Biology subjects

Mertz, J.

Publications and source records attributed to Mertz, J..

18 recordsLinked to original sources

bioRxiv2026

Subthreshold membrane depolarization powerfully engages intracellular calcium dynamics in the brain

Membrane voltage (Vm) regulates spike timing and intracellular signaling. While Vm is extensively modulated by behavior, it is unclear how subthreshold Vm dynamics engage intracellular signaling in the awake mammalian brain. We developed a bicistronic viral vector to express genetically encoded and color compatible voltage and calcium (Ca2+) indicators in the same neuron, and simultaneously recorded cellular Vm and Ca2+ dynamics in awake mice. We report that prolonged subthreshold Vm depolarization is closely accompanied by prominent large amplitude Ca2+ elevation, whereas isolated spikes are coupled with weak Ca2+ rise. Additionally, individual spikes differentially engage intracellular Ca2+ dynamics depending on post-spiking Vm depolarization, consistent with a prominent role of slow Vm depolarization in regulating cellular signaling. While brief intracranial electrical stimulation consistently leads to Vm depolarization and Ca2+ increase, longer stimulation disrupts Vm and Ca2+ coupling, highlighting a tightly regulated cellular mechanism that relays slow Vm depolarization to intracellular signaling.

neuroscience↗

bioRxiv2025

Relative phase of membrane potential theta oscillations between individual hippocampal neurons code space

The timing of spikes dictates a neurons impact on downstream circuits and behavior, and spike timing is determined by the membrane potential (Vm). However, due to technical challenges, it has been impossible to analyze the relative timing of Vm dynamics between neurons during behavior. Using large scale membrane voltage imaging, we simultaneously recorded Vm from many individual hippocampal neurons in animals engaged in a virtual spatial task. We found that relative phase of Vm theta oscillations across neurons exhibit gradual or discrete shifts depending on spatial position. This finding extends beyond previous studies showing Vm dynamics in single neurons or spiking activity in multiple neurons, revealing previously unknown evidence for consistent coding of space by spike-independent relative phase of Vm theta dynamics between neurons.

neuroscience↗

bioRxiv2025

Superresolution imaging of live samples by centroid reassignment microscopy

Superresolution imaging has become one of the most important recent advances in microscopy development. However, most superresolution methods are ill-adapted for live-sample imaging because they are unacceptably slow, susceptible to artifacts, or require the use of specialized fluorophores and labeling protocols. We introduce a superresolution method called centroid reassignment microscopy (CRM) that overcomes these limitations. CRM is a simple variation on confocal microscopy wherein the single-element detector and small pinhole are replaced by a centroid detector and larger pinhole. Superresolution is obtained by reassigning the detected fluorescence to its centroid location of as a function of the scanning excitation focus location. Our method bears resemblance to the method of image scanning microscopy, which involves the use of an array detector, with the advantage that CRM provides improved resolution for the same number of detected photons while being simpler to implement and less sensitive to noise. CRM is light-efficient, fast (single-frame), robust to defocus aberrations, and requires no changes whatsoever in standard fluorescence imaging protocols, making it uniquely attractive for superresolution imaging of live, dynamic samples.

bioengineering↗

bioRxiv2025

Three-photon population imaging of subcortical brain regions

Recording activity from large cell populations in deep neural circuits is essential for understanding brain function. Three-photon (3P) imaging is an emerging technology that allows for imaging of structure and function in subcortical brain structures. However, increased tissue heating, as well as the low repetition rate sources inherent to 3P imaging, have limited the fields of view (FOV) to areas of [≤] 0.3 mm2. Here we present a Large Imaging Field of view Three-photon (LIFT) microscope with a FOV of >3 mm2. LIFT combines high numerical aperture (NA) optimized sampling, using a custom scanning module, with deep learning-based denoising, to enable population imaging in deep brain regions. We demonstrate non-invasive calcium imaging in the mouse brain from >1500 cells across CA1, the surrounding white matter, and adjacent deep layers of the cortex, and show population imaging with high signal-to-noise in the rat cortex at a depth of 1.2 mm. The LIFT microscope was built with all off-the-shelf components and allows for a flexible choice of imaging scale and rate.

neuroscience↗

bioRxiv2024

Ultrasensitive in vivo infrared spectroscopic imaging via oblique photothermal microscopy

