A Low-Cost Modular Imaging System for Rapid, Multiplexed Immunofluorescence Detection in Clinical Tissues.

To image 4-plex immunofluorescence-stained tissue samples at a low cost with cellular level resolution and sensitivity and dynamic range required to detect lowly and highly abundant targets, here we describe a robust, inexpensive (<$9000), 3D printable portable imaging device (Tissue Imager). The Tissue Imager can immediately be deployed on benchtops for in situ protein detection in tissue samples. Applications for this device are broad, ranging from answering basic biological questions to clinical pathology, where immunofluorescence can detect a larger number of markers than the standard H&E or chromogenic immunohistochemistry (CIH) staining, while the low cost also allows usage in classrooms. After characterizing our platform's specificity and sensitivity, we demonstrate imaging of a 4-plex immunology panel in human cutaneous T-cell lymphoma (CTCL) formalin-fixed paraffin-embedded (FFPE) tissue samples. From those images, positive cells were detected using CellProfiler, a popular open-source software package, for tumor marker profiling. We achieved a performance on par with commercial epifluorescence microscopes that are >10 times more expensive than our Tissue Imager. This device enables rapid immunofluorescence detection in tissue sections at a low cost for scientists and clinicians and can provide students with a hands-on experience to understand engineering and instrumentation. We note that for using the Tissue Imager as a medical device in clinical settings, a comprehensive review and approval processes would be required.

SPIM-Flow: An Integrated Light Sheet and Microfluidics Platform for Hydrodynamic Studies of Hydra.

Selective plane illumination microscopy (SPIM), or light sheet microscopy, is a powerful imaging approach. However, access to and interfacing microscopes with microfluidics have remained challenging. Complex interfacing with microfluidics has limited the SPIM's utility for studying the hydrodynamics of freely moving multicellular organisms. We developed SPIM-Flow, an inexpensive light sheet platform that enables easy integration with microfluidics. We used SPIM-Flow to investigate the hydrodynamics of a freely moving Hydra polyp via particle tracking in millimeter-sized chambers. Initial experiments across multiple animals, feeding on a chip (Artemia franciscana nauplii used as food), and baseline behaviors (tentacle swaying, elongation, and bending) indicated the organisms' health inside the system. Fluidics were used to investigate Hydra's response to flow. The results suggested that the animals responded to an established flow by bending and swaying their tentacles in the flow direction. Finally, using SPIM-Flow in a proof-of-concept experiment, the shear stress required to detach an animal from a surface was demonstrated. Our results demonstrated SPIM-Flow's utility for investigating the hydrodynamics of freely moving animals.

Palmitoylation-regulated interactions of the pseudokinase calmodulin kinase-like vesicle-associated with membranes and Arc/Arg3.1.

Calmodulin kinase-like vesicle-associated (CaMKv), a pseudokinase belonging to the Ca2+/calmodulin-dependent kinase family, is expressed predominantly in brain and neural tissue. It may function in synaptic strengthening during spatial learning by promoting the stabilization and enrichment of dendritic spines. At present, almost nothing is known regarding CaMKv structure and regulation. In this study we confirm prior proteomic analyses demonstrating that CaMKv is palmitoylated on Cys5. Wild-type CaMKv is enriched on the plasma membrane, but this enrichment is lost upon mutation of Cys5 to Ser. We further show that CaMKv interacts with another regulator of synaptic plasticity, Arc/Arg3.1, and that the interaction between these two proteins is weakened by mutation of the palmitoylated cysteine in CamKv.

Differential Mobility and Self-Association of Arc/Arg3.1 in the Cytoplasm and Nucleus of Living Cells.

Arc, also known as Arg3.1, is an activity-dependent immediate-early gene product that plays essential roles in memory consolidation. A pool of Arc is located in the postsynaptic cytoplasm, where it promotes AMPA receptor endocytosis and cytoskeletal remodeling. However, Arc is also found in the nucleus, with a major portion being associated with promyelocytic leukemia nuclear bodies (PML-NBs). Nuclear Arc has been implicated in epigenetic control of gene transcription associated with learning and memory. In this study, we use a battery of fluorescence nanoimaging approaches to characterize the behavior of Arc ectopically expressed in heterologous cells. Our results indicate that in the cytoplasm, Arc exists predominantly as monomers and dimers associated with slowly diffusing particles. In contrast, nuclear Arc is almost exclusively monomeric and displays a higher diffusivity than cytoplasmic Arc. We further show that Arc moves freely and rapidly between PML-NBs and the nucleoplasm and that its movement within PML-NBs is relatively unobstructed.

Spatial transcriptomics using combinatorial fluorescence spectral and lifetime encoding, imaging and analysis.

