Biology subjects

Symvoulidis, P.

Publications and source records attributed to Symvoulidis, P..

4 recordsLinked to original sources

bioRxiv2024

Dense, Continuous Membrane Labeling and Expansion Microscopy Visualization of Ultrastructure in Tissues

Lipid membranes are key to the nanoscale compartmentalization of biological systems, but fluorescent visualization of them in intact tissues, with nanoscale precision, is challenging to do with high labeling density. Here, we report ultrastructural membrane expansion microscopy (umExM), which combines a novel membrane label and optimized expansion microscopy protocol, to support dense labeling of membranes in tissues for nanoscale visualization. We validated the high signal-to-background ratio, and uniformity and continuity, of umExM membrane labeling in brain slices, which supported the imaging of membranes and proteins at a resolution of [~]60 nm on a confocal microscope. We demonstrated the utility of umExM for the segmentation and tracing of neuronal processes, such as axons, in mouse brain tissue. Combining umExM with optical fluctuation imaging, or iterating the expansion process, yielded [~]35 nm resolution imaging, pointing towards the potential for electron microscopy resolution visualization of brain membranes on ordinary light microscopes.

bioengineering↗

bioRxiv2023

Imaging the voltage of neurons distributed across entire brains of larval zebrafish

Neurons interact in networks distributed throughout the brain. Although much effort has focused on whole-brain calcium imaging, recent advances in genetically encoded voltage indicators (GEVIs) raise the possibility of imaging voltage of neurons distributed across brains. To achieve this, a microscope must image at high volumetric rate and signal-to-noise ratio. We present a remote scanning light-sheet microscope capable of imaging GEVI-expressing neurons distributed throughout entire brains of larval zebrafish at a volumetric rate of 200.8 Hz. We measured voltage of [~]1/3 of the neurons of the brain, distributed throughout. We observed that neurons firing at different times during a sequence were located at different brain locations, for sequences elicited by a visual stimulus, which mapped onto locations throughout the optic tectum, as well as during stimulus-independent bursts, which mapped onto locations in the cerebellum and medulla. Whole-brain voltage imaging may open up frontiers in the fundamental operation of neural systems.

neuroscience↗

bioRxiv2022

Cell-type specific developmental defects in PTEN-mutant cortical organoids converge on abnormal circuit activity

De novo heterozygous loss-of-function mutations in PTEN are strongly associated with Autism spectrum disorders (ASD); however, it is unclear how heterozygous mutations in this gene affects different cell types during human brain development, and how these effects vary across individuals. Here, we used human cortical organoids from different donors to identify cell-type-specific developmental events that are affected by heterozygous mutations in PTEN. We profiled individual organoids by single-cell RNA-seq, proteomics and spatial transcriptomics, and revealed abnormalities in developmental timing in human outer radial glia progenitors and deep layer cortical projection neurons, which varied with the donor genetic background. Calcium imaging in intact organoids showed that both accelerated and delayed neuronal development phenotypes resulted in similar abnormal activity of local circuits, irrespective of genetic background. The work reveals donor-dependent, cell-type specific developmental phenotypes of PTEN heterozygosity that later converge on disrupted neuronal activity.

neuroscience↗

bioRxiv2019

Beyond early development: observing zebrafish over 6 weeks with hybrid optical and optoacoustic imaging

Zebrafish animal models have traditionally been used in developmental biology studies but have recently become promising models of cancer, tissue regeneration and metabolic disorders, as well as efficient platforms for functional genomics and phenotype-based drug discovery. Most studies of zebrafish have examined only the embryonic or larval stages of development, yet many questions in developmental biology and biomedicine require analysis of adults, when zebrafish are large and opaque. Conventional microscopy methods are highly sensitive to light scattering and therefore cannot be applied to zebrafish older than a few weeks. We describe a novel multi-modality system that can observe zebrafish from the larval stage to adulthood. Using a hybrid platform for concurrent selective plane illumination microscopy (SPIM) and optoacoustic mesoscopy we show continuous imaging of fish growth over 47 days of development at a similar object size-to-resolution ratio. Using multiple wavelength illumination over the visible and short-wavelength infrared regions, we reveal that the optoacoustic method can follow GFP-based contrast used in SPIM, enabling molecular imaging interrogation in adult fish. Moreover we optoacoustically reveal many other features of zebrafish based on optical contrast not present in SPIM, including contrast from endogenous blood, water and lipids. The hybrid method presented can extend optical imaging to adult zebrafish employed as model systems for studying long-term processes in development, cancer, diabetes and other disorders.

biophysics↗