In vivo IR spectroscopy faces challenges due to poor sensitivity in reflection mode and low resolution at micrometer scale. To break this barrier, we report an oblique photothermal microscope (OPTM) to enable ultrasensitive IR spectroscopic imaging of live subjects at sub-micron resolution. Classic photothermal measurement captures only a small fraction of probe photons through a pinhole to extract the photothermal signal. Instead, OPTM uses a differential split detector placed on the sample surface to collect 500-fold more photons and suppress the laser noise by 12 fold via balanced detection. Leveraging its improved sensitivity, OPTM enables low-dose IR imaging of skin without photodamage. Depth-resolved in vivo OPTM imaging of metabolic markers beneath mouse and human skin is shown. Furthermore, we demonstrate in vivo OPTM tracking of topical drug contents within mouse and human skin. Collectively, OPTM presents a highly sensitive imaging platform for in vivo and in situ molecular analysis.

bioengineering↗

bioRxiv2023

Multiplane HiLo microscopy with speckle illumination and non-local means denoising

SignificanceHiLo microscopy synthesizes an optically-sectioned image from two images, one obtained with uniform and another with patterned illumination, such as laser speckle. Speckle-based HiLo has the advantage of being robust to aberrations, but is susceptible to residual speckle noise that is difficult to control. We present a computational method to reduce this residual noise without compromising spatial resolution. In addition, we improve the versatility of HiLo microscopy by enabling simultaneous multiplane imaging (here 9 planes). AimOur goal is to perform fast, high contrast multiplane imaging with a conventional camera-based fluorescence microscope. ApproachMultiplane HiLo imaging is achieved with the use of a single camera and z-splitter prism. Speckle noise reduction is based on the application of a non-local means (NLM) denoising method to perform ensemble averaging of speckle grains. ResultsWe demonstrate the capabilities of multiplane HiLo with NLM denoising both with synthesized data and by imaging cardiac and brain activity in zebrafish larvae at 40 Hz frame rates. ConclusionsMultiplane HiLo microscopy aided by NLM denoising provides a simple tool for fast, opticallysectioned volumetric imaging that can be of general utility for fluorescence imaging applications.

neuroscience↗

bioRxiv2023

Resolution enhancement with deblurring by pixel reassignment (DPR)

Improving the spatial resolution of a fluorescence microscope has been an ongoing challenge in the imaging community. To address this challenge, a variety of approaches have been taken, ranging from instrumentation development to image post-processing. An example of the latter is deconvolution, where images are numerically deblurred based on a knowledge of the microscope point spread function. However, deconvolution can easily lead to noise-amplification artifacts. Deblurring by post-processing can also lead to negativities or fail to conserve local linearity between sample and image. We describe here a simple image deblurring algorithm based on pixel reassignment that inherently avoids such artifacts and can be applied to general microscope modalities and fluorophore types. Our algorithm helps distinguish nearby fluorophores even when these are separated by distances smaller than the conventional resolution limit, helping facilitate, for example, the application of single-molecule localization microscopy in dense samples. We demonstrate the versatility and performance of our algorithm under a variety of imaging conditions.

cell biology↗

bioRxiv2023

Large-scale deep tissue voltage imaging with targeted illumination confocal microscopy

Voltage imaging with cellular specificity has been made possible by the tremendous advances in genetically encoded voltage indicators (GEVIs). However, the kilohertz rates required for voltage imaging lead to weak signals. Moreover, out-of-focus fluorescence and tissue scattering produce background that both undermines signal-to-noise ratio (SNR) and induces crosstalk between cells, making reliable in vivo imaging in densely labeled tissue highly challenging. We describe a microscope that combines the distinct advantages of targeted illumination and confocal gating, while also maximizing signal detection efficiency. The resulting benefits in SNR and crosstalk reduction are quantified experimentally and theoretically. Our microscope provides a versatile solution for enabling high-fidelity in vivo voltage imaging at large scales and penetration depths, which we demonstrate across a wide range of imaging conditions and different GEVI classes.

neuroscience↗

bioRxiv2022

High-throughput deep tissue two-photon microscopy at kilohertz frame rates

Strategies to image biological phenomena at millisecond time scales are generally technically challenging and suffer from compromises between imaging field-of-view, depth penetration and excitation efficiency in thick tissue. We present a simple and cost-effective solution that enables a conventional video-rate two-photon microscope (2PM) to perform 2D scanning at kilohertz frame rates, while preserving all the benefits of standard 2PM, which we demonstrate by imaging neurovascular dynamics in mouse brains.

bioengineering↗

bioRxiv2022

Single and complex spikes relay distinct frequency-dependent circuit information in the hippocampus

