Abstract Background: Nanoscale collagen remodeling is a pivotal physical signature of tumor development and metastasis. Despite its importance, there has been a critical technical gap inresolving these subtle structural cues in a clinical setting without exogenous labels. Here, we report a first-of-its-kind label-free imaging mechanism based on bond-specific coherent interference in vibrational sum-frequency generation (VSFG) microscopy, providing unprecedented sensitivity to tumor-associated nanostructural variations. Methods: We utilized hyperspectral VSFG microscopy to investigate collagen I remodeling inlung tumor tissues. A novel biophysical model was developed to translate mode-specific interference patterns—specifically the I(NHs)/I(CH2,ss) intensity ratio—into quantitative collagen interfibrillar distances at the 20-50 nm level. To ensure immediate clinical impact, we conducted a timely validation of this mechanism on archived formalin-fixed paraffin-embedded(FFPE) tissues, benchmarking the results against optimal cutting temperature (OCT) cryosections and atomic force microscopy (AFM) nanomechanical mapping. Results: Our findings reveal that metastatic lung tumors exhibit dramatic spectral shifts driven bydistinctive interferences between vibrational modes. We demonstrate, for the first time, that thesespectral signatures can serve as a direct readout for collagen packing density at the sub-50 nmscale, which directly correlates with the increased tissue stiffness observed in tumor progression.Crucially, we provide the first evidence that the molecular-level structural cues detected by VSFGare preserved through harsh clinical fixation and embedding processes. The diagnostic metrics obtained from deparaffinized FFPE samples were statistically indistinguishable from fresh-frozenOCT controls, effectively removing the major barrier to applying this technology to clinical pathology. Conclusions: This study establishes a high-priority diagnostic platform that bridges the gapbetween nanoscale biophysics and clinical oncology. By demonstrating that VSFG can extract high-fidelity structural signatures from both fresh tissues and the vast global archives of FFPE samples, this work enables large-scale retrospective prognostic studies that were previously impossible. This discovery provides a powerful, label-free tool for future pathology and represents a significant advancement in our ability to monitor and understand the tumor microenvironment at the nanoscale. Citation Format: Jianyu Ren, Bin Yang, Chun-chieh Yu, Wei Xiong. Mode-specific coherent interference of vibrational sum-frequency generation imaging for detecting nanoscale collagen remodeling in lung tumors and clinical FFPE archives [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(8_Suppl):Abstract nr LB206.

Chemical imaging holds great promise for chemical, materials, and biological applications. However, its contrast often relies on subtle spectral differences arising from molecular-level changes. Here, we introduce label-free chemical imaging based on bond-specific coherent interference, which is highly sensitive to nanoscopic structural variations in collagen fibers. We demonstrated this idea using vibrational sum-frequency generation (VSFG) microscopy to identify lung tumors by detecting collagen I structural remodeling. Hyperspectral VSFG images reveal dramatic differences in the NH and CH x stretch regions between metastatic and tumor-free lung tissues. Based on these differences, key spectral signatures, such as the intensity ratio of NHS/CH2,Ss and CHS/CH2,Ss modes, were established to reliably distinguish metastatic tumor and tumor-free tissues with high fidelity. Theoretical modeling based on structural knowledge from Electron Microscopy indicates that distinctive interferences between VSFG vibrational modes make the intensity ratio directly related to interfibrillar distances at the 20-50 nm level. These findings suggest that collagen fibrils are more densely packed in tumors, corroborating the enhanced stiffness observed in tumor tissues. This result establishes an interference-based imaging mechanism that enhances chemical contrast and enables nanoscale structural readouts without labels. This method not only preserves sample integrity but also provides a powerful platform for fundamental biophysical studies and holds strong potential for future applications in oncology and pathology.

We present vibrational sum-frequency generation (VSFG) microscopy as a new label-free chemical imaging technique for tumor identification. This method combines the chemical-bond selectivity of vibrational spectroscopy with coherent interference of second-order coherent nonlinear optics. Using a fast line-scanning VSFG microscope, we obtained hyperspectral VSFG images of collagen I from both lung tissues bearing metastatic tumors and in tumor-free ones, which reveal drastic different spectral signatures: tumor samples exhibit large NH stretch (NHS) and CH stretch (CHS) versus the CH2 symmetric stretch (CH2,Ss), compared to healthy controls. We then identified two key spectral signatures to distinguish metastatic tumor and tumor-free tissues: the intensity ratio of NHS/CH2,Ss and CHS/CH2,Ss modes. These signatures demonstrated high fidelity in distinguishing between tumors and normal tissue in both mouse and human lung samples. Theoretical modeling indicates that distinctive interferences of spectral peaks are sensitive to interfibrillar distances, at 130 of nanometer level. These findings suggest that collagen fibrils are more densely packed in tumors, corroborating the enhanced stiffness observed in tumor tissues. VSFG microscopy offers a highly selective, label-free chemical imaging characterization that preserves sample integrity, making it a valuable tool for oncology, pathology and fundamental biophysical research.

