Biology subjects

Chu, C. J.

Publications and source records attributed to Chu, C. J..

At least 19 recordsLinked to original sources

bioRxiv2026

Sleep spindle deficits in childhood absence epilepsy improve with antiseizure treatment and disease resolution

Childhood absence epilepsy (CAE), the most common childhood epilepsy syndrome, is associated with bursts of generalized thalamocortical spike-wave discharges and cognitive impairment, yet the physiological mechanisms linking disease activity, treatment, and cognition remain unclear. Sleep spindles, generated by thalamocortical circuits, are associated with cognitive function and may be disrupted by epileptic activity. Here, we retrospectively studied 87 EEGs from 53 children with active and resolved CAE and 87 age- and sex-matched controls to evaluate sleep spindle activity across disease states and its association with antiseizure medication (ASM) exposure. We found that children with active CAE had reduced spindle rates across all cortical regions compared to those with resolved CAE and controls, with the largest reduction in the frontal region, where epileptic spike activity was also most prominent. Spindle rate was inversely correlated with epileptic spike rate, consistent with a shared thalamocortical circuitry. The association between ASM exposure and spindle rate varied by disease state. In active CAE, treated children had higher spindle rates than untreated children. In resolved CAE, treated children had lower spindle rates than untreated children. In contrast, ASM-related differences in spike rate were more limited, although a medication-specific reduction was observed in active CAE. These findings support sleep spindle rate as a candidate physiological marker of disease state and ASM exposure in children with CAE. Given the established role of sleep spindles in memory consolidation, these findings support testing of whether spindle disruption provides a mechanistic link between epileptic thalamocortical activity and cognitive vulnerability in CAE.

neuroscience↗

bioRxiv2025

Spatial proteomic mapping of the human and mouse retina using IBEX

PurposeTo generate a comparative spatial proteomic atlas of the human and mouse retina using a refined highly multiplexed immunohistochemistry technique called Iterative Bleaching Extends Multiplexity (IBEX). MethodsWe refined the IBEX workflow by integrating an antibody removal option alongside chemical bleaching. This dual strategy enabled removal of the entire antibody complex allowing for the flexible use of antibodies from the same host species across iterative cycles. Spatial proteomic data were cross-correlated to existing scRNAseq datasets. We also coupled this workflow with super-resolution imaging via deconvolution and applied it to the retina of healthy human, mouse and the Crb1rd8 mouse model. ResultsWe successfully imaged over 25 protein markers on human and mouse tissue sections, to generate spatial atlases of the major retinal cell populations. Cross-species protein expression was compared to scRNAseq datasets to identify protein and transcript disparities. Super-resolution IBEX delineated the ultrastructural features of the outer limiting membrane (OLM), identifying CD44 as a core structural component tightly co-localised with a highly organised F-actin belt within Muller glial endfeet. Using the Crb1rd8 mouse model, disruption of this complex was spatially associated with rosette formation and OLM structural failure. ConclusionSpatial proteomic atlases of the human and mouse retina were generated and correlated to transcriptional data, revealing insights into the arrangement of major retinal cell populations and OLM structure.

neuroscience↗

bioRxiv2025

A pathogenic CD4 T cell phenotype in experimental uveitis shares common features with other immune mediated inflammatory