Hippocampal neurons generate either single spikes or stereotyped bursts of spikes known as complex spikes. Although single and complex spikes co-occur in the same neuron, their contribution to information processing remains unclear. We analyzed hippocampal CA1 neurons in awake mice and in behaving rats, combining cellular membrane voltage imaging with optogenetics and extracellular recordings. We found that network-driven subthreshold membrane rhythms in the theta versus gamma frequencies preferably entrained complex versus single spikes in individual neurons. Optogenetic membrane perturbation revealed a causal link between subthreshold theta and gamma power and the initiation of complex versus single spikes. Further, single and complex spikes exhibited different place field properties and frequency-dependent coding during spatial navigation. Thus, individual hippocampal neurons do not integrate theta and gamma rhythms into a combined spike timing code, but instead, transmit frequency-specific information as distinct output modes of single versus complex spikes during spatial cognition.

neuroscience↗

bioRxiv2021

High-speed, multi-Z confocal microscopy for voltage imaging in densely labeled neuronal populations

Genetically encoded voltage indicators (GEVIs) hold great promise for monitoring neuronal population activity, but GEVI imaging in dense neuronal populations remains difficult due to a lack of contrast and/or speed. To address this challenge, we developed a novel confocal microscope that allows simultaneous multiplane imaging with high-contrast at near-kHz rates. This approach enables high signal-to-noise ratio voltage imaging in densely labeled populations and minimizes optical crosstalk during concurrent optogenetic photostimulation.

neuroscience↗

bioRxiv2021

Sensitivity optimization of a rhodopsin-based fluorescent voltage indicator

The ability to optically image cellular transmembrane voltage at millisecond-timescale resolution can offer unprecedented insight into the function of living brains in behaving animals. The chemigenetic voltage indicator Voltron is bright and photostable, making it a favorable choice for long in vivo imaging of neuronal populations at cellular resolution. Improving the voltage sensitivity of Voltron would allow better detection of spiking and subthreshold voltage signals. We performed site saturation mutagenesis at 40 positions in Voltron and screened for increased {Delta}F/F0 in response to action potentials (APs) in neurons. Using a fully automated patch-clamp system, we discovered a Voltron variant (Voltron.A122D) that increased the sensitivity to a single AP by 65% compared to Voltron. This variant (named Voltron2) also exhibited approximately 3-fold higher sensitivity in response to sub-threshold membrane potential changes. Voltron2 retained the sub-millisecond kinetics and photostability of its predecessor, with lower baseline fluorescence. Introducing the same A122D substitution to other Ace2 opsin-based voltage sensors similarly increased their sensitivity. We show that Voltron2 enables improved sensitivity voltage imaging in mice, zebrafish and fruit flies. Overall, we have discovered a generalizable mutation that significantly increases the sensitivity of Ace2 rhodopsin-based sensors, improving their voltage reporting capability.

neuroscience↗

bioRxiv2021

High-speed multifocus phase imaging in thick tissue

Phase microscopy is widely used to image unstained biological samples. However, most phase imaging techniques require transmission geometries, making them unsuited for thick sample applications. Moreover, when applied to volumetric imaging, phase imaging generally requires large numbers of measurements, often making it too slow to capture live biological processes with fast 3D index-of-refraction variations. By combining oblique back-illumination microscopy and a z-splitter prism, we perform phase imaging that is both epi-mode and multifocus, enabling high-speed 3D phase imaging in thick, scattering tissues with a single camera. We demonstrate here 3D qualitative phase imaging of blood flow in chick embryos over a field of view of 546 x 546 x 137 m3 at speeds up to 47 Hz.

bioengineering↗

bioRxiv2021

Large-scale voltage imaging in the brain using targeted illumination

Recent improvements in genetically encoded voltage indicators enabled optical imaging of action potentials and subthreshold membrane voltage dynamics from single neurons in the mammalian brain. To perform high speed voltage imaging, widefield microscopy remains an essential tool for recording activity from many neurons simultaneously over a large anatomical area. However, the lack of optical sectioning makes widefield microscopy more prone to background signal contamination, and thus far voltage imaging using fully genetically encoded voltage indicators remains limited to simultaneous sampling of a few cells over a restricted field-of-view. We here demonstrate a strategy for large scale voltage imaging using the fully genetically encoded voltage indicator SomArchon and targeted illumination. We implemented a simple, low-cost digital micromirror device based targeted illumination strategy to restrict illumination to the cells of interest, and systematically quantified the improvement of this microscopy design theoretically and experimentally with SomArchon expressing neurons in single layer cell cultures and in the brains of awake mice. We found that targeted illumination, in comparison to widefield illumination, increased SomArchon signal contrast and reduced background cross-contamination in the brain. Such improvement permitted the reduction of illumination intensity, and thus reduced fluorescence photobleaching and prolonged imaging duration. When coupled with a high-speed, large area sCMOS camera, we routinely imaged tens of spiking neurons simultaneously over minutes in the brain. Thus, the widefield microscopy design with an integrated targeted illumination system described here offers a simple solution for voltage imaging analysis of large neuron populations in behaving animals.

