our technique provides MIR spectroscopic performance comparable to that of a conventional infrared spectrometer in the molecular fingerprint region of 1450-1640 cm-1 and realizes wide-field molecular imaging of a silica-polystyrene bead mixture over a 100 µm×100 µm area at 1 frame per second with the spatial resolution of 430 nm and 2-3 orders of magnitude lower fluence of ∼10 pJ/µm2 compared to other high-speed label-free molecular imaging methods, reducing photodamages to the sample. With a high-energy MIR pulse source, our technique could enable high-speed, label-free, simultaneous, and in situ acquisition of quantitative morphology and MV contrast, providing new insights for studies of optically transparent complex dynamics.

Molecular contrast on phase-contrast microscope.

An optical microscope enables image-based findings and diagnosis on microscopic targets, which is indispensable in many scientific, industrial and medical settings. A standard benchtop microscope platform, equipped with e.g., bright-field and phase-contrast modes, is of importance and convenience for various users because the wide-field and label-free properties allow for morphological imaging without the need for specific sample preparation. However, these microscopes never have capability of acquiring molecular contrast in a label-free manner. Here, we develop a simple add-on optical unit, comprising of an amplitude-modulated mid-infrared semiconductor laser, that is attached to a standard microscope platform to deliver the additional molecular contrast of the specimen on top of its conventional microscopic image, based on the principle of photothermal effect. We attach this unit, termed molecular-contrast unit, to a standard phase-contrast microscope, and demonstrate high-speed label-free molecular-contrast phase-contrast imaging of silica-polystyrene microbeads mixture and molecular-vibrational spectroscopic imaging of HeLa cells. Our simple molecular-contrast unit can empower existing standard microscopes and deliver a convenient accessibility to the molecular world.

A deep learning-enabled portable imaging flow cytometer for cost-effective, high-throughput, and label-free analysis of natural water samples.

We report a deep learning-enabled field-portable and cost-effective imaging flow cytometer that automatically captures phase-contrast color images of the contents of a continuously flowing water sample at a throughput of 100 mL/h. The device is based on partially coherent lens-free holographic microscopy and acquires the diffraction patterns of flowing micro-objects inside a microfluidic channel. These holographic diffraction patterns are reconstructed in real time using a deep learning-based phase-recovery and image-reconstruction method to produce a color image of each micro-object without the use of external labeling. Motion blur is eliminated by simultaneously illuminating the sample with red, green, and blue light-emitting diodes that are pulsed. Operated by a laptop computer, this portable device measures 15.5 cm × 15 cm × 12.5 cm, weighs 1 kg, and compared to standard imaging flow cytometers, it provides extreme reductions of cost, size and weight while also providing a high volumetric throughput over a large object size range. We demonstrated the capabilities of this device by measuring ocean samples at the Los Angeles coastline and obtaining images of its micro- and nanoplankton composition. Furthermore, we measured the concentration of a potentially toxic alga (Pseudo-nitzschia) in six public beaches in Los Angeles and achieved good agreement with measurements conducted by the California Department of Public Health. The cost-effectiveness, compactness, and simplicity of this computational platform might lead to the creation of a network of imaging flow cytometers for large-scale and continuous monitoring of the ocean microbiome, including its plankton composition.

Microfluidic single-particle chemical analyzer with dual-comb coherent Raman spectroscopy.

Label-free particle analysis is a powerful tool in chemical, pharmaceutical, and cosmetic industries as well as in basic sciences, but its throughput is significantly lower than that of fluorescence-based counterparts. Here we present a label-free single-particle analyzer based on broadband dual-comb coherent Raman scattering spectroscopy operating at a spectroscopic scan rate of 10 kHz. As a proof-of-concept demonstration, we perform broadband coherent anti-Stokes Raman scattering measurements of polystyrene microparticles flowing on an acoustofluidic chip at a high throughput of >1000 particles per second. This high-throughput label-free particle analyzer has the potential for high-precision statistical analysis of a large number of microparticles including biological cells.

Edge sparsity criterion for robust holographic autofocusing.

Autofocusing is essential to digital holographic imaging. Previously used autofocusing criteria exhibit challenges when applied to, e.g., connected objects with different optical properties. Furthermore, in some of the earlier autofocusing criteria, the polarity, i.e., whether to search for the peak or the valley as a function of depth, changes for different types of samples, which creates another challenge. Here, we propose a robust and accurate autofocusing criterion that is based on the edge sparsity of the complex optical wavefront, which we termed the "sparsity of the gradient" (SoG). We demonstrated the success of SoG by imaging a wide range of objects, including resolution test targets, stained and unstained Papanicolaou smears, stained tissue sections, and blood smears.

Design for sequentially timed all-optical mapping photography with optimum temporal performance.

A recently developed ultrafast burst imaging method known as sequentially timed all-optical mapping photography (STAMP) [Nat. Photonics8, 695 (2014)10.1038/nphoton.2014.163] has been shown effective for studying a diverse range of complex ultrafast phenomena. Its all-optical image separation circumvents mechanical and electronic restrictions that traditional burst imaging methods have long struggled with, hence realizing ultrafast, continuous, burst-type image recording at a fame rate far beyond what is achievable with conventional methods. In this Letter, considering various design parameters and limiting factors, we present an optimum design for STAMP in terms of temporal properties including exposure time and frame rate. Specifically, we first derive master equations that can be used to predict the temporal performance of a STAMP system and then analyze them to realize optimum conditions. This Letter serves as a general guideline for the camera parameters of a STAMP system with optimum temporal performance that is expected to be of use for tackling problems in science that are previously unsolvable with conventional imagers.