Murine ocular autoimmunity develops through 3 stages; prodrome, primary peak and secondary regulation. During the prodromal phase, leukocytes accumulate within the retina and vitreous. Using the adoptive transfer model of experimental autoimmune uveitis, we can analyse the disease course by tracking the transferred cells during disease initiation being recruited to the ocular environment throughout peak of disease to secondary regulation. During initiation (the prodrome) of disease pathogenic transferred CD4+ T cells can be detected within the retina as well as an endogenous CD4+ infiltrate and as disease reaches peak, both transferred and endogenous CD4+ T cells can be found in large numbers in the retina. Active clinical disease resolves by day 21 but transferred CD4+ T cells persist within the retina when disease is in a clinically quiescent state. Concurrent transfer of RBP3 specific and OVA specific activated cells induces a similar clinical disease phenotype and time course. Both RBP3 and OVA specific cells are recruited during active clinical disease in equal measure showing that autoantigen specific CD4+ T cells induce susceptibility for recruitment of other activated CD4+ T cells. When analysing the endogenous and transferred CD4+ T cells by RNA sequencing, differences between the two sets of gene signatures highlight genes found in pathogenic T cells in other models, including upregulation of markers associated with cytokine interactions and NK cell mediated cytotoxicity. Due to the persistence of the original transferred population throughout clinical disease, in depth analysis of this population could suggest pathways contributing to ocular autoimmunity.

immunology↗

bioRxiv2025

Changes in slow oscillations and sleep spindles by auditory stimulation positively correlate with memory consolidation in children with epilepsy and controls

BackgroundSleep-dependent memory consolidation is supported by sleep spindles during stages 2 and 3 non-rapid eye movement sleep. Sleep spindles and sleep-dependent memory consolidation are both decreased in Rolandic epilepsy (RE). Non-invasive auditory stimulation evokes SOs and SO-spindle complexes in healthy adults but the impact on memory consolidation has been inconsistent. ObjectiveWe investigated the effects of auditory stimulation during sleep on SOs, SO-spindle complexes, and sleep-dependent memory consolidation in children with RE and controls. MethodsA prospective cross-over study was conducted in children with RE and control. Children completed two nap visits with auditory or sham stimulation. SOs and SO-spindle complexes rates were measured offline using validated detectors. Sleep-dependent memory consolidation was assessed using the motor sequence typing task. ResultsAuditory stimulation evoked SOs and SO-spindle complexes broadly with maximal effect over frontal electrodes. Compared to sham, stimulation delivered during background activity evoked SOs (29.8% increase, p<0.001) and SO-spindle complexes (16.8% increase, p<0.001) and stimulations delivered near the peak of an ongoing SO upstate maximally evoked SOs (51.3% increase, p<0.001) and SO-spindle complexes (32.3% increase, p<0.001). Changes in frontal SO (1.9% improvement per increase in SO/min; p<0.001) and SO-spindle complexes (9.5% improvement per increase in SO-spindle/min) event rates due to auditory stimulation positively predicted changes in sleep-dependent memory consolidation. ConclusionAuditory stimulation reliably modulates sleep oscillations when delivered on background activity and during the upstate of SOs. As increased event rates improve memory consolidation, stimulation paradigms to increase SO and SO-spindle complex rates are required to enhance memory.

neuroscience↗

bioRxiv2025

A hierarchical cascade of sleep rhythms drive memory consolidation in humans and are disrupted in epilepsy.

The cross-regional interplay of slow oscillations, sleep spindles, and ripples during sleep is believed to support systems memory consolidation but is understudied in humans. Using a validated behavioral task and intracranial neural recordings from orbitofrontal cortex, thalamus, and hippocampus in 19 epilepsy patients, we examined the cross-regional interplay of sleep-oscillations and their role in memory consolidation. Orbitofrontal slow oscillations robustly modulate sleep rhythms both within and across regions. Most combinations of oscillation rates predict overnight memory consolidation, but hippocampal ripple rate and coupled hippocampal-orbitofrontal ripples were the strongest positive predictors of memory consolidation. In contrast, epileptic spikes coupled to sleep oscillations strongly predicted reduced memory consolidation, with the strongest negative effect observed when epileptic spikes were coupled to slow oscillations. These findings provide direct evidence of the hierarchical cascade of sleep oscillations in human memory processing and reveal how epileptic spikes disrupt this process in patients with epilepsy.