neuroscience↗

bioRxiv2020

High-contrast multifocus microscopy with a single camera and z-splitter prism

We present a multifocus imaging strategy based on the use of a simple z-splitter prism that can be assembled from off-the-shelf components. Our technique enables a widefield image stack to be distributed onto a single camera and recorded simultaneously. We exploit the volumetric nature of our image acquisition by further introducing a novel extended-volume 3D deconvolution strategy to suppress far-out-of-focus fluorescence background to significantly improve the contrast of our recorded images, conferring to our system a capacity for quasi optical sectioning. By swapping in different z-splitter configurations, we can prioritize high speed or large 3D field-of-view imaging depending on the application of interest. Moreover, our system can be readily applied to a variety of imaging modalities in addition to fluorescence, such as phase-contrast and darkfield imaging. Because of its simplicity, versatility, and performance, we believe our system will be a useful tool for general biological or biomedical imaging applications.

bioengineering↗

bioRxiv2020

In vivo corneal and lenticular microscopy with asymmetric fundus retroillumination

We describe a new technique for non-contact in vivo corneal and lenticular microscopy. It is based on fundus retro-reflection and back-illumination of the crystalline lens and cornea. To enhance phase-gradient contrast, we apply asymmetric illumination by illuminating one side of the fundus. The technique produces micron-scale lateral resolution across a 1-mm diagonal field of view. We show representative images of the epithelium, the subbasal nerve plexus, large stromal nerves, dendritic immune cells, endothelial nuclei, and the anterior crystalline lens, demonstrating the potential of this instrument for clinical applications.

biophysics↗

bioRxiv2018

Non-mydriatic chorioretinal imaging in a transmission geometry and application to retinal oximetry

The human retina is typically imaged in a reflection geometry, where light is delivered through the pupil and images are formed from the light reflected back from the retina. In this configuration, artifacts caused by retinal surface reflex are often encountered, which complicate quantitative interpretation of the reflection images. We present an alternative illumination method, which avoids these artifacts. The method uses deeply penetrating near-infrared (NIR) light delivered transcranially from the side of the head, and exploits multiple scattering to redirect a portion of the light towards the posterior eye. This unique transmission geometry simplifies absorption measurements and enables flash-free, non-mydriatic imaging as deep as the choroid. Images taken with this new transillumination approach are applied to retinal oximetry.\n\nOCIS codes: (170.4460) Ophthalmic optics and devices; (170.2945) Illumination design; (170.1470) Blood or tissue constituent monitoring.

biophysics↗

bioRxiv2018

Axon Guidance Signaling Modulates Epithelial to Mesenchymal Transition in Stem Cell-Derived Retinal Pigment Epithelium

The critical role of epithelial to mesenchymal transition (EMT) in embryonic development, malignant transformation, and tumor progression has been well studied in normal and cancerous tissues and cells. Interestingly, EMT has also been reported to play a key role in the early progression of several retinal degenerative diseases, including scarring associated proliferative vitro-retinopathy (PVR), choroidal neo-vascularization induced \"wet\" age-related macular degeneration (AMD) and diabetic retinopathy (DR). Despite these studies, many questions remain unexplored regarding EMT-associated retinal pigment epithelium (RPE) degeneration and dysfunction. We hypothesize that RPE cells undergo EMT prior to cell death during the progression of atrophic \"dry\" AMD. Utilizing human stem cell-derived RPE (hRPE) as a model to study RPE EMT, we optimized two independent but complementary RPE EMT induction systems: 1) enzymatic dissociation of hRPE monolayer cultures and 2) co-treatment of hRPE monolayer cultures with transforming growth factor beta (TGF-{beta}) and the inflammatory cytokine, tumor necrosis factor alpha (TNF-). To further understand the molecular mechanisms of RPE EMT regulation, we performed an RNA-Sequencing (RNA-Seq) time course examination across 48 hours beginning with EMT induction. Our transcriptome profiling provides a comprehensive quantification of dynamic signaling events and associated biological pathways underlying RPE EMT and reveals an intriguing significance for widespread dysregulation of multiple axon guidance molecules in this process.

genomics↗