Elucidating organismal developmental processes requires a comprehensive understanding of cellular lineages in the spatial, temporal, and molecular domains. In this study, we introduce Zebrahub, a dynamic atlas of zebrafish embryonic development that integrates single-cell sequencing time course data with lineage reconstructions facilitated by light-sheet microscopy. This atlas offers high-resolution and in-depth molecular insights into zebrafish development, achieved through the sequencing of individual embryos across ten developmental stages, complemented by reconstructions of cellular trajectories. Zebrahub also incorporates an interactive tool to navigate the complex cellular flows and lineages derived from light-sheet microscopy data, enabling in silico fate-mapping experiments. To demonstrate the versatility of our multimodal resource, we utilize Zebrahub to provide fresh insights into the pluripotency of neuro-mesodermal progenitors (NMPs) and the origins of a joint kidney-hemangioblast progenitor population.

Vibrational sum-frequency generation (VSFG), a second-order nonlinear optical signal, has traditionally been used to study molecules at interfaces as a spectroscopy technique with a spatial resolution of ~100 µm. However, the spectroscopy is not sensitive to the heterogeneity of a sample. To study mesoscopically heterogeneous samples, we, along with others, pushed the resolution limit of VSFG spectroscopy down to ~1 µm level and constructed the VSFG microscope. This imaging technique not only can resolve sample morphologies through imaging, but also record a broadband VSFG spectrum at every pixel of the images. Being a second-order nonlinear optical technique, its selection rule enables the visualization of non-centrosymmetric or chiral self-assembled structures commonly found in biology, materials science, and bioengineering, among others. In this article, the audience will be guided through an inverted transmission design that allows for imaging unfixed samples. This work also showcases that VSFG microscopy can resolve chemical-specific geometric information of individual self-assembled sheets by combining it with a neural network function solver. Lastly, the images obtained under brightfield, SHG, and VSFG configurations of various samples briefly discuss the unique information revealed by VSFG imaging.

Recent developments in Oblique Plane Microscopy (OPM) have shown that it can achieve high spatio-temporal resolution. Here we describe a single objective light-sheet microscope based on oblique plane illumination that achieves: (i) large field of view and high-resolution imaging via a custom remote focusing objective; (ii) fast volumetric imaging by means of light-sheet stabilised stage scanning – a novel scanning modality that extends the imaging volume without compromising imaging speed nor quality; (iii) multi-view imaging by alternating the orientation of light-sheet illumination and detection to improve the image quality on large samples; (iv) simpler design and ergonomics by remote placement of coverslips to allow inverted imaging, enabling imaging across scales in a high-throughput format. Overall, we achieved a resolution of 450 nm laterally and 2 μm axially and a field of view of 3000 μm × 800 μm × 300 μm. We demonstrate the speed, field of view, resolution and versatility of our novel instrument by imaging various systems, including zebrafish whole brain activity, Drosophila egg chamber development, and zebrafish development – up to nine embryos simultaneously.

A new single-objective light-sheet microscope has been developed that uses novel optics and imaging protocols to increase resolution without compromising imaging speed and volume.

Receptor tyrosine kinase (RTK)-mediated activation of downstream effector pathways such as the RAS GTPase/MAP kinase (MAPK) signaling cascade is thought to occur exclusively from lipid membrane compartments in mammalian cells. Here, we uncover a membraneless, protein granule-based subcellular structure that can organize RTK/RAS/MAPK signaling in cancer. Chimeric (fusion) oncoproteins involving certain RTKs including ALK and RET undergo de novo higher-order assembly into membraneless cytoplasmic protein granules that actively signal. These pathogenic biomolecular condensates locally concentrate the RAS activating complex GRB2/SOS1 and activate RAS in a lipid membrane-independent manner. RTK protein granule formation is critical for oncogenic RAS/MAPK signaling output in these cells. We identify a set of protein granule components and establish structural rules that define the formation of membraneless protein granules by RTK oncoproteins. Our findings reveal membraneless, higher-order cytoplasmic protein assembly as a distinct subcellular platform for organizing oncogenic RTK and RAS signaling.