neuroscience↗

bioRxiv2025

Atypical Alpha Oscillatory EEG Dynamics in Children with Angelman Syndrome

ObjectivesBiomarkers of atypical brain development are crucial for advancing clinical trials and guiding therapeutic interventions in Angelman syndrome (AS). Electroencephalography (EEG) captures well-characterized developmental changes in peak alpha frequency (PAF) that reflect underlying neural circuit maturation and may provide a sensitive metric for mapping atypical neural trajectories in AS. MethodWe analyzed EEG recordings from 159 children with AS (ages 1-15 years) and 185 age-matched typically developing (TD) controls. PAF was quantified using a well-established curve-fitting method applied to 1/f-corrected power spectra. To validate robustness, we further evaluated PAF using an alternative prominence-based peak detection approach across varying detection thresholds. ResultsSignificant disruptions in PAF were evident in children with AS. While over 90% of EEGs from TD children exhibited a clear alpha peak, fewer than 50% of EEGs from children with AS showed a detectable PAF. Furthermore, when PAF was present, its frequency was significantly lower in AS children and did not show the typical age-related increases observed in TD children. Validation analyses confirmed consistently lower rates of PAF detection in AS across varying sensitivity thresholds, demonstrating the robustness of these results. ConclusionsPAF is a robust and developmentally sensitive marker of disrupted neural maturation in children with Angelman syndrome. As a quantifiable and sensitive measure of neural disruptions in AS, PAF has the potential to complement and enhance existing clinical trial outcome assessments by providing an objective index of underlying brain function. Future analyses will explore individual differences related to PAF in AS, to better understand mechanistic insights to guide targeted therapeutic strategies.

neuroscience↗

bioRxiv2024

Rebalancing Viral and Immune Damage versus Tissue Repair Prevents Death from Lethal Influenza Infection

Maintaining tissue function while eliminating infected cells is fundamental to host defense. Innate inflammatory damage contributes to lethal influenza and COVID-19, yet other than steroids, immunomodulatory drugs have modest effects. Among more than 50 immunomodulatory regimes tested in mouse lethal influenza infection, only the previously reported early depletion of neutrophils showed efficacy, suggesting that the infected host passes an early tipping point in which limiting innate immune damage alone cannot rescue physiological function. To re-balance the system late in infection, we investigated whether partial limitation of viral spread using oseltamivir (Tamiflu) together with enhancement of epithelial repair by blockade of interferon signaling or the limitation of further epithelial cell loss mediated by cytotoxic CD8+ T cells would prevent death. These treatments salvaged a large fraction of infected animals, providing new insight into the importance of repair processes and the timing of adaptive immune responses in survival of pulmonary infections.

immunology↗

bioRxiv2024

IL-23 drives uveitis by acting on a novel population of tissue-resident entheseal T cells

Recurrent acute anterior uveitis is a frequent extra-articular manifestation of the axial spondyloarthropathies (AxSpA); chronic inflammatory diseases affecting the spine, enthesis, peripheral joints, skin, and gastrointestinal tract. Pathology in AxSpA has been associated with local tissue-resident populations of interleukin (IL)-23 responsive lymphoid cells. Here we reveal a novel population of ocular T cells defined by CD3+CD4-CD8-{gamma}{delta}TCR+IL-23R+ that reside within the anterior uvea as an ocular entheseal analogue of the mouse eye. Localised cytokine expression demonstrates that uveal IL-23R+ IL-17A-producing cells are both necessary and sufficient to drive uveitis in response to IL-23. This T cell population is also present in humans, occupying extravascular tissues of the anterior uveal compartment. Consistent with the concept of IL-23 as a unifying mediator in AxSpA, we present evidence that IL-23 can also act locally on tissue resident T cells in the anterior compartment of the eye at sites analogous to the enthesis to drive ocular inflammation.

immunology↗

bioRxiv2024

Impaired sleep-dependent memory consolidation predicted by reduced sleep spindles in Rolandic epilepsy