Multiplexed mRNA profiling in the spatial context provides new information enabling basic research and clinical applications. Unfortunately, existing spatial transcriptomics methods are limited due to either low multiplexing or complexity. Here, we introduce a spatialomics technology, termed Multi Omic Single-scan Assay with Integrated Combinatorial Analysis (MOSAICA), that integrates in situ labeling of mRNA and protein markers in cells or tissues with combinatorial fluorescence spectral and lifetime encoded probes, spectral and time-resolved fluorescence imaging, and machine learning-based decoding. We demonstrate MOSAICA's multiplexing scalability in detecting 10-plex targets in fixed colorectal cancer cells using combinatorial labeling of five fluorophores with facile error-detection and removal of autofluorescence. MOSAICA's analysis is strongly correlated with sequencing data (Pearson's r = 0.96) and was further benchmarked using RNAscopeTM and LGC StellarisTM. We further apply MOSAICA for multiplexed analysis of clinical melanoma Formalin-Fixed Paraffin-Embedded (FFPE) tissues. We finally demonstrate simultaneous co-detection of protein and mRNA in cancer cells.

Rapid isolation of rare targets from large fluid volumes.

Rapidly isolating rare targets from larger, clinically relevant fluid volumes remains an unresolved problem in biomedicine and diagnosis. Here, we describe how 3D particle sorting can enrich targets at ultralow concentrations over 100-fold within minutes not possible with conventional approaches. Current clinical devices based on biochemical extraction and microfluidic solutions typically require high concentrations and/or can only process sub-milliliter volumes in time. In a proof-of-concept application, we isolated bacteria from whole blood as demanded for rapid sepsis diagnosis where minimal numbers of bacteria need to be found in a 1-10 mL blood sample. After sample encapsulation in droplets and target enrichment with the 3D particle sorter within a few minutes, downstream analyses were able to identify bacteria and test for antibiotic susceptibility, information which is critical for successful treatment of bloodstream infections.

Educated natural killer cells show dynamic movement of the activating receptor NKp46 and confinement of the inhibitory receptor Ly49A.

Educated natural killer (NK) cells have inhibitory receptors specific for self major histocompatibility complex (MHC) class I molecules and kill cancer cells more efficiently than do NK cells that do not have such receptors (hyporesponsive NK cells). The mechanism behind this functional empowerment through education has so far not been fully described. In addition, distinctive phenotypic markers of educated NK cells at the single-cell level are lacking. We developed a refined version of the image mean square displacement (iMSD) method (called iMSD carpet analysis) and used it in combination with single-particle tracking to characterize the dynamics of the activating receptor NKp46 and the inhibitory receptor Ly49A on resting educated versus hyporesponsive murine NK cells. Most of the NKp46 and Ly49A molecules were restricted to microdomains; however, individual NKp46 molecules resided in these domains for shorter periods and diffused faster on the surface of educated, compared to hyporesponsive, NK cells. In contrast, the movement of Ly49A was more constrained in educated NK cells compared to hyporesponsive NK cells. Either disrupting the actin cytoskeleton or adding cholesterol to the cells prohibited activating signaling, suggesting that the dynamics of receptor movements within the cell membrane are critical for the proper activation of NK cells. The faster and more dynamic movement of NKp46 in educated NK cells may facilitate a swifter response to interactions with target cells.

Organization of perinuclear actin in live tobacco cells observed by PALM with optical sectioning.

Actin performs a wide variety of different tasks. This functional diversity may be accomplished either by the formation of different isotypes or by suitable protein decoration that regulates structure and dynamics of actin filaments. To probe for such a potential differential decoration, the actin-binding peptide Lifeact was fused to different photoactivatable fluorescent proteins. These fusions were stably expressed in Nicotiana tabacum L. cv. Bright Yellow 2 cells to follow dynamic reorganization of the actin cytoskeleton during the cell cycle. The Lifeact-monomeric variant of IrisFP fusion protein was observed to indiscriminately label both, central and cortical, actin filaments, whereas the tetrameric Lifeact-photoswitchable red fluorescent protein fusion construct selectively labeled only a specific perinuclear sub-population of actin. By using photoactivated localization microscopy, we acquired super-resolution images with optical sectioning to obtain a 3D model of perinuclear actin. This novel approach revealed that the perinuclear actin basket wraps around the nuclear envelope in a lamellar fashion and repartitions toward the leading edge of the migrating nucleus. Based on these data, we suggest that actin that forms the perinuclear basket differs from other actin assemblies by a reduced decoration with actin binding proteins, which is consistent with the differential decoration model.

Dual color photoactivation localization microscopy of cardiomyopathy-associated desmin mutants.

Mutations in the DES gene coding for the intermediate filament protein desmin may cause skeletal and cardiac myopathies, which are frequently characterized by cytoplasmic aggregates of desmin and associated proteins at the cellular level. By atomic force microscopy, we demonstrated filament formation defects of desmin mutants, associated with arrhythmogenic right ventricular cardiomyopathy. To understand the pathogenesis of this disease, it is essential to analyze desmin filament structures under conditions in which both healthy and mutant desmin are expressed at equimolar levels mimicking an in vivo situation. Here, we applied dual color photoactivation localization microscopy using photoactivatable fluorescent proteins genetically fused to desmin and characterized the heterozygous status in living cells lacking endogenous desmin. In addition, we applied fluorescence resonance energy transfer to unravel short distance structural patterns of desmin mutants in filaments. For the first time, we present consistent high resolution data on the structural effects of five heterozygous desmin mutations on filament formation in vitro and in living cells. Our results may contribute to the molecular understanding of the pathological filament formation defects of heterozygous DES mutations in cardiomyopathies.