In the past few decades, aquatic animals have become popular model organisms in biology, spurring a growing need for establishing aquatic facilities. Zebrafish are widely studied and relatively easy to culture using commercial systems. However, a challenging aspect of maintaining aquatic facilities is animal feeding, which is both time- and resource-consuming. We have developed an open-source fully automatic daily feeding system, Zebrafish Automatic Feeder (ZAF). ZAF is reliable, provides a standardized amount of food to every tank, is cost-efficient and easy to build. The advanced version, ZAF+, allows for the precise control of food distribution as a function of fish density per tank, and has a user-friendly interface. Both ZAF and ZAF+ are adaptable to any laboratory environment and facilitate the implementation of aquatic colonies. Here we provide all blueprints and instructions for building the mechanics, electronics, fluidics, as well as to setup the control software and its user-friendly graphical interface. Importantly, the design is modular and can be scaled to meet different user needs. Furthermore, our results show that ZAF and ZAF+ do not adversely affect zebrafish culture, enabling fully automatic feeding for any aquatic facility.

In the developing embryos, the cortical polarity regulator Par-3 is critical for establishing Notch signaling asymmetry between daughter cells during asymmetric cell division (ACD). How cortically localized Par-3 establishes asymmetric Notch activity in the nucleus is not understood. Here, using in vivo time-lapse imaging of mitotic radial glia progenitors in the developing zebrafish forebrain, we uncover that during horizontal ACD along the anteroposterior embryonic axis, endosomes containing the Notch ligand DeltaD (Dld) move toward the cleavage plane and preferentially segregate into the posterior (subsequently basal) Notchhi daughter. This asymmetric segregation requires the activity of Par-3 and dynein motor complex. Using label retention expansion microscopy, we further detect Par-3 in the cytosol colocalizing the dynein light intermediate chain 1 (Dlic1) onto Dld endosomes. Par-3, Dlic1, and Dld are associated in protein complexes in vivo. Our data reveal an unanticipated mechanism by which cytoplasmic Par-3 directly polarizes Notch signaling components during ACD.

{\mu}Manager, an open-source microscopy acquisition software, has been an essential tool for many microscopy experiments over the past 15 years, but is not easy to use for experiments in which image acquisition and analysis are closely coupled. This is because {\mu}Manager libraries are written in C++ and Java, whereas image processing is increasingly carried out with data science and machine learning tools most easily accessible through the Python programming language. We present Pycro-Manager, a tool that enables rapid development of such experiments, while also providing access to the wealth of existing tools within {\mu}Manager through Python.

Drosophila oocytes develop together with 15 sister germline nurse cells (NCs), which pass products to the oocyte through intercellular bridges. The NCs are completely eliminated during stages 12-14, but we discovered that at stage 10B, two specific NCs fuse with the oocyte and extrude their nuclei through a channel that opens in the anterior face of the oocyte. These nuclei extinguish in the ooplasm, leaving 2 enucleated and 13 nucleated NCs. At stage 11, the cell boundaries of the oocyte are mostly restored. Oocytes in egg chambers that fail to eliminate NC nuclei at stage 10B develop with abnormal morphology. These findings show that stage 10B NCs are distinguished by position and identity, and that NC elimination proceeds in two stages: first at stage 10B and later at stages 12-14.

We present an oblique plane microscope (OPM) that uses a bespoke glass-tipped tertiary objective to improve the resolution, field of view, and usability over previous variants. Owing to its high numerical aperture optics, this microscope achieves lateral and axial resolutions that are comparable to the square illumination mode of lattice light-sheet microscopy, but in a user friendly and versatile format. Given this performance, we demonstrate high-resolution imaging of clathrin-mediated endocytosis, vimentin, the endoplasmic reticulum, membrane dynamics, and Natural Killer-mediated cytotoxicity. Furthermore, we image biological phenomena that would be otherwise challenging or impossible to perform in a traditional light-sheet microscope geometry, including cell migration through confined spaces within a microfluidic device, subcellular photoactivation of Rac1, diffusion of cytoplasmic rheological tracers at a volumetric rate of 14 Hz, and large field of view imaging of neurons, developing embryos, and centimeter-scale tissue sections.

We present a single-objective light-sheet microscope, also known as an oblique-plane microscope, that uses a bespoke glass-tipped tertiary objective and improves the resolution, field of view, usability, and stability over previous variants. Owing to its high numerical aperture optics, this microscope achieves the highest lateral resolution in light-sheet fluorescence microscopy, and its axial resolution is similar to that of Lattice Light-Sheet Microscopy. Given this performance, we demonstrate high-resolution imaging of clathrin-mediated endocytosis, vimentin, the endoplasmic reticulum, membrane dynamics, and natural killer cell-mediated cell death. Furthermore, we image biological phenomena that would be otherwise challenging or impossible to perform in a traditional light-sheet microscope geometry, including cell migration through a confined space within a microfluidic device, photoactivation of PI3K, and diffusion of cytoplasmic rheological tracers at a volumetric rate of 14 Hz.