Background and ObjectivesSleep spindles are prominent thalamocortical brain oscillations during sleep that have been mechanistically linked to sleep-dependent memory consolidation in animal models and healthy controls. Sleep spindles are decreased in Rolandic epilepsy and related sleep-activated epileptic encephalopathies. We investigate the relationship between sleep spindle deficits and deficient sleep dependent memory consolidation in children with Rolandic epilepsy. MethodsIn this prospective case-control study, children were trained and tested on a validated probe of memory consolidation, the motor sequence task (MST). Sleep spindles were measured from high-density EEG during a 90-minute nap opportunity between MST training and testing using a validated automated detector. ResultsTwenty-three children with Rolandic epilepsy (14 with resolved disease), and 19 age- and sex-matched controls were enrolled. Children with active Rolandic epilepsy had decreased memory consolidation compared to control children (p=0.001, mean percentage reduction: 25.7%, 95% CI [10.3, 41.2]%) and compared to children with resolved Rolandic epilepsy (p=0.007, mean percentage reduction: 21.9%, 95% CI [6.2, 37.6]%). Children with active Rolandic epilepsy had decreased sleep spindle rates in the centrotemporal region compared to controls (p=0.008, mean decrease 2.5 spindles/min, 95% CI [0.7, 4.4] spindles/min). Spindle rate positively predicted sleep-dependent memory consolidation (p=0.004, mean MST improvement of 3.9%, 95% CI [1.3, 6.4]%, for each unit increase in spindles per minute). DiscussionChildren with Rolandic epilepsy have a sleep spindle deficit during the active period of disease which predicts deficits in sleep dependent memory consolidation. This finding provides a mechanism and noninvasive biomarker to aid diagnosis and therapeutic discovery for cognitive dysfunction in Rolandic epilepsy and related sleep activated epilepsy syndromes.

neuroscience↗

bioRxiv2024

Hippocampal ripples mediate motor learning during brief rest breaks in humans

Critical aspects of motor learning and memory happen offline, during both wake and sleep. When healthy young people learn a motor sequence task, most of their performance improvement happens not while typing, but offline, during interleaved rest breaks. In contrast, the performance of patients with dense amnesia due to hippocampal damage actually gets worse over the rest breaks and improves while typing. These findings indicate that an intact hippocampus is necessary for offline motor learning during wake, but do not specify its mechanism. Here, we studied epilepsy patients (n=17) undergoing direct intracranial EEG monitoring of the hippocampus as they learned the same motor sequence task. Like healthy young people, they showed greater speed gains across rest breaks than while typing. They also showed higher hippocampal ripple rates during these rest breaks that predicted offline gains in speed. This suggests that motor learning during brief rest breaks during wake is mediated by hippocampal ripples. These results expand our understanding of the role of hippocampal ripples beyond declarative memory to include enhancing motor procedural memory. Significance StatementIn patients with epilepsy undergoing direct intracranial EEG monitoring of the hippocampus, we found a higher rate of ripples during the brief rest breaks of a motor sequence task than during task execution. These offline hippocampal ripples predicted the amount of performance improvement over the break. We conclude that the hippocampus contributes to motor learning during brief rest breaks, and propose that this offline learning is mediated by ripples.

neuroscience↗

bioRxiv2024

Photothrombosis induced cortical stroke produces electrographic epileptic biomarkers in mice