In modern microscopy imaging systems, optical components are carefully designed to obtain diffraction-limited resolution. However, live imaging of large biological samples rarely attains this limit because of sample induced refractive index inhomogeneities that create unknown temporally variant optical aberrations. Importantly, these aberrations are also spatially variant, thus making it challenging to correct over wide fields of view. Here, we present a framework for deep-learning based wide-field optical aberration sensing and correction. Our model consists of two modules which take in a set of three phase-diverse images and (i) estimate the wavefront phase in terms of its constituent Zernike polynomial coefficients and (ii) perform blind-deconvolution to yield an aberration-free image. First, we demonstrate our framework on simulations that incorporate optical aberrations, spatial variance, and realistic modelling of sensor noise. We find that our blind deconvolution achieves a 2-fold improvement in frequency support compared to input images, and our phase-estimation achieves a coefficient of determination () of at least 80% when estimating astigmatism, spherical aberration and coma. Second, we show that our results mostly hold for strongly varying spatially-variant aberrations with a 30% resolution improvement. Third, we demonstrate practical usability for light-sheet microscopy: we show a 46% increase in frequency support even in imaging regions affected by detection and illumination scattering.

Abstract The spatial organization of a cell includes lipid membrane-based compartments and an emerging class of subcellular structures collectively described as biomolecular condensates1. Lipid membranes act as a biologically active scaffold to concentrate signaling molecules in multiple signal transduction pathways that regulate normal physiology and pathologic conditions such as cancer2. Notably, receptor tyrosine kinase (RTK)-mediated RAS GTPase/MAP kinase (MAPK) pathway signaling is thought to occur exclusively from lipid membrane compartments in mammalian cells3,4. Here, we uncover a membraneless, protein granule-based subcellular structure that can organize RTK/RAS/MAPK signaling. Chimeric (fusion) oncoproteins involving certain RTKs including ALK and RET undergo de novo assembly into cytoplasmic protein granules that locally concentrate the RAS activating complex GRB2/SOS1 and activate RAS in a lipid membrane-independent manner to initiate MAPK signaling. We show that formation of higher-order membraneless protein granules is both necessary and sufficient for RAS/MAPK signaling output in cells. These large-scale protein assemblies are functionally distinct from lower-order oligomerization of cytoplasmic RTK fusion oncoproteins. Our findings reveal membraneless, higher-order cytoplasmic protein assembly as a subcellular platform to activate RAS GTPases and a distinct principle by which cells can organize kinase-mediated oncogenic signaling.

We designed an epi-illumination SPIM system that uses a single objective and has a sample interface identical to that of an inverted fluorescence microscope with no additional reflection elements. It achieves subcellular resolution and single-molecule sensitivity, and is compatible with common biological sample holders, including multi-well plates. We demonstrated multicolor fast volumetric imaging, single-molecule localization microscopy, parallel imaging of 16 cell lines and parallel recording of cellular responses to perturbations.

Understanding how cells spatially organize signaling events is important in normal biology and pathological conditions such as cancer. Here, we uncover a membraneless, protein granule-based subcellular structure that can organize receptor tyrosine kinase (RTK)-mediated RAS/MAPK pathway signaling, which is thought to occur exclusively from lipid-membrane compartments in mammalian cells. De-novo assembly of cytoplasmic protein granules by certain RTKs, including oncogenic gene fusions involving ALK and RET, is dependent on multimerization domains in the RTK fusion partners. Protein granule formation is both necessary and sufficient to locally concentrate the RAS activating complex GRB2/SOS1 to initiate MAPK pathway signaling. Our findings reveal membraneless, higher-order protein assembly as a principle by which cells can organize kinase-mediated proliferative signals. One Sentence Summary Kinase/RAS signaling via protein granules

Spectral imaging is a powerful technique used to simultaneously study multiple fluorophore labels with overlapping emissions. Here, we present a computational spectral imaging method, which uses sample spatial fluorescence information as a reconstruction constraint. Our method addresses both the under-sampling issue of compressive spectral imaging and the low throughput issue of scanning spectral imaging. With simulated and experimental data, we have demonstrated the reconstruction precision of our method in two and three-color imaging. We have experimentally validated this method for differentiating cellular structures labeled with two red-colored fluorescent proteins, tdTomato and mCherry, which have highly overlapping emission spectra. Our method has the advantage of totally free wavelength choice and can also be combined with conventional filter-based sequential multi-color imaging to further improve multiplexing capability.