ObjectiveInterictal epileptiform spikes, high-frequency ripple oscillations, and their co-occurrence (spike ripples) in human scalp or intracranial voltage recordings are well-established epileptic biomarkers. While clinically significant, the neural mechanisms generating these electrographic biomarkers remain unclear. To reduce this knowledge gap, we introduce a novel photothrombotic stroke model in mice that reproduces focal interictal electrographic biomarkers observed in human epilepsy. MethodsWe induced a stroke in the motor cortex of C57BL/6 mice unilaterally (N=7) using a photothrombotic procedure previously established in rats. We then implanted intracranial electrodes (2 ipsilateral and 2 contralateral) and obtained intermittent local field potential (LFP) recordings over several weeks in awake, behaving mice. We evaluated the LFP for focal slowing and epileptic biomarkers - spikes, ripples, and spike ripples - using both automated and semi-automated procedures. ResultsDelta power (1-4 Hz) was higher in the stroke hemisphere than the non-stroke hemisphere in all mice (p<0.001). Automated detection procedures indicated that compared to the non-stroke hemisphere, the stroke hemisphere had an increased spike ripple (p=0.006) and spike rates (p=0.039), but no change in ripple rate (p=0.98). Expert validation confirmed the observation of elevated spike ripple rates (p=0.008) and a trend of elevated spike rate (p=0.055) in the stroke hemisphere. Interestingly, the validated ripple rate in the stroke hemisphere was higher than the non-stroke hemisphere (p=0.031), highlighting the difficulty of automatically detecting ripples. Finally, using optimal performance thresholds, automatically detected spike ripples classified the stroke hemisphere with the best accuracy (sensitivity 0.94, specificity 0.94). SignificanceCortical photothrombosis-induced stroke in commonly used C57BL/6 mice produces electrographic biomarkers as observed in human epilepsy. This model represents a new translational cortical epilepsy model with a defined irritative zone, which can be broadly applied in transgenic mice for cell type specific analysis of the cellular and circuit mechanisms of pathologic interictal activity. Key PointsO_LICortical photothrombosis in mice produces stroke with characteristic intermittent focal delta slowing. C_LIO_LICortical photothrombosis stroke in mice produces the epileptic biomarkers spikes, ripples, and spike ripples. C_LIO_LIAll biomarkers share morphological features with the corresponding human correlate. C_LIO_LISpike ripples better lateralize to the lesional cortex than spikes or ripples. C_LIO_LIThis cortical model can be applied in transgenic mice for mechanistic studies. C_LI

neuroscience↗

bioRxiv2024

Iterative Bleaching Extends Multiplexity (IBEX) imaging facilitates simultaneous identification of all cell types in the vertebrate retina

The vertebrate retina is a complex multicellular tissue made up of distinct neuron types and glia, arranged in a stereotypic layered organisation to facilitate vision. Understanding how these cell types come together to form precise circuits during development requires the ability to simultaneously discriminate between multiple cell types and their spatial position in the same tissue. Currently, we have a limited capacity to resolve all constitutive cell types and their relationships to one another due to our limited ability to combine multiple cellular markers. To extend this capacity, we have adapted a highly multiplexed immunohistochemistry technique known as Iterative Bleaching Extends Multiplexity (IBEX) and applied it to the development of the zebrafish (Danio rerio) retina. IBEX allows for multiple rounds of cellular labelling to be performed, before imaging and integration of data, resulting in the ability to visualise multiple markers on the same tissue. We have optimised IBEX in zebrafish using fluorescent micro-conjugation of known antibody markers to label the complete retina with up to 11 cell-specific antibodies. We have further adapted the IBEX technique to be compatible with fluorescent transgenic reporter lines, in situ hybridisation chain reaction (HCR), and wholemount immunohistochemistry (WMIHC). We then took advantage of IBEX to explore the multicellular relationships in the developing retina between glial cells and neurons and photoreceptor subtypes. Finally, we tested IBEX on retinas from the emerging ageing model, the killifish (Nothobranchius furzeri), and developmental model, the African clawed frog (Xenopus laevis), demonstrating the usefulness of the technique across multiple species. The techniques described here can be applied to any tissue in any organism where antibodies are readily available to efficiently explore cellular relationships in the context of development, ageing or disease.

developmental biology↗

bioRxiv2023

Preliminary evidence of a relationship between sleep spindles and treatment response in epileptic encephalopathy

ObjectiveEpileptic encephalopathy with spike wave activation in sleep (EE-SWAS) is a challenging neurodevelopmental disease characterized by abundant epileptiform spikes during non-rapid eye movement (NREM) sleep accompanied by cognitive dysfunction. The mechanism of cognitive dysfunction is unknown, but treatment with high-dose diazepam may improve symptoms. Spike rate does not predict treatment response, but spikes may disrupt sleep spindles. We hypothesized that in patients with EE-SWAS: 1) spikes and spindles would be anticorrelated, 2) high-dose diazepam would increase spindles and decrease spikes, and 3) spindle response would be greater in those with cognitive improvement. MethodsConsecutive EE-SWAS patients treated with high-dose diazepam that met criteria were included. Using a validated automated spindle detector, spindle rate, duration, and percentage were computed in pre- and post-treatment NREM sleep. Spikes were quantified using a validated automated spike detector. Cognitive response was determined from chart review. ResultsSpindle rate was anticorrelated with spike rate in the channel with the maximal spike rate (p=0.002) and averaged across all channels (p=0.0005). Spindle rate, duration, and percentage each increased, and spike rate decreased, after high-dose diazepam treatment (p[≤]2e-5, all tests). Spindle rate, duration, and percentage (p[≤]0.004, all tests) were increased in patients with cognitive improvement after treatment, but not those without. Changes in spike rate did not distinguish between groups. InterpretationThese findings confirm thalamocortical disruption in EE-SWAS, identify a mechanism through which benzodiazepines may support cognitive recovery, and introduce sleep spindles as a promising mechanistic biomarker to detect treatment response in severe epileptic encephalopathies.

neuroscience↗

bioRxiv2023

Human thalamic recordings reveal that epileptic spikes block sleep spindle production during non-rapid eye movement sleep

In severe epileptic encephalopathies, epileptic activity contributes to progressive cognitive dysfunction. Several epileptic encephalopathies share the trait of spike-wave activation during non-rapid eye movement sleep (EE-SWAS), a state dominated by sleep oscillations known to coordinate offline memory consolidation. How epileptic activity impacts these thalamocortical sleep oscillations has not been directly observed in humans. Using a unique dataset of simultaneous human thalamic and cortical recordings in subjects with and without EE-SWAS, we reconcile prior conflicting observations about how epileptic spikes coordinate with sleep oscillations and provide direct evidence for epileptic spike interference of sleep spindle production. We find that slow oscillations facilitate both epileptic spikes and sleep spindles during stage 2 sleep (N2) at different phases of the slow oscillation. We show that sleep activated cortical epileptic spikes propagate to the thalamus (thalamic spike rate is increased after a cortical spike, p[~]0). Thalamic spikes increase the spindle refractory period (p<1.5e-21). In patients with EE-SWAS, the abundance of thalamic spikes result in downregulation of spindles for 30 seconds after each thalamic spike (p=3.4e-11) and decreased overall spindle rate across N2 (p=2e-7). These direct human thalamocortical observations identify a novel mechanism through which epileptiform spikes could impact cognitive function, wherein sleep-activated epileptic spikes inhibit thalamic sleep spindles in epileptic encephalopathy.

neuroscience↗

bioRxiv2023

A parsimonious model of the universal 1/f-like behavior of neural field spectra

Consistent observations across recording modalities, experiments, and neural systems find neural field spectra with 1/f-like scaling, eliciting many alternative theories to explain this universal phenomenon. We show that a general dynamical system with stochastic drive and minimal assumptions generates 1/f-like spectra consistent with the range of values observed in vivo, without requiring a specific biological mechanism or collective critical behavior.

neuroscience↗

bioRxiv2023

Different methods to estimate the phase of neural rhythms agree, but only during times of low uncertainty

Rhythms are a common feature of brain activity. Across different types of rhythms, the phase has been proposed to have functional consequences, thus requiring its accurate specification from noisy data. Phase is conventionally specified using techniques that presume a frequency band-limited rhythm. However, in practice, observed brain rhythms are typically non-sinusoidal and amplitude modulated. How these features impact methods to estimate phase remains unclear. To address this, we consider three phase estimation methods, each with different underlying assumptions about the rhythm. We apply these methods to rhythms simulated with different generative mechanisms and demonstrate inconsistency in phase estimates across the different methods. We propose two improvements to the practice of phase estimation: (1) estimating confidence in the phase estimate, and (2) examining the consistency of phase estimates between two (or more) methods. Significant StatementRhythms in the brain can coordinate the activity of individual neurons and communication within brain networks, making these rhythms a target for therapeutic interventions. Brain rhythms manifest in diverse ways, appearing with sinusoidal and non-sinusoidal waveforms, multiple peak frequencies, and variable durations. Across this diversity of rhythms, an important feature to characterize is the rhythms phase. In this manuscript, we demonstrate the ambiguity inherent in estimating phase from neural data. We propose estimating uncertainty in the phase and comparing multiple phase estimators improves phase estimation in practice.

neuroscience↗

bioRxiv2022

Normative sleep spindle database and findings from 772 healthy children from birth through 18 years

Work in the last two decades has identified sleep spindles, discrete "sigma band" oscillations during stage 2 sleep, as a key oscillatory mechanism required for off-line memory consolidation. Although, sleep spindles are known to evolve concomitant with brain maturation and reflect cognitive function across the lifespan, the details of this developmental trajectory are unknown. To address this, we curated a database of sleep electroencephalograms from 772 developmentally normal children to characterize spindles from birth through 18 years. After validating an automated spindle detector against ~20,000 hand-marked spindles across ages, we demonstrate that sleep spindle features follow distinct age-specific patterns in distribution, rate, duration, frequency, estimated refractory period, and inter-hemispheric spindle lag. These data expand our current knowledge of normal physiological brain development and provide a large normative database to detect deviations in sleep spindles to aid discovery, biomarker development, and diagnosis in pediatric neurodevelopmental disorders.

neuroscience↗

bioRxiv2020

IBEX: A versatile multi-plex optical imaging approach for deep phenotyping and spatial analysis of cells in complex tissues

The diverse composition of mammalian tissues poses challenges for understanding the cell-cell interactions required for organ homeostasis and how spatial relationships are perturbed during disease. Existing methods such as single-cell genomics, lacking a spatial context, and traditional immunofluorescence, capturing only 2-6 molecular features, cannot resolve these issues. Imaging technologies have been developed to address these problems, but each possesses limitations that constrain widespread use. Here we report a new method that overcomes major impediments to highly multi-plex tissue imaging. Iterative Bleaching Extends multi-pleXity (IBEX) uses an iterative staining and chemical bleaching method to enable high resolution imaging of >65 parameters in the same tissue section without physical degradation. IBEX can be employed with various types of conventional microscopes and permits use of both commercially available and user-generated antibodies in an open system to allow easy adjustment of staining panels based on ongoing marker discovery efforts. We show how IBEX can also be used with amplified staining methods for imaging strongly fixed tissues with limited epitope retention and with oligonucleotide-based staining, allowing potential cross-referencing between flow cytometry, Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq), and IBEX analysis of the same tissue. To facilitate data processing, we provide an open source platform for automated registration of iterative images. IBEX thus represents a technology that can be rapidly integrated into most current laboratory workflows to achieve high content imaging to reveal the complex cellular landscape of diverse organs and tissues. Significance StatementSingle cell flow cytometry and genomic methods are rapidly increasing our knowledge of the diversity of cell types in metazoan tissues. However, suitably robust methods for placing these cells in a spatial context that reveal how their localization and putative interactions contribute to tissue physiology and pathology are still lacking. Here we provide a readily accessible pipeline (IBEX) for highly multi-plex immunofluorescent imaging that enables a fine-grained analysis of cells in their tissue context. Additionally, we describe extensions of the IBEX workflow to handle hard to image tissue preparations and a method to facilitate direct integration of the imaging data with flow cytometry and sequencing technologies.

immunology↗